Available online at

http://jurnal.permi.or.id/index.php/mionline
ISSN 1978-3477, eISSN 2087-8575 DOI: 10.5454/mi.9.3.4
Vol 9, No 3, September 2015, p 120-128

Optimization of Surfactin Production by Bacillus amyloliquefaciens MD4-12

Using Response Surface Methodology

AHMAD WIBISANA1*, WAHONO SUMARYONO2,3, TJAHJANI MIRAWATI SUDIRO4, AND

PRATIWI PUDJILESTARI SUDARMONO4
1
Biotech Center-Badan Pengkajian dan Penerapan Teknologi, Gedung 630,
Kawasan Puspiptek Serpong, Tangerang Selatan, Indonesia;
2
Center of Pharmaceutical and Medical Technology, Badan Pengkajian dan Penerapan Teknologi,
Gedung Laptiab Kawasan Puspiptek Serpong Tangerang Selatan, Indonesia;
3
Faculty of Pharmacy Universitas Pancasila, Jalan Srengseng Sawah Jagakarsa Jakarta Selatan, 12640, Indonesia;
4
Department of Microbiology, Faculty of Medicine, Universitas Indonesia/Cipto Mangunkusumo General Hospital,
Jakarta, Jalan Pegangsaan Timur 16 Jakarta, 10320, Indonesia

Surfactin is a lipopeptide biosurfactant that show potential biomedical application due to its activities such as
antiviral, antibacterial, antifungi, anticancer, and antimycoplasma. Bacillus amyloliquefaciens MD4-12, isolated
from oil-contaminated soil, produced promising yield of surfactin in McKeen medium. The production of
surfactin was influenced by many fermentation process parameters such as carbon, nitrogen, minerals and also
environmental conditions such as pH and agitation. Therefore, to obtain high yield of surfactin by B.
amyloliquefaciens MD4-12, optimization of process production was conducted in shake flask fermentation using
response surface methodology. McKeen medium composition was used as basal medium. Screening of the best
carbon and nitrogen source were selected in preliminary experiments followed by selection of the influencing
significant parameters on surfactin production using Plackett-Burman design. Selected parameters were
optimized by central composite design and for the data analysis was used response surface methodology. The
result showed that the optimum medium composition contained (g L-1) 45.0 glucose, 6.33 urea, 1.0 monosodium
glutamate, 1.85 MgSO4.7H2O, 0.4 KCl, 0.5 K2HPO4, and 0.5 mL trace elements. The surfactin yield at optimal
condition was 1.25 g L-1, increased 2.4 times compared to condition prior to optimization.

Key words: lipopeptide, optimization, response surface methodology, surfactin

Surfaktin adalah senyawa biosurfaktan lipopeptida yang sangat potensial untuk aplikasi di bidang biomedis
karena mempunyai berbagai aktifitas biologi seperti antivirus, antibakteri, antijamur, antikanker, dan
antimikoplasma. Bacillus amyloliquefaciens MD4-12, yang diisolasi dari tanah yang tercemar minyak
menunjukkan kemampuan yang tinggi dalam menghasilkan surfaktin menggunakan medium dasar McKeen.
Produksi surfaktin secara fermentasi dipengaruhi oleh berbagai faktor seperti sumber karbon, sumber nitrogen,
mineral serta kondisi lingkungan seperti pH dan agitasi. Dengan menggunakan medium McKeen sebagai
medium dasar, maka pada penelitian ini dilakukan optimasi produksi surfaktin menggunakan metode permukaan
respon. Pada percobaan awal dilakukan pemilihan sumber karbon dan nitrogen terbaik, dan selanjutnya diikuti
dengan pemilihan faktor-faktor yang berpengaruh secara signifikan untuk produksi surfaktin menggunakan
rancangan Plackett-Burman. Faktor-faktor yang terpilih selanjutnya dioptimasi dengan menggunakan central
composite design dan analisis data dilakukan dengan menggunakan metode permukaan respon. Hasil penelitian
menunjukkan bahwa komposisi medium yang optimal terdiriatas (g L-1) glukosa 45,0; urea 6,33; monosodium
glutamat1,0; MgSO4.7H2O 1,85; KCl 0,4,; K2HPO4 0,5 dan mikroelemen 0.5 mL. Pada kondisi optimum
perolehan surfaktin adalah 1,25 g L-1, meningkat 2,4 kali dibandingkan dengan kondisi sebelum optimasi.

Kata kunci: lipopeptida, metode permukaan respon, optimasi, surfaktin

Biosurfactants are microbial metabolites that work al. 2010; Meena et al. 2015). Their important
as surface active agents produced by some characteristics such as biological activities, emulsifying,
microorganisms, including bacteria, yeast and fungi. foaming, soaping and dispersing acquire them for
Interests in biosurfactants are growing due to their multiple applications. Based on their chemical
broad possible applications such as in the pharmaceuti- structures, biosurfactants are divided into five major
cal, cosmetic, agricultural and food industries, classes, i.e. lipopeptides, glycolipids, phospholipids,
environmental protection and crude oil drilling (Banat et neutral lipids, and polymeric compounds. One of the
most important families of lipopeptide class is surfactin.
Surfactin is a cyclic molecules consisting of a fatty acid
*Corresponding author; Phone: +62-21- 7560208, Fax: +62-
21-75603120, Email: awibisana2000@gmail.com of variable length (hydrophobic moiety) linked to a short
Volume 9, 2015 Microbiol Indones 121

peptide chain (hydrophilic moiety) of seven amino taken from Palembang, Indonesia. Furthermore, it was
acids. It has ability to reduce surface tension of water identified as B. amyloliquefaciens MD4-12 by
from 72 mN m-1 to 27 mN m-1 (Cooper et al. 1981). morphological, biochemical, and 16S rRNA sequence
Several biological activities have been reported for analysis.
biomedical application, such as antiviral, antibacterial, Inoculum and Culture Conditions. The strain
antifungi, antitumor, antiinflammatory, antiadhesive, was stored lyophilized and working stock cultures were
antimycoplasma and thrombolytic activity (Huang et al. maintained on nutrient agar slant and stored at 4 °C. For
2006; Sabate et al.2013; Liu et al. 2012; Kim et al. 2007; surfactin production, two-stage inoculum were
Zhang et al. 2015; Zeraik et al. 2010, Lim et al. 2005; prepared as following: one isolated colony from fresh
Meena et al. 2015). culture grown onto nutrient agar medium and incubated
A large variety of Bacillus has been reported to for 24 h, was dispensed in 5 mL of nutrient broth
produce surfactin, nevertheless microbial exploration medium and incubated overnight at 37 °C. The culture
is still needed to obtain high productivity strain. (4 mL) was used to inoculate 100 mL nutrient broth
Bacillus amyloliquefaciens MD4-12 isolated from oil- medium in 500 mL Erlenmeyer flasks and incubated in
contaminated soil showed promising ability to produce a rotatory shaker at 200 rpm and 37 °C (±0.5) for 16 h.
surfactin. According to previous studies, the surfactin At this condition, absorbance around 3 measured
production was influenced by several nutritional spectrophotometrically at 600 nm was reached. For
factors including the nature and concentration of fermentation, 2 mL of culture was inoculated to 250 mL
carbon and nitrogen substrate, the concentration of Erlenmeyer flask containing 50 mL of production
metal ion such as Mg, Fe, Zn, and Mn in the medium medium. Modified McKeen medium composition was
and environmental conditions such as temperature, pH, used as the production medium consist of (g L-1) 20.0
agitation and aeration (Sen 1997; Yi et al. 2013; Sen et glucose, 2.0 monosodium glutamate, 3.0 yeast extract,
al. 1997; Al-Ajlani et al. 2007; Abdel-Mawgoud et al. 1.0 MgSO4.7H2O, 1.0 KCl with addition of 1 mL trace
2008; Amani et al. 2013; Singh et al. 2014a). To element solution (in 100 mL deionized water : 0.64 g
achieve high yield of surfactin production by B. MnSO4.4H2O, 0.16 g CuSO4.5H2O, and 0.015 g
amyloliquefaciens MD4-12, optimization of nutrient FeSO4.7H2O). pH was adjusted at 7.0. Fermentation
supply and environmental condition is very important. was conducted for 28 h.
Statistical technique, including combination of Experimental Design and Data Analysis.
Plackett-Burman design (PBD), central composite Preliminary experiments were conducted to screen
design (CCD) and response surface methodology carbon and nitrogen source. The effect of different
(RSM) are the most widely method used for carbon sources was evaluated by substitution of
optimization of biological processes. The Plackett- glucose in McKeen medium composition with sucrose,
Burman experimental design is a two-level factorial fructose, maltose, dextrose, mannitol, sorbitol,
design, used to determine the critical physicochemical galactose, xylose, and starch. The best carbon source
parameters. Interaction effects among the variables are generated the highest surfactin production was used for
not considered (Mnif et al. 2012). To screen N variables further study. Furthermore, the effect of the different
using PBD, N+1 experiments are needed. The nitrogen source addition was studied similarly by
significant variables obtained by PBD can be further substitution of yeast extract with ammonium nitrate,
optimized using CCD and (RSM). RSM has been used ammonium sulphate, sodium nitrate, soy flour,
extensively in biological process optimization due to peptone, casein hydrolysate, and urea.
its efficiency and accuracy for building of a model, The next step was to screen the significant factors
evaluating the effects of factors and predicting affecting surfactin production by B. amyloliquefaciens
optimum conditions. The objective of the present study MD4-12 using the PBD, as noted in Table 1. All
was to optimize of surfactin production by B. components in the production medium (seven factors)
amyloliquefaciens MD4-12 using response surface and some environmental conditions (two-factor) were
methodology. used to determine the factors that influence
significantly. This experiment required 12 runs as
MATERIALS AND METHODS presented in Table 2. The factors with confidence levels
above 95% were considered as significant effect on
Microorganisms. A surfactin-producing microor- surfactin production.
ganism has been isolated from oil-contaminated soil The selected significant influence factor obtained
122 WIBISANA ET AL. Microbiol Indones

from PBD experiments was optimized with CCD and experimental design and results were presented in Table
RSM as presented in Table 4. Thirty experiments 2. The ANOVA of the experiments (Table 3) showed that
consisted of sixteen factorial points, eight star points the p-value (Prob>F) of the model was 0.0402, implied
and six center points were conducted to optimize four that the model was significant. The magnitude of each
significant factors. For each factor was studied at three factor affecting on surfactin production was indicated by
level concentration (low, middle and high levels) coded p-value. Among the factors screened, the concentration
with (-1, 0, +1). All experiments were conducted of glucose, urea, MgSO4.7H2O, and KCl showed
triplicate and the final result was expressed as the significantly effecting on surfactin production with p-
average value. values, 0.0438, 0.0100, 0.0221, and 0.0262 with the
Statistical Analysis. Design Expert (Version 7.0, coefficient of the model 0.056, 0.12, -0.080, and 0.074
Stat-Ease Inc., USA) software was used to generate the respectively. The other factors have p-value (Prob>F) >
experimental designs and data analysis. The quality of 0.05 that means no significantly affecting on surfactin
polynomial model equation was evaluated statistically production.
using analysis of variance (ANOVA) to determine the Optimization Using Response Surface Methodo-
coefficient of determination, R2 and adjusted R2. The logy. Optimization of four significant factors on
statistical significance of the model and the regression surfactin production was conducted using RSM and the
coefficient were determined using the F-test. results were presented in Table 4. The other factors
Surfactin Analysis. The gravimetric method as were kept at their low level. The experiments result was
described by Liu et al. (2012) was used for modeled with a second-order polynomial equation to
determination of surfactin concentration with minor explain the effect of each factor on surfactin
modification. The broth culture was centrifuged at 10 production, as follows:
000 rpm for 10 min to obtain the cell free supernatant
(CFS). An aliquot of the CFS was acidified to pH 2.0 Y = 1.18 - 0.034 X1 - 4.758E-003 X2 + 0.069 X3 - 0.080
using 6 N HCl, left overnight at 4 °C, and then X4 + 0.057 X1 X2 + 0.018 X1 X3 - 0.11 X1 X4 -
centrifuged at 10 000 rpm for 20 min. The resulting 2.814E-003 X2 X3 + 0.047 X2 X4 + 8.887E-005 X3
supernatant was discarded and the remaining pellet was X4 - 0.041 X12 - 0.077 X22 - 0.065 X32 + 0.023 X42
collected, and then extracted three times with methanol.
The dry pellet was obtained by removal of methanol Where Y was the estimated surfactin production
using a rotary vacuum evaporator. The net weight of and X1, X2, X3 and X4 were coded value for glucose, urea,
pellet was determined and calculated as crude surfactin. MgSO4.7H2O and KCl concentration, respectively. The
Structural characterization of the crude surfactin by results of the second-order response surface model in
mass spectrophotometry revealed that it contained the form of analysis of variance (ANOVA) are given in
surfactin as the major component (data not shown). Table 5. It can be seen that two linear (X3 and X4) and
three quadratic (X1, X2, and X3) terms were significant,
RESULTS with the p-values being very small (p<0.05). The
quadratic effect of X4 was not significant (p>0.05). The
Selection of Carbon Source and Nitrogen significant interaction of variables was shown by X1.X2;
Source. Among the carbon sources used in this X1.X4, and X2.X4 terms (p<0.05). The other interaction
experiment, the use of glucose yielded highest of factors was insignificantly effected on surfactin
surfactin production, i.e. 0.320 g L-1. Furthermore, production. By considering the only significant factors,
from the experiments result to screen the nitrogen the model for surfactin production could be written as
source showed that the use of urea yielded the highest following equation:
surfactin production, i.e. 0.436 g L-1. Therefore,
glucose and urea were used as carbon and nitrogen Y = +1.18 - 0.069 X3 - 0.080 X4 + 0.057 X1 X2 - 0.11
source for the next study. X1 X4 - + 0.047 X2 X4 - 0.041 X12 - 0.077 X22 -
Plackett-Burman Experimental. The experiments 0.065 X32
for selection of nine critical factors affected surfactin
production by B. amyloliquefaciens MD4-12 namely According to the equation, the yield of surfactin
medium composition (seven factors) and environment could be estimated as a linear effect of magnesium
conditions (initial medium pH and fermentation sulphate and potassium chloride concentration and
temperature) were conducted using PBD. The squared effect of glucose, urea and magnesium
Volume 9, 2015 Microbiol Indones 123

Table 1 Plackett-Burman design for screening of significant parameters on surfactin production by Bacillus amyloliquefaciens
MD4-12

Level
Variable Code Unit
-1 +1

Carbon source A g L-1 15 45

Nitrogen source B g L-1 1 9

MSG C g L-1 1 4

MgSO4.7H2O D g L-1 0.5 2

K2HPO4 E g L-1 0.5 2.5

KCl F g L-1 0.3 1

Trace element G mL 0.5 2

pH H 6 8

Temperature I °C 25 35

Table 2 The Plackett-Burman design and the experimental results for the selection of important parameters on the surfactin
production

Variables
Std Surfactin
A B C D E F G H I
g L-1 g L-1 g L-1 g L-1 g L-1 g L-1 mL °C g L-1

1 45 9 1 2 2.5 1 0.5 6 25 0.366

2 15 9 4 0.5 2.5 1 2 6 25 0.413

3 45 1 4 2 0.5 1 2 8 25 0.133

4 15 9 1 2 2.5 0.3 2 8 35 0.048

5 15 1 4 0.5 2.5 1 0.5 8 35 0.207

6 15 1 1 2 0.5 1 2 6 35 0.116

7 45 1 1 0.5 2.5 0.3 2 8 25 0.085

8 45 9 1 0.5 0.5 1 0.5 8 35 0.572

9 45 9 4 0.5 0.5 0.3 2 6 35 0.508

10 15 9 4 2 0.5 0.3 0.5 8 25 0.180

11 45 1 4 2 2.5 0.3 0.5 6 35 0.039

12 15 1 1 0.5 0.5 0.3 0.5 6 25 0.064

124 WIBISANA ET AL. Microbiol Indones

Table 3 ANOVA of the factors affecting on surfactin production using Plackett-Burman design

Coeficient Sum of Mean p-value

Source Squares df F Value
model Square Prob > F

Model 0.39 9 0.043 24.26 0.0402*

Glucose 0.056 0.038 1 0.038 21.32 0.0438*

Urea 0.17 1 0.17 98.32 0.0100*

Yeast Extract 0.019 4.376E-003 1 4.376E-003 2.48 0.2563

MgSO4.7H2O -0.080 0.077 1 0.077 43.83 0.0221*

K2HPO4 -0.035 0.014 1 0.014 8.15 0.1039

KCl 0.074 0.065 1 0.065 36.72 0.0262*

Trace element -0.011 1.355E-003 1 1.355E-003 0.77 0.4737

Initial pH -0.024 6.651E-003 1 6.651E-003 3.76 0.1920

Temperature 0.021 5.243E-003 1 5.243E-003 2.97 0.2272

*) significant at the level > 95.0% (for p-value < 0.05).

sulphate concentration. respectively.

ANOVA of the model for surfactin production Validation of the Optimized Condition. Based on
showed that the regression model was highly the model, the optimized medium composition consists
significant and the lack of fit was insignificant (Table of (g L-1): 45.00 glucose, 6.33 urea, 1 monosodium
5). The high determination coefficient (R2) showed the glutamate, 1.85 MgSO4.7H2O, 0.40 KCl, 0.5 K2HPO4,
goodness of fit of the second order model, and it implied and 0.5 mL trace element. The model predicts the
that only 9.58% the total variations were not explained maximum yield of surfactin as 1.35 g L-1. Under
by the model. The high adjusted determination optimized condition, validation experiments were
coefficient (adj R2) was also satisfactory to confirm the conducted in triplicates. The result of experiments
significance of the model. A lower value of the showed that the yield of average surfactin was 1.25 g L-1,
coefficient of variation (CV) indicates that experiments suggesting that experimental and predicted values of
were precise and reliable (Box et al. 1978). The signal surfactin yield were in a good agreement. The usage of
to noise ratio (adequate precision) for the model was optimized medium determined by response surface
higher than 4, indicating a good fit. methodology for surfactin production could increase
Visualization of the interaction between the 2.4-fold over the yield in non-optimized medium.
response and experimental levels of each variable and
the type of interactions between test variables in order DISCUSSION
to deduce the optimum conditions provided by the 3D
response surface. Fig 1 depicted the 3D response B. amyloliquefaciens MD4-12 is a potential strain
surface showing the interactive effect of the significant for surfactin production. The selection of potential
variable interaction. The other insignificant variables, strain is necessary for future mutation or bioengineering
i.e. concentration of monosodium glutamate, K2HPO4, studies. The significant influence factors on surfactin
and trace element were kept at a minimum level (1 g L-1, production by B. amyloliquefaciens MD4-12 consist of
0.5 g L-1, and 0.5 mL L-1, respectively) and initial pH of glucose, urea, MgSO4.7H2O and KCl selected by PBD
medium and temperature were 7.0 and 30 °C, experiments. B. amyloliquefaciens MD4-12 can use
Volume 9, 2015 Microbiol Indones 125

Table 4 Central composite design and the experimental results for optimization of important parameters on surfactin
production

Std X1:Glucose X2:Urea X3:MgSO4.7H2O X4:KCl Surfactin

(g L-1) (g L-1) (g L-1) (g L-1) (g L-1)

1 30.00 5.00 0.70 0.40 1.088

2 45.00 5.00 0.70 0.40 1.261

3 30.00 9.00 0.70 0.40 0.922

4 45.00 9.00 0.70 0.40 1.064

5 30.00 5.00 2.00 0.40 1.170

6 45.00 5.00 2.00 0.40 1.313

7 30.00 9.00 2.00 0.40 1.033

8 45.00 9.00 2.00 0.40 1.175

9 30.00 5.00 0.70 1.00 1.190

10 45.00 5.00 0.70 1.00 0.556

11 30.00 9.00 0.70 1.00 1.000

12 45.00 9.00 0.70 1.00 0.868

13 30.00 5.00 2.00 1.00 1.180

14 45.00 5.00 2.00 1.00 0.790

15 30.00 9.00 2.00 1.00 1.103

16 45.00 9.00 2.00 1.00 0.988

17 22.50 7.00 1.35 0.70 0.953

18 52.50 7.00 1.35 0.70 1.020

19 37.50 3.00 1.35 0.70 0.783

20 37.50 11.00 1.35 0.70 0.864

21 37.50 7.00 0.05 0.70 0.630

22 37.50 7.00 2.65 0.70 1.008

23 37.50 7.00 1.35 0.10 1.388

24 37.50 7.00 1.35 1.30 1.061

25 37.50 7.00 1.35 0.70 1.169

26 37.50 7.00 1.35 0.70 1.079

27 37.50 7.00 1.35 0.70 1.159

126 WIBISANA ET AL. Microbiol Indones

Table 5 ANOVA for respon surface with quadratic regression model

Coeficient Sum of Mean F Value p-value

Source Estimates Squares Square Prob > F
df

Model 0.91 14 0.065 10.11 < 0.0001*

Intercept 1.18

X1-Glucose -0.0337 0.027 1 0.027 4.26 0.0567

X2-Urea -0.0048 5.433E-004 1 5.433E-004 0.085 0.7747

X3-MgSO4.7H2O 0.06930 0.12 1 0.12 18.02 0.0007*

X4-KCl -0.0798 0.15 1 0.15 23.89 0.0002*

X1.X2 0.05693 0.052 1 0.052 8.11 0.0122*

X1.X3 0.01790 5.127E-003 1 5.127E-003 0.80 0.3847

X1.X4 -0.1136 0.21 1 0.21 32.29 < 0.0001*

X2.X3 -0.0028 1.267E-004 1 1.267E-004 0.020 0.8899

X2.X4 0.04682 0.035 1 0.035 5.48 0.0334*

X3.X4 8.89E -15 1.264E-007 1 1.264E-007 1.976E-05 0.9965

2
X1 -0.0412 0.046 1 0.046 7.26 0.0166*
2
X2 -0.0767 0.16 1 0.16 25.23 0.0002*
2
X3 -0.0647 0.11 1 0.11 17.97 0.0007*
2
X4 0.0225 0.014 1 0.014 2.17 0.1610

Residual 0.096 15 6.396E-003

Lack of Fit 0.082 10 8.195E-003 2.93 0.1236

Pure Error 0.014 5 2.798E-003

*) significant at the level > 95.0% (for p value < 0.05); R2 = 0.9042; Adj R2 = 0.8148; Coeficient variation (C.V. %) =
7.63; Signal to noise ratio (adequate precision) = 14.605

several carbon sources for surfactin production. Mawgoud et al. 2008; Ghribi et al. 2011). In this study,
However, the nature and concentration of carbon source the usage of glucose concentration higher than 40 g L-1
influenced on surfactin yield (Abdel-Mawgoud et al. still showed increasing of surfactin production and at
2008; Yi et al. 2013; Singh et al. 2014b). The best glucose concentration 45 g L-1 reached maximum
carbon source for surfactin production by B. surfactin yield. Although in this study the concentration
amyloliquefaciens MD4-12 is glucose since this carbon of glucose required higher but keep in mind that
source is easy to assimilate. The concentration of surfactin produced was also higher than previous
glucose for maximum surfactin production was reported.
reported at 40 g L-1 and at glucose concentration higher The nature of nitrogen source also plays an
than 40 g L-1, the surfactin yield decreased (Abdel- important role in the production of surface-active
Volume 9, 2015 Microbiol Indones 127

1.35 1.35

1.255 1.2525

1.16 1.155

Surfactin
Surfactin

1.065 1.0575

0.97 0.96

30.00
30.00
33.75 33.75
37.50 9.00 37.50
X1: Glucose 41.25 X1: Glucose 1.00
8.00 41.25
7.00 0.85
45.00 6.00 0.70
5.00 X2: Urea 45.00 0.55
0.40 X4: KCl

A B

1.35

1.2075

1.065
Surfactin

0.9225

0.78

5.00
6.00
7.00
X2: Urea
1.00
8.00 0.85
0.70
9.00 0.55
0.40 X4: KCl

C
Fig 1 3D respon surface plot for effect combination of (a) glucose and urea (b) glucose and KCl and (c) urea and KCl on
surfactin production.

compounds by microorganisms (Singh et al. 2014b). In optimum condition of MgSO4.7H2O and KCl were 1.85
this study the use of urea showed highest surfactin and 0.4 g L-1, respectively, which is equal to
production. The optimum urea concentration was 6.33 concentration of Mg2+ and K+ of 7.5 and 5.3 mM,
g L-1. The adequate C/N ratio is an important factor for respectively. MgSO4 concentration influenced cell
efficiently surfactin synthesis. growth as well as the surfactin production, while KCl
The other significant factors effecting surfactin was reported to relate with the permeation pressure by
production in this study were MgSO4.7H2O and KCl. offering a buffer environment in cooperation with
These results correspond to those reported in literature KH2PO4 for cell growth (Yi et al. 2013). Maintaining the
(Wei et al. 2007). Under optimal condition of the metal environmental condition is important to support
ion Mg2+ and K+ (2.4 and 10 mM, respectively), surfactin biomass growth and surfactin production as well as
yield was improved significantly. In this study the inhibit byproduct formation in the fermentation process.
128 WIBISANA ET AL. Microbiol Indones

In this study, after optimization of significant doi:10.1016/j/febslet.207.01.059.

factors by RSM the surfactin yield could be markedly Lim JH, Park BK, Kim MS, Hwang MH, Rhee MH, Park SC,
enhanced (2.4-fold). Using glucose, urea, MgSO4.7H2O Yun HI. 2005. The anti-thrombotic activity of
and KCl at optimal concentration and keeping other surfactins. J Vet Sci. 6(4):353-5.
factors at their minimum value, the yield of surfactin Liu X, Ren B, Gao H, Liu M, Dai H, Song F, Yu Z, Wang S,
Hu J, Kokare CR, Zhang L. 2012. Optimization for the
reached 1.25 g L-1, which closely corresponds to the production of surfactin with a new ynergisticantifungal
model estimates (1.35 g L-1). The surfactin yields activity. PLoS ONE7(5). doi:10.1371/journal.pone.003
obtained in this study was higher than some of reported 4430.
yields in literature and also have shorter fermentation Meena KR, Kanwar SS. 2015. Lipopeptides as the antifungal
cycle (Sen 1997; Abdel-Mawgoud et al. 2008; Al- and antibacterial agents: applications in food afety and
Ajlani et al. 2007). therapeutics. BioMed Res Int. doi:10.1155/2015/473050.
Mnif I, Chaabouni-Ellouze S, Ghribi D. 2012. Optimization
of the nutritional parameters for enhanced production
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