Journal of Environmental Sciences 2011, 23(Supplement) S63–S65

Production of surfactin using pentose carbohydrate by Bacillus subtilis

Abdul Wahab Khan1,3, Mohammad Shahedur Rahman1,3, Umme Salma Zohora1,3,Masahiro Okanami2, Takashi Ano1,2,∗

1. Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan.E-mail: khan.abdulwahab@gmail.com

2. Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University,930 Nishimitani, Kinokawa-city, Wakayama, 649-6493, Japan

3. Institute of Biological Resources, Anise Corporation, N605 Building #C, 1-12 Minamiwatarida-cho,Kawasaki-ku, Kawasaki-shi 210-0855, Japan

AbstractInterest in microbial surfactants has been steadily increasing in recent years due to their diversity, mass production possibility,

selectivity, performance under extreme conditions and potential applications in environmental protection. In this study two pentose

sugars (xylose and arabinose) were investigated for the submerged fermentation (SmF) of Bacillus subtilis in surfactant production

medium for bio-surfactant surfactin production. An excellent vegetative growth of B. subtilis (× 1010 CFU/mL) was observed for xylose

and arabinose containing medium which were comparable to glucose supplemented medium. Low growth (× 108 CFU/mL) was found

when medium was not supplemented with any of the sugars. Surfactin production in xylose, arabinose and glucose containing medium

was 2700, 2600 and 2000 mg/L, respectively, whereas, medium without any sugar showed low surfactin (700 mg/L) production. These

results clearly indicate the effect of pentose sugars on production of surfactin. Gradual depletion of the xylose and arabinose were

confirmed by HPLC analysis during the growth phase of the strain that ultimately produced the surfactin.

Key words: xylose, arabinose; Bacillus subtilis; surfactin, submerged fermentation

Introduction

Surfactants are surface active agents. They are amphi-

pathic molecules with both hydrophilic and hydrophobic

(generally hydrocarbon) moieties. For this reason, surfac-

tants partition preferentially at the interface between fluid

phases with different degrees of polarity and hydrogen

bonding such as oil-water or air-water interfaces. These

characteristics confer surfactant to use as an excellent

detergent, emulsifying agent, foaming agent, and dispers-

ing agent, which make it some of the most versatile

process chemicals. Almost all surfactants currently in use

are chemically derived from petroleum; however, inter-

est in microbial surfactants has been steadily increasing

in recent years due to their diversity, environmentally

friendly nature, the possibility of their production through

fermentation, and their potential applications in the envi-

ronmental protection, crude oil recovery, health care, and

food-processing industries (Banat, 1995; Fiechter, 1992;

Mulligan, 2005). Usually chemically defined medium and

hexose are used as carbon source for biosurfactin produc-

tion. The demand for hexose carbon source is increasing

gradually even for ethanol production. Naturally we

have abundant hemi-cellulose resources. Hemi-cellulose is

mainly composed of pentose sugars like xylose and ara-

binose. Escherichia coli (Lawlis et al., 1984) and Bacillus

* Corresponding author. E-mail: tano@waka.kindai.ac.jp

(Rygus et al., 1991) are already reported for utilization of

xylose.

B. subtilis MI113 (pC115) is a recombinant of plasmid

pC112 containing the Ipa-14 gene related to surfactin

production from B. subtilis RB14 (Ohno et al., 1995). Due

to the presence of lpa-14, B. subtilis MI113 is capable

of producing bio-surfactant surfactin. This study was con-

ducted to investigate the suitability of pentose sugar, xylose

and arabinose as carbon source in SmF of B. subtilis MI113

for surfactin production for the first time.

1 Materials and methods

1.1 Experimental setup

B. subtilis MI113 (pC115) strain was transfer red from

culture stock into 5 mL of modified L-medium (1%

Polypepton (Nippon Pharmaceuticals Co., Tokyo), 0.5%

yeast extract (Oriental Yeast Co., Tokyo), 0.5% NaCl, pH

7.0) and incubated overnight with 120 strokes per minute

(spm) at 37°C to prepare the preculture. SmF was conduct-

ed in 200 mL Erlenmeyer flasks which were containing

xylose or arabinose as pentose sugar in surfactin produc-

tion medium (8% Polypepton S (Nippon Pharmaceuticals

Co., Tokyo), 6.7% xylose or arabinose (Kanto Chemical

Co., Tokyo), 0.5% K2HPO4 (Kanto Chemical Co., Tokyo),

0.05% MgSO4·7H2O (Kanto Chemical Co., Tokyo), 25

S64 Journal of Environmental Sciences 2011, 23(Supplement) S63–S65 / Abdul Wahab Khan et al. Vol. 23

mg/L FeSO4·7H2O (Kanto Chemical Co., Tokyo), 22 mg/L

MnSO4·7H2O (Kanto Chemical Co., Tokyo) and 184 mg/L

CaCl2 (Kanto Chemical Co., Tokyo)). Whereas glucose

containing medium and without any sugar containing

medium were used as positive control and negative control

respectively. All the flasks were autoclaved at 121°C for

20 min. After sterilization, medium was cooled at room

temperature and the preculture (1%, V/V) was then added.

The flasks were incubated at 37°C at 120 r/min for four

days.

1.2 Determination of microbial growth, pH and sur-factin concentration

Samples were collected from the culture mix at specific

intervals to analyze. Samples were serially diluted and

spread on agar plate for total CFU count. At the same

time samples were heated at 80°C for 10 min before

spreading for spore count. For iturin A extraction, 100

μL of sample was added into 900 μL of surfactin extrac-

tion buffer (acetonitrile: 3.8 mmol/L trifluoroacetic acid

(TFA) (80:20; V/V)). The mixture was vortexed at room

temperature for 20 min and then centrifuged at 15,000 × gfor 10 min at 4°C. The supernatant was filtrated through

0.20 μm polytetrafluoroethylene (PTFE) membrane filter

(Advantec, Tokyo, Japan) and 20 μL of the filtrate was

injected into HPLC column for surfactin ditection. A

mixture of acetonitrile and 3.8 mmol/L trifluoroacetic acid

(80:20, V/V) was used in mobile phase through ODS

column (GL science ODS-2 4.6 mm φ × 250 mm) at a

flow rate of 1.5 mL/min at 30°C.

1.3 Determination of carbon depletion rate in medium

For the determination of residual sugar concentration,

100 μL of sample was diluted in 900 μL of buffer (CH3CN:

3.8 mmol/L trifluoroacetic acid (TFA) solution = 4:1

(V/V)). The mixture was vortexed at room temperature for

20 min and then centrifuged at 15,000 × g for 10 min

at 4°C. After centrifugation, the supernatant was filtered

by 0.20 μm PTFE membrane filters. The filtrate of 20

μL was injected into HPLC column for determination of

residual sugar concentration. A mixture of 10 mmol/L

CH3CN: H2O = 3:1 (V/V) was used in mobile phase

through ODS column (column: Chromolith Performance

RP-18eb (MERCK) 4.6 mm φ × 100 mm) at a flow rate of

1 mL/min at 40°C.

2 Results and discussions

2.1 Pentose sugar as carbon source for B. subtilis MI113growth

Xylose, arabinose and glucose containing medium

showed similar vegetative growths which were 100 times

higher compared to without any sugar containing surfactin

production medium during their four days of cultivation

(Fig. 1). When additional sugar was not added, the inherent

carbon content of the Polypepton S supported vegetative

growth of this strain. The growth difference between the

cultures with or without sugar clearly shows the effect

00 1 2 3 4

Cultivation time (day)C

olo

ny f

orm

ing u

nit

s (m

L)

0

10

20

30

40

50

60

70

80

Res

idual

sugar

in m

ediu

m (

g/L

)

101

102

103

104

105

106

107

108

109

1010

1011

0

Total cell in:

Glucose No sugarXylose

Spore in

Arabinose

Glucose No sugarXylose Arabinose

Glucose No sugarXylose Arabinose

Residual sugar

Fig. 1 B. subtilis MI113 growth and residual sugar concentration in

medium.

of different sugars. On the other hand, similar vegetative

growths in arabinose, xylose and glucose attribute that

B. subtilis MI113 (pC115) can utilize pentose sugars

arabinose and xylose. The residual sugar concentration

in the medium decreased with respect to cultivation time

for all culture medium. Among the three sugars (glucose,

xylose and arabinose), arabinose was found to be easily

and rapidly assimilated by Bacillus, whereas, assimilation

of xylose was slow (Fig. 1). Bacillus took almost the

double time for complete depletion of xylose compared

to arabinose and glucose. The depletion rate of carbon

source also affects the Bacillus spore formation in the

medium (Fig. 1). During the fermentation medium pH

was observed. The glucose, xylose, arabinose and without

sugar containing medium showed their maximum pH on

their 4th, 3rd, 3rd and 2nd day of cultivation which were 8,

7.5, 8 and 9 respectively (Fig. 2).

2.2 Pentose sugar as carbon source in surfactin produc-tion medium

A number of carbon substrates have been used by many

researchers for biosurfactant production. The nature of

carbon substrate influence and affect the quantity and

quality of the biosurfactant production (Raza et al., 2007).

The hexose carbon source glucose and the pentose carbon

source xylose and arabinose also produce different amount

of biosurfactant. In this investigation the maximum biosur-

factant production was observed on day 4 of cultivation

which were 2000, 2700, 2600 and 700 mg/L (Fig. 3)

for glucose, xylose, arabinose and no sugar containing

medium, respectively. Lee and Kim (1993) reported that

in batch culture and in fed-batch culture of Torulopsisbombicola about 37% and 60% of the carbon input was

incorporated into biosurfactant. Thus the low production

in medium without any sugar was due to the lack of

carbon source in medium. Differences in biosurfactant

Supplement Production of surfactin using pentose carbohydrate by Bacillus subtilis S65

6.0

6.5

7.5

8.5

9.0

9.5

0 1 2 3 4

Cultivation time (day)

pH

7.0

8.0

Glucose No sugarXylose Arabinose

Fig. 2 pH profile of different medium during submerged fermentation.

0

500

1000

1500

2000

2500

3000

Glucose Xylose Arabinose No sugar

Surf

acti

n c

once

ntr

atio

n (

mg/L

)

Fig. 3 Surfactin production in different sugar containing medium by B.subtilis MI113.

productions between hexose and pentose are very clear

whereas two pentose carbon sources showed similar

production. Therefore it can be said that pentose sugars are

more suitable for B. subtilis MI113 (pC115) for surfactin

production.

3 Conclusions

The depletion of pentose sugar in the medium attributes

the utilization of pentose sugar by B. subtilis MI113

(pC115). The remarkable enhanced production of surfactin

confers that hemicellulose originated pentose sugar xylose

and arabinose might be a good alternative of hexose sugar

like glucose. Further experiments are required to optimize

the pentose sugar content in medium and fermentation

parameters for successful utilization of pentose sugar.

References

Banat I M, 1995. Biosurfactants production and possible uses in

microbial enhanced oil recovery and oil pollution remedia-

tion: a review. Bioresource Technology, 51: 1–12.

Fiechter A, 1992. Biosurfactants: moving towards industrial

application. Trends Biotechnology, 10: 208–217.

Lawlis V B, Dennis M S, Chen E Y, Smith D H, Henner D

J, 1984. Cloning and sequencing of the xylose isomerase

and xylulose kinase genes of Escherichia coli. Applied andEnvironmental Microbiology, 47: 15–21.

Lee L H, Kim J H, 1993. Distribution of substrate carbon

in sophorose lipid production by Torulopsis bombicola.

Biotechnology Letter, 15: 263–266.

Mulligan C N, 2005. Environmental applications for biosurfac-

tants. Environmental Pollution, 133(2): 183–198.

Ohno A, Ano T, Shoda M, 1995. Production of a lipopeptide an-

tibiotic, surfactin, by recombinant Bacillus subtilis in solid

state fermentation. Biotechnology and Bioengineering, 47:

209–214.

Rygus T, Scheler A, Allmansberger R, Hillen W, 1991. Molecular

cloning, structure, promoters and regulatory elements for

transcription of the Bacillus megaterium encoded regulon

for xylose utilization. Archives of Microbiology, 155: 535–

542.

Raza Z A, Rehman A, Khan M S, Khalid Z M, 2007. Improved

production of Biosurfactant by Pseudomonas aeruginosamutant using vegetable oil refinery wastes. Biodegradation,

18(1): 115–121.