antimicrobial bio surf act ants from marine bacillus circulans extra cellular synthesis and...

ORIGINAL ARTICLE

Antimicrobial biosurfactants from marine Bacillus circulans:extracellular synthesis and purificationS. Mukherjee, P. Das, C. Sivapathasekaran and R. Sen

Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal, India

Introduction

Biosurfactants are the surface-active molecules produced

as a result of metabolism in several micro-organisms and

occur in nature as glycolipids, lipopeptides, lipoproteins

and polymeric biosurfactants (Desai and Banat 1997;

Mukherjee et al. 2006). Besides their potential application

in industrial emulsification and bioremediation, these

molecules have recently been reported to possess several

properties of therapeutic and biomedical importance, e.g.

antimicrobial and anti-adhesive action against several

pathogenic micro-organisms (Singh and Cameotra 2004;

Rodrigues et al. 2006). Most extensively studied class of

biosurfactants; the lipopeptides are produced mainly by

Bacillus species. Surfactin, the antibiotic lipopeptide, pro-

duced mostly by Bacillus subtilis is the most well known

member of this class (Arima et al. 1968). Other members

of this group: lichenysin, iturin, arthrofactin and pumilac-

idin also possess antimicrobial properties. Although bio-

surfactants have been widely studied in past few years, the

marine environment still remains mostly unexplored and

only a few reports have been there regarding biosurfactant

production by marine micro-organisms (Passeri et al.

1992; Maneerat et al. 2006; Das et al. 2008a,b). Several

downstream processing strategies have been reported for

the biosurfactant purification based on their physical and

chemical properties (Mukherjee et al. 2006). The forma-

tion of molecular aggregates called micelles by the micro-

bial surfactants is one of these and has been exploited for

their separation using membrane ultra filtration (Sen and

Swaminathan 2005). However, another effective separa-

tion procedure, the size exclusion chromatography that

separates molecules based on the difference in their

molecular weight, has not been exploited for biosurfactant

Keywords

activity, antimicrobial, Bacillus, fermentation,

production.

Correspondence

Ramkrishna Sen, Department of

Biotechnology, Indian Institute of Technology,

Kharagpur, West Bengal, India.

E-mail: [email protected]

Authors S. Mukherjee and P. Das contributed

equally to this work.

2008 ⁄ 0394: received 6 March 2008, revised 8

September 2008 and accepted 9 September

2008

doi:10.1111/j.1472-765X.2008.02485.x

Abstract

Aims: To purify the biosurfactant produced by a marine Bacillus circulans

strain and evaluate the improvement in surface and antimicrobial activities.

Methods and Results: The study of biosurfactant production by B. circulans

was carried out in glucose mineral salts (GMS) medium using high perfor-

mance thin layer chromatography (HPTLC) for quantitative estimation. The

biosurfactant production by this strain was found to be growth-associated

showing maximum biosurfactant accumulation at 26 h of fermentation. The

crude biosurfactants were purified using gel filtration chromatography with

Sephadex� G-50 matrix. The purification attained by employing this technique

was evident from UV–visible spectroscopy and TLC analysis of crude and puri-

fied biosurfactants. The purified biosurfactants showed an increase in surface

activity and a decrease in critical micelle concentration values. The antimicro-

bial action of the biosurfactants was also enhanced after purification.

Conclusions: The marine B. circulans used in this study produced biosurfac-

tants in a growth-associated manner. High degree of purification could be

obtained by using gel filtration chromatography. The purified biosurfactants

showed enhanced surface and antimicrobial activities.

Significance and Impact of the Study: The antimicrobial biosurfactant pro-

duced by B. circulans could be effectively purified using gel filtration and can

serve as new potential drugs in antimicrobial chemotherapy.

Letters in Applied Microbiology ISSN 0266-8254

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009) 281–288 281

purification. In this paper, we are reporting the produc-

tion of a lipopeptide biosurfactant by a marine Bacillus

circulans and its purification by size exclusion chromatog-

raphy. The results suggest that significant purification of

biosurfactants can be achieved using this chromatographic

technique evident by the increase in its surface activity

and antimicrobial action.

Materials and methods

Micro-organism, media composition and cultivation

conditions

A B. circulans isolated from the seawater sample obtained

from Andaman and Nicobar Islands, India was used in

this study (Das et al. 2008a). Zobell Marine broth 2216

(HiMedia, Mumbai, India) was used for the preparation

of primary inoculum. For preparation of the inoculum,

cultures were grown in this medium for 10–12 h at 37�C

(OD 600 nm: 1Æ2–1Æ4). This was used for inoculating

glucose mineral salts (GMS) production medium at 2%

(v ⁄ v). The GMS media had the following composition

per litre: 20 g glucose, 3Æ3 g NH4NO3, 2Æ2 g K2HPO4,

0Æ14 g KH2PO4, 0Æ01 g NaCl, 0Æ6 g MgSO4, 0Æ04 g CaCl2,

0Æ2 g FeSO4 and 0Æ5 ml l)1 of a stock solution containing

the following trace elements per litre: 2Æ32 g ZnSO4Æ7H2O,

1Æ78 g MnSO4Æ4H2O, 0Æ56 g H3BO3, 1Æ0 g CuSO4Æ5H2O,

0Æ39 g Na2MoO4Æ2H2O, 0Æ42 g CoCl2Æ6H2O, 1Æ0 g EDTA,

0Æ004 g NiCl2Æ6H2O and 0Æ66 g KI.

Study of growth and biosurfactant production

The growth and production of biosurfactants were mon-

itored during fermentation in GMS production media

described earlier. The growth was monitored by measur-

ing the optical density (OD) values at 600 nm and also

by the amount of dry biomass production. The sugar

concentration was measured spectrophotometrically at

540 nm by the anthrone reaction. The biosurfactant pro-

duction was monitored as a function of reduction in

surface tension. The surface tension measurements were

obtained using a DCAT-11 digital surface tensiometer

(DataPhysics Instruments, Filderstadt, Germany) using

Wilhelmy plate method. The quantitative analysis of

biosurfactants was done chromatographically using high

performance thin layer chromatography (HPTLC). For

HPTLC analysis, 10 ll of each of the clarified different

hour’s samples was spotted onto a 20 · 10 cm pre-

coated silica gel HPTLC plate (Merck, Germany)

containing green fluorescent F254. These samples were

spotted under a flow of nitrogen gas with the help of a

Linomat-5 TLC spotting device (CAMAG, Switzerland)

having a robotic arm. After sample application on these

plates, they were developed in a solvent system contain-

ing chloroform, methanol and water in a ratio of

65:25:4, respectively. The developing jars (CAMAG) were

saturated with solvent system for 15–20 min prior to the

development. After development, these plates were air-

dried to remove solvent and a densitometric scan at

210 nm was performed with the help of a TLC Scanner

3 (CAMAG) for detection and quantification of biosurf-

actant. The quantification of biosurfactant was done

against a calibration curve for the pure biosurfactant.

The isolation procedure of the pure biosurfactant has

been described later.

Isolation of the crude biosurfactant and its purification

The surface-active molecules produced by the micro-

organism were isolated chemically by acidification of the

cell free broth (Sen and Swaminathan 1997). Briefly, after

about 28 h of growth the culture broth was centrifuged at

10 000 g for 20 min in a tabletop centrifuge (Eppendorf,

Hamburg, Germany) to pellet the cells. Concentrated HCl

was added to the cell free supernatant until it attained a

pH value of 2. The acidified cell free culture broth was

then stored at 4�C overnight for precipitation of sur-

face-active compounds. The precipitate was centrifuged

at 10 000 g for 20 min to get the crude biosurfactant as

pellet. The biosurfactant pellet was re-suspended in

water and the pH was raised to 7Æ5 to solubilize biosurf-

actants. Above a certain minimum concentration known

as the critical micelle concentration (CMC), the biosurf-

actants form aggregates or micelles due to mutual inter-

action of their hydrophobic part. These aggregates or

micelles contain a large number of individual surfactant

molecules and form bulky structures with higher effec-

tive molecular mass, which is a multiple of mass of

individual surfactant molecules. This property of biosurf-

actants to form bulky molecular aggregates has been uti-

lized effectively for their purification by size exclusion or

gel filtration chromatography. The crude water-soluble

biosurfactants were centrifuged at 10 000 g for 5 min to

exclude any insoluble matter. This clarified and concen-

trated solution of crude biosurfactants was then applied

to a Sephadex� G-50 column (10 mm · 300 mm, Amer-

sham Biosciences) pre-equilibrated with Milli-Q water

and eluted with slightly alkaline (pH 8Æ0) degassed

Milli-Q water (Millipore). Fractions, each of 1 ml, were

collected with a flow rate maintained at 1 ml min)1.

The absorbance of the fractions was monitored at

210 nm using a UV–visible spectrophotometer (Perkin-

Elmer, USA). The purified biosurfactant fractions were

pooled and lyophilized in a Savant freeze dryer (model:

micro modulyo 230, Thermo Scientific) to get the pure

biosurfactant as a dry powder.

Purification of a biosurfactant with antimicrobial potential S. Mukherjee et al.

282 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009) 281–288

ª 2009 The Authors

Determination of critical micelle concentration

The critical micelle concentration (CMC) is the minimum

concentration of surfactants at which the surface tension

reaches its minimum value and at this concentration the

surfactant molecules form molecular aggregates called

micelles. The CMC value of any surfactant is an indicator

of its surfactant capacity. Thus, a powerful surfactant has

a lesser CMC value than a weak one. The CMC value also

indicates the degree of purity attained by the surfactant

during downstream processing and thus, the CMC value

decrease as the degree of purification increases. The CMC

values of the crude and the purified biosurfactant were

determined by gradual addition of biosurfactant to pure

water. For this, concentrated solutions of crude and puri-

fied biosurfactant (5 g l)1) were prepared in de-ionized

water. Biosurfactants were gradually added to Milli-Q

water (Millipore) from this aqueous solution so that the

final concentration of biosurfactant increases by

5Æ0 mg l)1 with each addition. The change in surface ten-

sion of the water was noted with each addition in a

DCAT digital surface tensiometer (DataPhysics). The

minimum value of biosurfactant at which the surface ten-

sion is lowered abruptly reaching its minimum value was

considered as the CMC for the biosurfactant sample.

UV–visible spectroscopy

UV–visible spectroscopy was performed to check the pur-

ity attained by the biosurfactants after gel permeation.

For this purpose the first few column fractions (fractions

7–12) containing purified biosurfactant in micelle form

were collected, pooled and lyophilized. Similarly the latter

fractions with contaminants (fraction 19–22) were also

pooled and lyophilized. Equal amounts of crude biosurf-

actants, purified biosurfactants (fractions 7–12) and con-

taminants (fractions 19–22) were dissolved in water and

their absorption properties were checked in UV and visi-

ble range. The UV–visible spectra absorption scans of

these samples were performed in a Perkin-Elmer double

beam UV–visible spectrophotometer. Samples were taken

in quartz cuvette and scan was performed from 700 to

190 nm range by acquiring data at intervals of 1 nm.

A background spectrum was obtained for pure water and

was subtracted from the sample spectra. For comparison

of the absorption properties overlapping spectra were

obtained for all the samples.

Antimicrobial action of biosurfactants

The antimicrobial action of the chemically isolated crude

and gel filtration purified biosurfactant was evaluated

against several pathogenic bacterial, yeast and fungal

strains listed in Table 1. For antimicrobial tests,

1 mg ml)1 solution of crude and purified biosurfactants

were prepared in methanol. The antimicrobial action

against bacterial strains were checked by agar well diffu-

sion test on Mueller–Hinton agar medium (Hi-Media).

For fungal strains agar plates were prepared containing

their respective growth supporting solid medium. Crude

and gel filtration purified biosurfactant solutions were

poured into the different wells on these plates. Methanol

was poured into one of the wells as a negative control.

The bacterial test strains were incubated at 37�C while

the fungal test strains were incubated at 28�C. After

growth, the microbial inhibition zones (halo diameter)

around the wells were measured using an antibiotic zone

scale (HiMedia, Mumbai, India).

Results

Growth, biosurfactant production and isolation

The organism showed a typical growth and biosurfactant

production pattern in the GMS production media. The

concentration of bacteria expressed as dry bacterial bio-

mass was obtained as a function of OD and could be

expressed with the standard equation, i.e. dry biomass

(g l)1) = 0Æ38 · OD600 nm. After an initial lag period of

about 4 h the organism’s growth proceeded at a slow rate

till about 12–14 h. At around 14 h the microbial growth

Table 1 Antimicrobial action of crude and purified biosurfactants on

various strains of bacteria and fungi

Organism

Halo diameter

Crude

biosurfactant

(50 lg)

Purified

biosurfactant

(50 lg)

Gram positive bacteria

Micrococcus flavus + + + + + +

Bacillus pumilis + + + + +

Mycobacterium smegmatis + + + + +

Gram negative bacteria

Escherichia coli + + + + +

Serratia marcescens + + + + +

Proteus vulgaris + + +

Klebsiella aerogenes + + +

Pseudomonas sp. + + + + +

Fungal strains

Aspergillus niger + + + + + +

Aspergillus flavus + + + +

Candida albicans + + + + + +

Halo diameters: +,10–13 mm; + +, 14–17 mm; + + +, 18–21 mm;

+ + + +, 21–above.

The results showed an increase in antimicrobial action upon purifi-

cation.

S. Mukherjee et al. Purification of a biosurfactant with antimicrobial potential

ª 2009 The Authors


was slowed before the start of the major growth phase of

this micro-organism at about 16 h. After this the organ-

ism enters into the exponential phase of its growth which

continues up to about 28 h (Fig. 1). Although the bio-

surfactant production begins as early as 10 h as evident

from the reduction in surface tension of the medium, sig-

nificant foaming of the medium was observed only after

about 14 h of incubation. The surface tension of the

media was reduced to a minimum of 28 dynes ⁄ cm at

about 16 h of incubation upon reaching the critical

micelle concentration (CMC) after which it remained

more or less constant at this value (Fig. 1). Significant

production began at 16 h and continued up to 26 h as

indicated from quantitative analysis of biosurfactants by

HPTLC. A sudden rise in biosurfactant concentration was

noticed after 16 h of fermentation. From a relatively low

concentration of 0Æ072 g l)1 at 16 h, the biosurfactant

concentration increased steadily to 0Æ4225 g l)1 at 18 h.

The biosurfactant concentration reached its maximum

value of �1 g l)1 at 26 h of fermentation after which the

biosurfactant concentration started to decrease in the

medium (Fig. 1). After about 36 h of fermentation the

biosurfactant concentration was reduced to about

0Æ5 g l)1. The bacterium also showed a glucose utilization

profile corresponding to its growth and biosurfactant pro-

duction. The glucose concentration in the production

medium was reduced from the initial value of 20 g l)1 to

about 16 g l)1 in first 16 h. However, the concentration

was reduced from 16 g l)1 to about 9 g l)1 in the next

2 h of fermentation and finally reached a value of about

0Æ9 g l)1 at 28 h (Fig. 1). No sugar was detected in the

media after 36 h of fermentation. The pH of the culture

medium increased slightly to 7Æ5 from an original value

of 7Æ0 and remained more or less constant at this value.

Isolation and purification of crude biosurfactants

The biosurfactant produced in the production media

could be isolated by acidification of the cell free culture

broth with concentrated HCl. After overnight acidificat-

ion at 4�C the crude biosurfactant was separated as

precipitate. The precipitate could be obtained by centrifu-

gation. The pH of this crude biosurfactant pellet was

raised to 7Æ5 and the concentrated biosurfactant solution

was applied to gel filtration using Sephadex� G-50 for

further purification. With a flow rate of 1 ml min)1 the

biosurfactant aggregates in form of micelles were eluted

early in the fractions 7–12. The contaminating com-

pounds comprising of other bacterial metabolites and iso-

lated surfactant molecules were eluted in latter fractions,

i.e. fractions 19–22 (Fig. 2). The purification attained was

checked by measuring the critical micelle concentration

(CMC) values and by thin layer chromatographic (TLC)

analysis of the crude and gel filtration purified biosurfac-

tants. In the CMC experiments the minimum surface

tension of 28Æ78 mN m)1 was obtained after adding

40 mg l)1 of crude biosurfactants. On the other hand

using column purified biosurfactant, a minimum surface

tension of 27Æ89 mN m)1 was obtained after addition of

25 mg l)1 biosurfactant to the pure water (Fig. 3). Analy-

sis of the crude and gel permeation purified biosur-

factants by TLC (Fig. 4) showed that significant level

of purification was achieved upon gel filtration. In the

lane containing the purified biosurfactants, the different

4035302520151050

3·0

0·6

0·4

0·2

0·0

1·0

1·2

1·4

1·6

1·8

2·0

2·2

2·4

2·6

2·8

0·8

Time (h)

Bio

mas

s/B

iosu

rfac

tant

(gl

–1)

20

30

40

50

60

70

Surface tension (m

Nm

–1)

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

Glu

cose

(gl

–1)

Figure 1 The growth of marine Bacillus

circulans as a function of reduction in surface

tension (d) of glucose mineral salts (GMS)

production medium, biomass ( ) and biosurf-

actant (h) production with time. It also shows

glucose utilization (·) as a function of accu-

mulation of biosurfactant in the GMS produc-

tion medium.



ª 2009 The Authors

surfactant fractions were seen as individual spots and

were found to be devoid of any smearing pattern caused

due to presence of other contaminating small molecules

produced during metabolism. The thin layer chromato-

graphy of the later fractions (column fractions 19–22)

also showed presence of high level of impurities being

concentrated in these column fractions. The impurities

present in the crude biosurfactants had the property to

absorb at higher wavelengths and showed fluorescence

under a 366-nm lamp. The lane containing the pure bio-

surfactants did not show fluorescence under this wave-

length, while the contaminants separated in latter column

fractions showed high absorption under this light. The

UV–visible spectrum scan also confirmed this fact, where

the pure biosurfactants absorbed only in the far UV

region (Fig. 5). On the other hand the crude and the col-

umn separated contaminants absorbed at higher wave-

lengths (�340–400 nm). The intensity of absorption in

this region increased in contaminants compared to crude

biosurfactants.

Antimicrobial action of the biosurfactant

The biosurfactant was found to possess inhibitory action

against most strains tested. It was found to be active both

against Gram positive and negative bacteria and fungal

strains (Table 1). Good inhibitory activity was seen

against Gram positive bacteria like Micrococcus flavus,

–0·1

0·0

0·1

0 25 302015105

0·2

0·3

0·4

0·5

0·6

0·7

Abs

orba

nce

(210

nm

)

Fraction number b (1 ml each)

Figure 2 Purification of the crude biosurfactants by size exclusion

chromatography using Sephadex� G-50 matrix. The pure biosurfac-

tants aggregated in form of micelles are eluted in the earlier column

fractions due to their bulky nature, while the contaminants are eluted

in the later column fractions.

200 40 60 8020

30

40

50

60

70

Sur

face

tens

ion

(mN

m–1

)

Biosurfactant concentration (mg l–1)

Figure 3 Determination of the critical micelle concentration (CMC) of

crude (d) and purified ( ) biosurfactants. The minimum amount of

biosurfactant required to reach CMC is decreased with increase in the

purity of biosurfactants.

1 2

Figure 4 Thin layer chromatogram showing the crude (lane 1) and

size exclusion purified (lane 2) biosurfactants. The purification attained

by the biosurfactant is evident from appearance of smear free distinct

biosurfactant spots in lane 2. The plate was developed with a solvent

system comprising chloroform: methanol: water (65:25:4) and visual-

ized under a 254-nm UV lamp.


ª 2009 The Authors


Bacillus pumilis and Mycobacterium smegmatis and Gram

negative bacteria like Escherichia coli, Serratia marcescens,

Proteus vulgaris, Pseudomonas sp. and Klebsiella aerogenes.

Among fungal strains, it showed good inhibitory action

against Aspergillus niger, A. flavus and Candida albicans.

The inhibition zones were found to be largest in case of

Gram-positive bacteria such as M. flavus, B. pumilis and

fungus such as A. niger and C. albicans. As a general

observation, the inhibition zone diameter was found to

be larger and more well defined when same concentration

of gel permeation purified biosurfactants were used

instead of the crude biosurfactants.

Discussion

A marine B. circulans producing extracellular biosur-

factants was isolated and identified from Andaman and

Nicobar Islands, India. Bacillus species have been widely

reported as producers of extracellular biosurfactants,

mostly lipopeptides (Vater et al. 2002). The biosurfactant

product used in this study was also identified as lipo-

peptide by FTIR and TLC analysis (data not shown). The

production of the biosurfactants by this strain showed a

direct relationship with the cell growth, i.e. biosurfactant

accumulated in the medium as the cells entered into the

exponential phase of their growth and its concentration in

the medium increased gradually thereafter. The concentra-

tion of the biosurfactant in medium increased from a very

low value of 0Æ07 g l)1 at the beginning of the exponential

phase to a maximum of 1Æ0 g l)1 by the end of growth

phase. This type of biosurfactant production profile is

similar to that reported for B. subtilis BBK06 (Chen et al.

2006), B. licheniformis JF-2 (Lin et al. 1993), Pseudomonas

sp. strain PP2 (Prabhu and Phale 2003) and Bacillus

subtilis LB5a (Nitschke and Pastore 2004). However, it is

quite contrasting to growth characteristic reported for

surfactin production by Bacillus subtilis ATCC 21332

(Davis et al. 1999; Nitschke and Pastore 2004) in which

biosurfactant accumulation starts as the cells reach their

stationary phase. Different nutritional and ecological roles

have been postulated for biosurfactants, which explain the

production of these molecules by microbes in the different

stages of their growth cycle. In the growth associated type

of production, these molecules behave more like a primary

metabolite and seem to be directly involved in the normal

growth and nutrient uptake process while in the other case

they behave as secondary metabolite and seem to have

some ecological role rather than growth, like those of anti-

biotics and pigments. The glucose uptake by the bacteria

shows a sharp rise after 16 h along with a sudden hike in

biosurfactant concentration. This indicates that besides the

cell growth, a considerable amount of the carbon is

diverted towards the metabolic pathway involving biosurf-

actant production. The decline observed in the biosurf-

actant concentration during late stationary and death

phase may be explained by enzymatic hydrolysis and

uptake of these molecules caused due to substrate scarcity

in the medium. Although the production of any protease

and subsequent enzymatic degradation of biosurfactants

have not been investigated in the present work, a similar

mechanism has been reported for B. subtilis 21332 produc-

ing lipopeptide biosurfactants using cassava substrates

(Nitschke and Pastore 2004). Another explanation of this

decline in biosurfactant level may be the inhibitory effect

of these molecules on cell growth above a certain concen-

tration, which induces the subsequent degradation of these

molecules. The slight increase in pH of the production

medium during fermentation is similar to that reported

for surfactin production by B. subtilis 21332 (Nitschke and

Pastore 2004). In the present work, biosurfactants have

been successfully purified with help of size exclusion chro-

matography. The formation of molecular aggregates called

micelles by biosurfactant molecules in aqueous solutions

facilitates their separation from the contaminants. Biosurf-

actants in the form of micelles, due to their bulky struc-

tures, were eluted in early column fractions, while other

contaminating small molecules were eluted in latter frac-

tions due to their small size and inability to form such

aggregates. Although the micelle forming behaviour of

biosurfactants has been exploited for their purification by

membrane ultrafiltration (Sen and Swaminathan 2005), to

the best of our knowledge, this is the first report of

purification of lipopeptide biosurfactants by size exclusion

chromatography. As evident from the CMC values, con-

siderable purification was attained by application of this

technique for purification. The CMC values were nearly

halved when the biosurfactants were subjected to column

400 500 600 700·00·00

190·0 300

0·5

1·0

1·5

2·0

2·5

3·00

nm

A

Cont

P

Cr

Figure 5 UV–visible spectra of the crude biosurfactant (Cr), purified

biosurfactant (P) and the contaminants (Cont) separated in size exclu-

sion chromatography. The contaminants present in the crude biosurf-

actants absorb at higher wavelengths (�340–400 nm) while the pure

biosurfactants absorb only in the far UV region of the spectrum.



ª 2009 The Authors

purification. The minimum surface tension value obtained

in CMC experiments was lower in case of column-purified

biosurfactants than those obtained for crude chemically

isolated biosurfactants. This indicates an increase in sur-

face activity of these molecules upon purification. The

purification attained by the biosurfactants was also evident

from TLC analysis in which the purified biosurfactants

showed well-resolved spots with less smear caused due to

contaminating metabolites. The crude biosurfactants

absorbed in higher wavelengths (300–400 nm) while the

pure biosurfactants absorbed only in far UV region. The

UV–visible scan of the crude biosurfactant, purified

biosurfactant and contaminants proved that fluorescing

property of biosurfactants at higher wavelengths (366-nm

lamp) is due to the contaminating molecules present in it.

The absorption scan of the contaminants confirms this

fact as these show significant absorption in range �340–

400 nm. The purified biosurfactants did not show any

absorbance or fluorescence in these wavelengths. The

crude and purified biosurfactants showed profound anti-

microbial activity against a panel of pathogenic and semi-

pathogenic bacterial and fungal strains. The purification

attained by the biosurfactant was evident from the

increase in the antimicrobial action upon purification

reflected in larger inhibition zones produced by pure bio-

surfactant. The biosurfactant from this strain showed good

inhibitory action against Gram-negative bacteria. This is

in contrast to reports in which Bacillus lipopeptides have

been found to be active mostly against Gram-positive bac-

teria having little or no effect on Gram negatives (Singh

and Cameotra 2004). This may be due to production of

different biosurfactant isoforms, which shows an antago-

nistic effect on both Gram-positive and Gram-negative

bacteria. It has been reported earlier that different iso-

forms of the biosurfactant are being produced depending

on the micro-organism, substrate used and the culture

conditions employed (Mukherjee and Das 2005). Good

inhibitory action against fungal strains such as A. niger

and C. albicans suggests the potential use of these mole-

cules against infection involving these pathogens. In this

study, a marine micro-organism producing antimicrobial

lipopeptide during the exponential phase of its growth has

been isolated and identified. Results suggested that size

exclusion chromatography could be used as an effective

means for purifying bacterial lipopeptide facilitating their

use in drug industry as new and potent antimicrobial

agents.

Acknowledgements

S.M. acknowledges CSIR, New Delhi and P.D. acknowl-

edge IIT, Kharagpur for the financial assistances. R.S. and

S.C. acknowledge the Department of Biotechnology

(DBT), Govt. of India for the project grant (BT ⁄ PR-

6827 ⁄ AAQ ⁄ 03 ⁄ 263 ⁄ 2005) in marine biotechnology.

Authors also gratefully acknowledge members of medical

biotechnology and biomaterials laboratories for their

immense help during the course of investigation. We

thank Subhasish Das for the photographs.

References

Arima, K., Kakinuma, A. and Tamura, G. (1968) Surfactin, a

crystalline peptidelipid surfactant produced by Bacillus

subtilis: isolation, characterization and its inhibition of

fibrin clot formation. Biochem Biophys Res Commun 31,

488–494.

Chen, C.Y., Baker, S.C. and Darton, R.C. (2006) Batch produc-

tion of biosurfactant with foam fractionation. J Chem Tech

Biotechnol 81, 1923–1931.

Das, P., Mukherjee, S. and Sen, R. (2008a) Antimicrobial

potentials of a lipopeptide biosurfactant derived from a

marine Bacillus circulans. J Appl Microbiol 104, 1675–1684.

Das, P., Mukherjee, S. and Sen, R. (2008b) Improved bioavail-

ability and biodegradation of a model polyaromatic hydro-

carbon by a biosurfactant producing bacterium of marine

origin. Chemosphere 72, 1229–1234.

Davis, D.A., Lynch, H.C. and Varley, J. (1999) The production

of surfactin in batch culture by Bacillus subtilis ATCC

21332 is strongly influenced by the conditions of nitrogen

metabolism. Enz Microb Technol 25, 322–329.

Desai, J.D. and Banat, I.M. (1997) Microbial production of

surfactants and their commercial potential. Microbiol Mol

Biol Rev 61, 47–64.

Lin, S.C., Sharma, M.M. and Georgiou, G. (1993) Production

and deactivation of biosurfactant by Bacillus licheniformis

JF-2. Biotechnol Prog 9, 138–145.

Maneerat, S., Bamba, T., Harada, K., Kobayashi, A., Yamada, H.

and Kawai, F. (2006) A novel crude oil emulsifier secreted

in the culture supernatant of a marine bacterium, Myroides

sp. Strain SM1. Appl Microbiol Biotechnol 70, 254–259.

Mukherjee, A.K. and Das, K. (2005) Correlation between

diverse cyclic lipopeptides production and regulation of

growth and substrate utilization by Bacillus subtilis

strains in a particular habitat. FEMS Microbiol Ecol 54,

479–489.

Mukherjee, S., Das, P. and Sen, R. (2006) Towards commercial

production of microbial surfactants. Trends Biotechnol 24,

509–515.

Nitschke, M. and Pastore, G.M. (2004) Biosurfactant produc-

tion by Bacillus subtilis using cassava-processing effluent.

Appl Biochem Biotechnol 112, 163–172.

Passeri, A., Schmidt, M., Haffner, T., Wray, V., Lang, S. and

Wagner, F. (1992) Marine biosurfactants. IV. Production,

characterization and biosynthesis of an anionic glucose

lipid from the marine bacterial strain MM1. Appl Microbiol

Biotechnol 37, 281–286.


ª 2009 The Authors


Prabhu, Y. and Phale, P.S. (2003) Biodegradation of phenan-

threne by Pseudomonas sp. strain PP2: novel metabolic

pathway, role of biosurfactant and cell surface hydropho-

bicity in hydrocarbon assimilation. Appl Microbiol Biotech-

nol 61, 342–351.

Rodrigues, L., Banat, I.M., Teixeira, J. and Oliveira, R. (2006)

Biosurfactants: Potential applications in medicine. J Anti-

microb Chemother 57, 609–618.

Sen, R. and Swaminathan, T. (1997) Application of

response-surface methodology to evaluate the

optimum environmental conditions for the enhanced

production of surfactin. Appl Microbiol Biotechnol 47,

358–363.

Sen, R. and Swaminathan, T. (2005) Characterization of con-

centration and purification parameters and operating con-

ditions for the small-scale recovery of surfactin. Proc

Biochem 40, 2953–2958.

Singh, P. and Cameotra, S.S. (2004) Potential applications of

microbial surfactants in biomedical sciences. Trends Bio-

technol 22, 142–146.

Vater, J., Kablitz, B., Wilde, C., Franke, P., Mehta, N. and

Cameotra, S.S. (2002) Matrix-assisted laser desorption ion-

ization-time of flight mass spectrometry of lipopeptide

biosurfactants in whole cells and culture filtrates of Bacillus

subtilis C-1 isolated from petroleum sludge. Appl Environ

Microbiol 68, 6210–6219.



ª 2009 The Authors

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