Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Antimicrobial biosurfactants from marine Bacillus circulans: extracellular synthesis and purication
S. Mukherjee, P. Das, C. Sivapathasekaran and R. Sen
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal, India

Keywords activity, antimicrobial, Bacillus, fermentation, production. Correspondence Ramkrishna Sen, Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal, India. E-mail: rksen@yahoo.com

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 performance 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 puried using gel ltration chromatography with Sephadex G-50 matrix. The purication attained by employing this technique was evident from UVvisible spectroscopy and TLC analysis of crude and puried biosurfactants. The puried biosurfactants showed an increase in surface activity and a decrease in critical micelle concentration values. The antimicrobial action of the biosurfactants was also enhanced after purication. Conclusions: The marine B. circulans used in this study produced biosurfactants in a growth-associated manner. High degree of purication could be obtained by using gel ltration chromatography. The puried biosurfactants showed enhanced surface and antimicrobial activities. Signicance and Impact of the Study: The antimicrobial biosurfactant produced by B. circulans could be effectively puried using gel ltration and can serve as new potential drugs in antimicrobial chemotherapy.

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

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 emulsication 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, produced 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 pumilacidin also possess antimicrobial properties. Although biosurfactants 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 purication based on their physical and chemical properties (Mukherjee et al. 2006). The formation of molecular aggregates called micelles by the microbial surfactants is one of these and has been exploited for their separation using membrane ultra ltration (Sen and Swaminathan 2005). However, another effective separation procedure, the size exclusion chromatography that separates molecules based on the difference in their molecular weight, has not been exploited for biosurfactant
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purication. In this paper, we are reporting the production of a lipopeptide biosurfactant by a marine Bacillus circulans and its purication by size exclusion chromatography. The results suggest that signicant purication 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 1012 h at 37C (OD 600 nm: 1214). 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, 33 g NH4NO3, 22 g K2HPO4, 014 g KH2PO4, 001 g NaCl, 06 g MgSO4, 004 g CaCl2, 02 g FeSO4 and 05 ml l)1 of a stock solution containing the following trace elements per litre: 232 g ZnSO47H2O, 178 g MnSO44H2O, 056 g H3BO3, 10 g CuSO45H2O, 039 g Na2MoO42H2O, 042 g CoCl26H2O, 10 g EDTA, 0004 g NiCl26H2O and 066 g KI. Study of growth and biosurfactant production The growth and production of biosurfactants were monitored during fermentation in GMS production media described earlier. The growth was monitored by measuring 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 production 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 claried different hours samples was spotted onto a 20 10 cm precoated silica gel HPTLC plate (Merck, Germany) containing green uorescent F254. These samples were spotted under a ow of nitrogen gas with the help of a Linomat-5 TLC spotting device (CAMAG, Switzerland) having a robotic arm. After sample application on these
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plates, they were developed in a solvent system containing chloroform, methanol and water in a ratio of 65:25:4, respectively. The developing jars (CAMAG) were saturated with solvent system for 1520 min prior to the development. After development, these plates were airdried to remove solvent and a densitometric scan at 210 nm was performed with the help of a TLC Scanner 3 (CAMAG) for detection and quantication of biosurfactant. The quantication 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 purication The surface-active molecules produced by the microorganism were isolated chemically by acidication of the cell free broth (Sen and Swaminathan 1997). Briey, 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 acidied cell free culture broth was then stored at 4C overnight for precipitation of surface-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 75 to solubilize biosurfactants. Above a certain minimum concentration known as the critical micelle concentration (CMC), the biosurfactants form aggregates or micelles due to mutual interaction of their hydrophobic part. These aggregates or micelles contain a large number of individual surfactant molecules and form bulky structures with higher effective molecular mass, which is a multiple of mass of individual surfactant molecules. This property of biosurfactants to form bulky molecular aggregates has been utilized effectively for their purication by size exclusion or gel ltration chromatography. The crude water-soluble biosurfactants were centrifuged at 10 000 g for 5 min to exclude any insoluble matter. This claried and concentrated solution of crude biosurfactants was then applied to a Sephadex G-50 column (10 mm 300 mm, Amersham Biosciences) pre-equilibrated with Milli-Q water and eluted with slightly alkaline (pH 80) degassed Milli-Q water (Millipore). Fractions, each of 1 ml, were collected with a ow rate maintained at 1 ml min)1. The absorbance of the fractions was monitored at 210 nm using a UVvisible spectrophotometer (PerkinElmer, USA). The puried biosurfactant fractions were pooled and lyophilized in a Savant freeze dryer (model: micro modulyo 230, Thermo Scientic) to get the pure biosurfactant as a dry powder.

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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 purication increases. The CMC values of the crude and the puried biosurfactant were determined by gradual addition of biosurfactant to pure water. For this, concentrated solutions of crude and puried 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 nal concentration of biosurfactant increases by 50 mg l)1 with each addition. The change in surface tension of the water was noted with each addition in a DCAT digital surface tensiometer (DataPhysics). The minimum value of biosurfactant at which the surface tension is lowered abruptly reaching its minimum value was considered as the CMC for the biosurfactant sample. UVvisible spectroscopy UVvisible spectroscopy was performed to check the purity attained by the biosurfactants after gel permeation. For this purpose the rst few column fractions (fractions 712) containing puried biosurfactant in micelle form were collected, pooled and lyophilized. Similarly the latter fractions with contaminants (fraction 1922) were also pooled and lyophilized. Equal amounts of crude biosurfactants, puried biosurfactants (fractions 712) and contaminants (fractions 1922) were dissolved in water and their absorption properties were checked in UV and visible range. The UVvisible spectra absorption scans of these samples were performed in a Perkin-Elmer double beam UVvisible 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 ltration puried 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 puried biosurfactants were prepared in methanol. The antimicrobial action against bacterial strains were checked by agar well diffusion test on MuellerHinton agar medium (Hi-Media). For fungal strains agar plates were prepared containing their respective growth supporting solid medium. Crude and gel ltration puried 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 37C while the fungal test strains were incubated at 28C. 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 biomass was obtained as a function of OD and could be expressed with the standard equation, i.e. dry biomass (g l)1) = 038 OD600 nm. After an initial lag period of about 4 h the organisms growth proceeded at a slow rate till about 1214 h. At around 14 h the microbial growth
Table 1 Antimicrobial action of crude and puried biosurfactants on various strains of bacteria and fungi
Halo diameter Crude biosurfactant (50 lg) Puried biosurfactant (50 lg)

Organism Gram positive bacteria Micrococcus avus Bacillus pumilis Mycobacterium smegmatis Gram negative bacteria Escherichia coli Serratia marcescens Proteus vulgaris Klebsiella aerogenes Pseudomonas sp. Fungal strains Aspergillus niger Aspergillus avus Candida albicans

++ ++ ++ ++ ++ + + ++ ++ ++ ++

++++ +++ +++ + + + + + ++ ++ + + ++

++++ ++ ++++

Halo diameters: +,1013 mm; + +, 1417 mm; + + +, 1821 mm; + + + +, 21above. The results showed an increase in antimicrobial action upon purication.

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was slowed before the start of the major growth phase of this micro-organism at about 16 h. After this the organism enters into the exponential phase of its growth which continues up to about 28 h (Fig. 1). Although the biosurfactant production begins as early as 10 h as evident from the reduction in surface tension of the medium, signicant 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). Signicant 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 0072 g l)1 at 16 h, the biosurfactant concentration increased steadily to 04225 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 05 g l)1. The bacterium also showed a glucose utilization prole corresponding to its growth and biosurfactant production. 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 rst 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 nally reached a value of about 09 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 75 from an original value of 70 and remained more or less constant at this value. Isolation and purication of crude biosurfactants The biosurfactant produced in the production media could be isolated by acidication of the cell free culture broth with concentrated HCl. After overnight acidication at 4C the crude biosurfactant was separated as precipitate. The precipitate could be obtained by centrifugation. The pH of this crude biosurfactant pellet was raised to 75 and the concentrated biosurfactant solution was applied to gel ltration using Sephadex G-50 for further purication. With a ow rate of 1 ml min)1 the biosurfactant aggregates in form of micelles were eluted early in the fractions 712. The contaminating compounds comprising of other bacterial metabolites and isolated surfactant molecules were eluted in latter fractions, i.e. fractions 1922 (Fig. 2). The purication attained was checked by measuring the critical micelle concentration (CMC) values and by thin layer chromatographic (TLC) analysis of the crude and gel ltration puried biosurfactants. In the CMC experiments the minimum surface tension of 2878 mN m)1 was obtained after adding 40 mg l)1 of crude biosurfactants. On the other hand using column puried biosurfactant, a minimum surface tension of 2789 mN m)1 was obtained after addition of 25 mg l)1 biosurfactant to the pure water (Fig. 3). Analysis of the crude and gel permeation puried biosurfactants by TLC (Fig. 4) showed that signicant level of purication was achieved upon gel ltration. In the lane containing the puried biosurfactants, the different

Biomass/Biosurfactant (gl )

30 28 26 24 22 20 18 16 14 12 10 08 06 04 02 00 0 5 10 15 20 Time (h) 25 30 35

30 70 28 26 24 60 22 20 18 16 14 12 10 8 6 4 2 0 Surface tension (mNm1)

50

40

Glucose (gl1)
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 biosurfactant (h) production with time. It also shows glucose utilization () as a function of accumulation of biosurfactant in the GMS production medium.

30

20 40

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07 06 Absorbance (210 nm) 05 04 03 02 01 00 01 0 15 25 10 20 5 Fraction number b (1 ml each) 30

Figure 2 Purication of the crude biosurfactants by size exclusion chromatography using Sephadex G-50 matrix. The pure biosurfactants 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.

70

Surface tension (mNm1)

60

50

40

Figure 4 Thin layer chromatogram showing the crude (lane 1) and size exclusion puried (lane 2) biosurfactants. The purication 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 visualized under a 254-nm UV lamp.

30

20

20 40 60 Biosurfactant concentration (mg l1)

80

Figure 3 Determination of the critical micelle concentration (CMC) of crude (d) and puried ( ) biosurfactants. The minimum amount of biosurfactant required to reach CMC is decreased with increase in the purity of biosurfactants.

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 chromatography of the later fractions (column fractions 1922) 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 uorescence

under a 366-nm lamp. The lane containing the pure biosurfactants did not show uorescence under this wavelength, while the contaminants separated in latter column fractions showed high absorption under this light. The UVvisible spectrum scan also conrmed this fact, where the pure biosurfactants absorbed only in the far UV region (Fig. 5). On the other hand the crude and the column separated contaminants absorbed at higher wavelengths (340400 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 avus,
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300 25 20 A 15 10 05 000 1900 300 400 500 600 7000

Cont P Cr

nm

Figure 5 UVvisible spectra of the crude biosurfactant (Cr), puried biosurfactant (P) and the contaminants (Cont) separated in size exclusion chromatography. The contaminants present in the crude biosurfactants absorb at higher wavelengths (340400 nm) while the pure biosurfactants absorb only in the far UV region of the spectrum.

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. avus and Candida albicans. The inhibition zones were found to be largest in case of Gram-positive bacteria such as M. avus, 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 dened when same concentration of gel permeation puried biosurfactants were used instead of the crude biosurfactants. Discussion A marine B. circulans producing extracellular biosurfactants was isolated and identied 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 identied as lipopeptide 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 concentration of the biosurfactant in medium increased from a very low value of 007 g l)1 at the beginning of the exponential phase to a maximum of 10 g l)1 by the end of growth phase. This type of biosurfactant production prole 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
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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 antibiotics 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 biosurfactant production. The decline observed in the biosurfactant 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 producing 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 concentration, 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 puried with help of size exclusion chromatography. The formation of molecular aggregates called micelles by biosurfactant molecules in aqueous solutions facilitates their separation from the contaminants. Biosurfactants in the form of micelles, due to their bulky structures, were eluted in early column fractions, while other contaminating small molecules were eluted in latter fractions due to their small size and inability to form such aggregates. Although the micelle forming behaviour of biosurfactants has been exploited for their purication by membrane ultraltration (Sen and Swaminathan 2005), to the best of our knowledge, this is the rst report of purication of lipopeptide biosurfactants by size exclusion chromatography. As evident from the CMC values, considerable purication was attained by application of this technique for purication. The CMC values were nearly halved when the biosurfactants were subjected to column

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purication. The minimum surface tension value obtained in CMC experiments was lower in case of column-puried biosurfactants than those obtained for crude chemically isolated biosurfactants. This indicates an increase in surface activity of these molecules upon purication. The purication attained by the biosurfactants was also evident from TLC analysis in which the puried biosurfactants showed well-resolved spots with less smear caused due to contaminating metabolites. The crude biosurfactants absorbed in higher wavelengths (300400 nm) while the pure biosurfactants absorbed only in far UV region. The UVvisible scan of the crude biosurfactant, puried biosurfactant and contaminants proved that uorescing property of biosurfactants at higher wavelengths (366-nm lamp) is due to the contaminating molecules present in it. The absorption scan of the contaminants conrms this fact as these show signicant absorption in range 340 400 nm. The puried biosurfactants did not show any absorbance or uorescence in these wavelengths. The crude and puried biosurfactants showed profound antimicrobial activity against a panel of pathogenic and semipathogenic bacterial and fungal strains. The purication attained by the biosurfactant was evident from the increase in the antimicrobial action upon purication reected in larger inhibition zones produced by pure biosurfactant. 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 bacteria 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 antagonistic effect on both Gram-positive and Gram-negative bacteria. It has been reported earlier that different isoforms 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 molecules 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 identied. 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. acknowledge IIT, Kharagpur for the nancial assistances. R.S. and S.C. acknowledge the Department of Biotechnology

(DBT), Govt. of India for the project grant (BT PR6827 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
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