Biochemical Engineering Journal 42 (2008) 254–260

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Biochemical Engineering Journal

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Enhanced production of biosurfactant by a marine bacterium on statisticalscreening of nutritional parameters

Soumen Mukherjee1, Palashpriya Das1, C. Sivapathasekaran, Ramkrishna Sen ∗

Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal 721302, India

a r t i c l e i n f o

Article history:Received 27 May 2008Received in revised form 30 June 2008Accepted 7 July 2008

Keywords:FermentationSubmerged cultureChromatographyOptimizationPlackett–Burman design

a b s t r a c t

Marine microorganisms can serve as rich sources for novel biosurfactants with diverse biological activi-ties. In the present investigation, the nutritional requirement for the growth and biosurfactant productionby a marine bacterium was determined using a Plackett–Burman-based statistical screening procedure.Six out of the eleven factors of a reported production medium were found to be critically affecting the bio-surfactant metabolism. Glucose, the carbon substrate of the medium was the most influential factor withan effect contribution of 78.13% and a very low p-value of <0.001. Glucose, NH4NO3 and FeSO4·7H2O hada direct proportional correlation with biosurfactant production while, K2HPO4, KH2PO4 and MgSO4·7H2Oshowed inversely proportional relationship with biosurfactant production in the selected experimentalrange. A simpler modified medium (MM) was formulated based on the statistical screening results. Mod-ified medium combination (MM-1) having the following composition in g l−1: glucose 30; NH4NO3 6.0;

−2 −2

Biosurfactant K2HPO4 1.1; MgSO4·7H2O 0.3; KH2PO4 2.8 × 10 and FeSO4. 7H2O 6 × 10 showed 84.7% increase in bio-surfactant yield over the reported medium (SM). Fourier transform infrared spectroscopy and thin layerchromatography studies showed that the biosurfactants produced in the modified medium (MM) were

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. Introduction

Biosurfactants or microbial surfactants are surface-activeolecules produced by a wide variety of bacteria. These constitutediverse group of versatile molecules and are found in a vari-

ty of chemical structures such as glycolipids, lipopeptides andipoproteins, fatty acids, neutral lipids, phospholipids, and poly-

eric and particulate structures. Some of the features, which makehem promising alternatives to chemically synthesized surfactants,re their lower toxicity, higher biodegradability and better environ-ental compatibility. They are also found to be stable at extremes

f pH, salinity and temperature [1,2]. These molecules have recentlyeen reported to possess several commercial applications [2,3] asell as properties of therapeutic and biomedical importance. Some

f these properties are antibacterial, antifungal, antiviral proper-ies; inhibition of blood clot formation and anti-adhesive action

gainst many pathogenic microbes [4–6]. Despite such advantageshese molecules have not been commercialized due to low yieldsnd high production cost in production process. One of the strate-ies to improve production is to optimize the growth media in order

∗ Corresponding author. Tel.: +91 3222 283752; fax: +91 3222 278707.E-mail address: rksen@yahoo.com (R. Sen).

1 These authors contributed equally to this work.

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369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2008.07.003

e reported medium (SM).© 2008 Elsevier B.V. All rights reserved.

o get maximum production [2]. Formulation of an optimized pro-uction medium involves selection of the right nutrients at theirorrect levels to provide an ideal microenvironment for support-ng growth and metabolite production. Statistical experimentalesigns such as response-surface methodology [7], factorial [8,9]nd Taguchi [10] designs have been used to optimize media compo-ents and increase the product yields. These statistical optimizationrocedures minimize the number of experiments saving time and

abor. However, in most cases, the choice of media componentssed in such experimentation and the range of their concentra-ion levels are chosen on the basis of earlier literature reports. Tonsure an effective optimization of the medium it is advisable tocreen medium constituents for their significant effect on a partic-lar production process. Plackett–Burman statistical experimentalesigns [11], which can screen up to n variables with n + 1 experi-ent, are often helpful in this regard. These experimental designs

elp to screen the critically influential variables, thus reducinghe number of factors to be considered in optimization experi-

ents. This makes the subsequent optimization procedure muchimpler. In the present work, a 12-run Plackett–Burman experimen-

al design has been used to screen the medium components, whichignificantly affect biosurfactant production by a marine Bacillus sp.ased on the results, a modified simple medium was devised whichhowed significant enhancement in biosurfactant productionevels.

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. Materials and methods

.1. Microorganism

The microorganism used in this study was isolated from aater sample obtained from the Andaman and Nicobar Islands,

ndia. This culture was identified as a Bacillus sp. on the basisf various morphological, physiological and biochemical tests byicrobial Type Culture Collection (MTCC), Chandigarh, India. The

ulture was maintained on Zobell marine agar (HiMedia, Mumbai,ndia).

.2. Preparation of the seed culture

The seed culture for inoculating the production media wasrepared in two stages. The first stage of seed culture prepara-ion was done in Zobell marine broth (HiMedia, Mumbai, India).riefly, 2 ml of sterile marine broth was inoculated with a bac-erial colony previously maintained on marine agar plates. Aftervernight growth, this primary inoculum was transferred to 20 mlf sterile glucose mineral salts medium (GMSM) the compositionf which is described in the next section. This secondary inocu-um was grown up to the mid log phase (OD600: 2.3–2.5) and wassed to inoculate the final production media at 2% (v/v) level. Alleed cultures were grown at 37 ◦C and 180 rpm shaking speed in aotary incubator shaker (New Brunswick Scientific Co., Inc., Edison,J, USA).

.3. Standard production media and culture conditions

The media suggested by Makkar and Cameotra [12] with slightodifications was selected as the standard medium, as the organ-

sm showed growth and biosurfactant production in this medium.he composition of this medium per liter is as follows: 20 g glu-ose, 3.3 g NH4NO3, 2.2 g K2HPO4, 0.14 g KH2PO4, 0.01 g NaCl, 0.6 ggSO4, 0.04 g CaCl2, 0.2 g FeSO4 supplemented with 0.5 ml l−1 oftrace-salts solution containing the following trace elements per

iter: 2.32 g ZnSO4·7H2O, 1.78 g MnSO4·4H2O, 0.56 g H3BO3, 1.0 guSO4·5H2O, 0.39 g Na2MoO4·2H2O, 0.42 g CoCl2·6H2O, 1.0 g EDTA,.004 g NiCl2·6H2O and 0.66 g KI. All the fermentations were car-ied out in 250 ml Erlenmeyer flasks with 50 ml working volume.he cultures were incubated in a rotary incubator shaker (Newrunswick Scientific Co., Inc., Edison, NJ, USA) for 28 h at 37 ◦C and80 rpm.

.4. Determination of the critical medium components

The production medium described earlier was a mixture ofarious inorganic salts which supported growth of the organism.owever, all the salts present in the media may not be necessary

or the survival, growth or biosurfactant production as nutrientequirement varies amongst the organisms. In order to determineedia constituents absolutely necessary for the biosurfactant pro-

uction, experiments were designed in which the medium was kepteficient in one or the other component. For initial screening of

mportant factors, nine different combinations of media were pre-ared which were deficient in glucose, NH4NO3, K2HPO4, KH2PO4,aCl, MgSO4, CaCl2, FeSO4 and trace-salts solution, respectively.he media were sterilized by autoclaving at 121 ◦C and 1.3 kg cm−2

ressure maintained for 15 min in a horizontal autoclave (Natsteel,

umbai, India). The fermentations were carried out for 28 h and

he surface tension of the culture supernatant was measured withhe help of a digital surface-tensiometer (DCAT, DataPhysics Instru-

ents, GmbH, Filderstadt, Germany) working on the principle ofilhelmy plate method. The biosurfactant concentration was mea-

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ering Journal 42 (2008) 254–260 255

ured with help of high performance thin layer chromatographyHPTLC) described later in the text. All experiments were performedn triplicate and the values shown represent mean ± standard devi-tion of the values.

.5. Statistical experimental design: Plackett–Burmanxperiments

The significance of the various media constituents towards bio-urfactant production was further tested using Plackett–Burmantatistical experimental designs [11]. This method is based uponhe existence of Hadamard matrices, which are square matrices ofrder N with entries at two levels, +1 and −1. These matrices arerthogonal such that for each column the number of +1 is equal tohe number of −1. This experimental design is suitable for screen-ng the effect of a large number of factors in an experiment anddeal for the determination of main effects. The design assumes thathere are no interactions between different media constituents, xi,n the range of variables under consideration and a linear approachs considered sufficient for screening:

= ˇ0 +∑

ˇixi (i = 1, . . . , k) (1)

here Y is the estimated target function or response and ˇi are theegression coefficients.

With this experimental design, N factors can be screened withnly N + 1 experiments and screening up to 100 variables [13]s possible with the help of this design. In the present work,ffect of 11 major factors of the glucose mineral salts mediumn biosurfactant production was determined. Factors (media-omponents) taken into consideration were glucose (C6H12O6),mmonium nitrate (NH4NO3), magnesium sulfate (MgSO4), di-otassium hydrogen phosphate (K2HPO4), potassium di-hydrogenhosphate (KH2PO4), iron sulfate (FeSO4·7H2O), calcium chlorideCaCl2), sodium chloride (NaCl), copper sulfate (CuSO4·5H2O), zinculfate (ZnSO4·7H2O) and manganese sulfate (MnSO4·4H2O). Thetatistical software package Design-Expert® v. 7.1.3. (Stat Ease Inc.,inneapolis, USA) was used to generate the Plackett–Burman

xperimental design consisting of a set of 12 experimental runsn which the factors were kept either at their high (+) or low (−)evels. These media with the desired combinations of factors wererepared and sterilized by autoclaving at 121 ◦C and 1.3 kg cm−2

ressure maintained for 15 min in a horizontal autoclave (Natsteel,umbai, India). These statistically designed media were inoculatedith the seed culture at 2% (v/v) level and incubated at conditions

tated earlier for 28 h. After fermentation the biosurfactant concen-ration in the culture broth was estimated using HPTLC describedater in the text. All the experiments were performed in triplicatend at two different occasions and the responses considered fornalysis represent mean of these responses. The effect of each vari-ble was determined by the standard equation:

ffect = 2[∑

R(H) − ∑R(L)]

N(2)

here R(H) = all responses when component was at high levels,(L) = all responses when component was in low levels, N = totalumber of runs.

The standard error (S.E.) of the concentration effect is the squareoot of the variance of an effect and the significance level (p-value)

f each concentration effect is determined using student’s t-test:

(xi) = E(xi)S.E.

(3)

here, E(xi) is the effect of the variable xi.

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.6. Formulation of a modified production medium (MM)

Based on the results of Plackett–Burman experiments themportant nutritional factors involved in the biosurfactant produc-ion process by this organism could be identified. Five different

edia combinations were prepared containing different levels ofhese components. MM-1: +1 levels of the medium componentsositively affecting production and −1 levels of the medium com-onents negatively affecting production; MM-2: −1 levels of theedia components affecting positively and +1 levels of medium

omponents affecting negatively; MM-3: all components at theirasic (0) levels; MM-4: all components at their +1 levels and MM-: all components at their −1 levels. 50 ml of each medium wasrepared in triplicate in 250 ml Erlenmeyer flasks and sterilized.he fermentation was carried out for 28 h at 37 ◦C at 180 rpm. Theiosurfactant concentration was measured with HPTLC analysis.ll the experiments were performed in triplicate and the values

ndicate mean ± S.D.

.7. Chemical characterization of the biosurfactant

The chemical characterization of the biosurfactants producedn standard medium (SM) and modified medium (MM) wasone using FTIR spectroscopy (described later in text) and post-hromatographic detection after TLC described herein. Briefly,he biosurfactants obtained from standard medium and modi-ed medium were spotted in triplicate on pre-coated silica gellates (Merck, Darmstadt, Germany) and developed using the suit-ble solvent system (Chloroform: Methanol: Water/65:25:4). Afterhromatography one of the TLC plates were treated with iodineapors for detection of any lipids. Similarly the other plate wasreated with ninhydrin solution (2% ninhydrin in absolute alco-ol) followed by heating at 110 ◦C for detection of peptides and freemino acids. The third plate was visualized under a 254 nm UV lampo detect biosurfactant spots. All the plates were photographed forhe comparison and characterization of the biosurfactant fractions.

.8. Analytical methods

.8.1. Surface tension measurementsThe surface tension (ST) of the culture supernatants was

easured with a digital surface tensiometer (DCAT, DataPhysicsnstruments, GmbH, Filderstadt, Germany) working on the princi-les of Wilhelmy plate method [14]. The validity of these readingsas confirmed by taking surface tension readings of pure water

70.78 ± 0.02) before each sample reading.

.8.2. High-performance thin layer chromatographyHigh-performance thin layer chromatography was used for the

uantitative estimation of the biosurfactant as reported previously

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able 1he effect of mineral salts on growth and biosurfactant production

issing component in MSM Final ST (mN m−1) a X (

lucose 51.31 ± 0.03 NDH4NO3 45.15 ± 0.03 ND2HPO4 47.23 ± 0.02 NDgSO4·7H2O 54.35 ± 0.01 ND

H2PO4 30.90 ± 0.03 4.0aCl 28.10 ± 0.02 3.7aCl2 30.12 ± 0.01 2.2eSO4·7H2O 40.43 ± 0.01 0.8race-salts solution 28.87 ± 0.01 3.8ontrol (with all components) 28.68 ± 0.02 4.1

he critical components could be recognized as the bacteria failed to grow and produce bio= Biosurfactant.a Surface tension of the cell free supernatant after 28 h of fermentation.

ering Journal 42 (2008) 254–260

5]. Briefly for HPTLC analysis, 5 �l of the culture supernatant waspotted on pre-coated silica gel HPTLC plates (Merck, Darmstadt,ermany) with the help of a Linomat-5 (CAMAG, Switzerland) TLCpotter. After sample application, these plates were dried and theneveloped in a solvent system containing chloroform, methanolnd water (65:25:4). The densitometric scan of these TLC platesas performed at 210 nm with the help of a TLC Scanner 3 (CAMAG,

witzerland). The amount of the biosurfactant was estimated by theeak area determined in the densitometric analysis by comparingith a calibration curve for the purified biosurfactant product. Theure biosurfactant product used for the preparation of the calibra-ion curve was obtained through preparative TLC. Briefly, the crudeiosurfactant was spotted and developed with the same solventystem as mentioned before. The biosurfactant spots were thenisualized under UV lamp and scrapped off from the plates. Theilica containing these biosurfactant fractions was then dissolvedn a mixture of chloroform and methanol in the ratio of 65:35. Theilica was discarded after centrifugation and the supernatant wasept for evaporation of the solvent. The resulting pure biosurfactantas dissolved in water and lyophilized for further use.

.8.3. Fourier transform infrared spectroscopy (FTIR)Fourier transform infrared spectroscopy was used for compar-

son of the nature of the biosurfactant produced using standardeported medium (SM) and modified medium. The FTIR analysisas performed by using a Nexus-870 FTIR spectrometer (Thermo

lectron Co., Yokohama, Japan) with lyophilized samples dispersedn the pellets of KBr. The lyophilized biosurfactant samples wereround with spectral grade KBr (Merck, Darmstadt, Germany) andressed into pellets under approximately 5–6 tons/cm2 pressuresing a hydraulic press (Specac, Orpington, Kent, UK). Spectraere normally acquired at 4 cm−1 resolution over the range of00–4000 cm−1. All data were corrected for the background spec-rum.

. Results and discussion

.1. Critical media components

The marine Bacillus sp. was cultivated in the modified glucoseineral salts medium [12] described earlier with one or the otherajor components missing in it. The biosurfactant production by

his bacterium started during exponential phase and continued upo the stationary phase. The time needed to achieve maximum

oncentration of biosurfactant was found to be 28 h. Therefore, allhe cultures were incubated for 28 h for monitoring biosurfactantroduction. After 28 h of incubation no growth and biosurfactantroduction was seen in the medium deficient in glucose, NH4NO3,gSO4·7H2O and K2HPO4, respectively. A very little growth

g l−1) P (g l−1) YP/X YP/S

ND – –ND – –ND – –ND – –

5 ± 0.11 1.15 ± 0.13 0.28 0.051 ± 0.16 1.06 ± 0.14 0.28 0.059 ± 0.23 0.81 ± 0.07 0.35 0.040 ± 0.15 ND 0.00 0.001 ± 0.06 1.03 ± 0.12 0.27 0.052 ± 0.11 1.24 ± 0.06 0.30 0.06

surfactants in the absence of important components. ND, not detected; X = Biomass;

S. Mukherjee et al. / Biochemical Engineering Journal 42 (2008) 254–260 257

Table 2The various media components included in Plackett–Burman experiments and theircorresponding higher and lower concentration levels

Variables code Media constituents High level (+1)(g l−1)

Low level (−1)(g l−1)

A Glucose 30 10B NH4NO3 6 1.5C K2HPO4 5.5 1.1D MgSO4·7H2O 1.5 0.3E KH2PO4 0.42 2.8 × 10−2

F NaCl 0.03 2.0 × 10−3

G CaCl2 0.12 8.0 × 10−3

H FeSO4·7H2O 6 × 10−2 4.0 × 10−3

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Factors (Coded) Biosurfactantyield (g l−1)

Runs A B C D E F G H I J K

1 − + − + + + – – − + − 1.652 − + + − + + + − − − + 0.553 + − + + − + + + − − − 1.704 − + − + + − + + + − − 0.805 − − + − + + − + + + − 0.406 − − − + − + + − + + + 0.257 + − − − + − + + − + + 2.358 + + − − − + − + + − + 2.509 + + + − − − + − + + − 2.21

10 − + + + − − − + − + + 0.8011 + − + + + − − − + − + 1.1812 − − − − − − − − − − − 0.70

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CuSO4·5H2O 15 × 10−4 1.0 × 10−4

ZnSO4·7H2O 34 × 10−4 2.3 × 10−4

MnSO4·4H2O 26 × 10−4 1.78 × 10−4

ithout any biosurfactant production was seen in the mediumeficient in FeSO4·7H2O. On the other hand this microorganismas able to grow and produce biosurfactant in the medium defi-

ient in CaCl2, NaCl, KH2PO4 and microsalts solution, respectively.lthough in these cases, the bacterium was able to utilize otheredium components, grow and produce biosurfactants, the level

f production was lowered in all these media compared to theontrol media which contained all the nutrients. Table 1 showshe final biomass, biosurfactant concentration and the final sur-ace tension of the selective nutrient deficient media after 28 h ofncubation. The results suggest that at least five components, i.e.lucose, NH4NO3, K2HPO4, MgSO4·7H2O and FeSO4·7H2O has to bencluded in any optimized medium for the growth and biosurfac-ant production by this bacterium.

.2. Effect of medium components on biosurfactant production:lackett–Burman screening

Initial experiments performed with media deficient in one orhe other major medium components showed that five factorsiz. glucose, NH4NO3, K2HPO4, MgSO4·7H2O and FeSO4·7H2O werebsolutely important for biosurfactant production. Although theritical elements affecting growth could be identified with thesexperiments, it was desirable to identify all the components signif-cantly altering biosurfactant production. A media component mayot be absolutely essential for the growth and production process,till its concentration levels in the production media can affect theroduction process. The Plackett–Burman experiments were per-

ormed to screen these components and to find out their probableptimal levels. Table 2 represents the independent variables andheir respective high and low values used in the statistical screen-ng study. Table 3 represents the Plackett–Burman experimentalesign for 12 trials at two levels of concentration for each vari-

3

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able 4tatistical parameters for the various components included in the Plackett–Burman scree

ariable Media component Effect SSa

Glucose 1.35 5.45NH4NO3 0.32 0.31K2HPO4 −0.24 0.17MgSO4·7H2O −0.39 0.45KH2PO4 −0.20 0.13NaCl −0.17 0.082CaCl2 0.10 0.033FeSO4·7H2O 0.33 0.34CuSO4·5H2O −0.068 0.014ZnSO4·7H2O 0.038 0.004MnSO4·4H2O 0.028 0.002

a Sum of squares.b p-values < 0.05 were considered to be significant.

ig. 1. Pareto chart showing the relative levels of significance of various media com-onents in coded. The components A (glucose), B (NH4NO3) and H (FeSO4·7H2O)howed significant positive effect while C (K2HPO4), D (MgSO4·7H2O) and EKH2PO4) showed significant effect.

ble along with the responses (biosurfactant yield in g l−1). Theesponses were analyzed with help of the statistical software pack-ge Design-Expert® v. 7.1.3. (Stat Ease Inc., Minneapolis, USA).

.2.1. Significant variablesTable 4 shows the statistical parameters obtained after the

esign-Expert analysis. As clear from the table, six out of theleven variables included in this study were found to be statistically

ning study

Contribution (%) F-Value p-Valueb Significance

78.13 404.75 <0.001 Yes4.45 23.04 0.008 Yes2.37 12.29 0.024 Yes6.48 33.57 0.004 Yes1.81 9.36 0.037 Yes1.17 6.06 0.069 No0.47 2.53 >0.1 No4.82 24.99 0.007 Yes0.20 1.07 >0.1 No0.063 0.33 >0.1 No0.034 0.18 >0.1 No

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58 S. Mukherjee et al. / Biochemical E

ignificant in the biosurfactant production process. Fig. 1 showshe Pareto-chart of the effects displaying the relative levels ofignificance. Glucose, the carbon source in the medium, showshe highest level of significance with a % contribution of 78.13.n F-value of 404.75 and a very low p-value of less than 0.001emonstrate its significance and large effect on the biosurfactantroduction. This implies that besides supporting growth and nor-al metabolism, a large part of carbon is diverted towards the

iosurfactant production pathway. Glucose shows a positive effectf 1.35 on biosurfactant production. This positive correlation thatxisted between glucose concentration and biosurfactant produc-ion implies that a higher amount is more effective in increasingields in the experimental limits chosen. The carbon source haseen reported in literature as a vital limiting factor in biosurfactantroduction process. The biosurfactant yields can change drasti-ally depending upon the concentration of carbon in the medium7] justifying its high significance in these experiments. Ammo-ium nitrate (NH4NO3), the source of nitrogen in the medium alsohows its significance in production process although at a muchower level with 4.45% contribution. It gives an F-value of 23.04nd p-value of 0.0087 displaying its significance in the process. Aositive effect of this nitrogen source predicts an increase in pro-uction upon increasing its concentration in the medium. Besidesrowth, nitrogen is an important constituent of the peptide partf the lipopeptide biosurfactants. This justifies its significance inhe production process of biosurfactants, lipopeptides in particular./N ratio has been reported to significantly affect the biosurfactantields; a low C/N ratio being more effective in increasing bio-urfactant concentration [15]. Our results suggest a similar trendince an increase in nitrogen concentration implies a low C/N ratio.itrogen limitation has been reported to enhance biosurfactantroduction with an initial high carbon and nitrogen in the mediums also found in our case [16]. The two potassium phosphate saltsncluded in this study, i.e. KH2PO4 and K2HPO4 also showed signif-cant effects in biosurfactant production process. Both these saltsad negative effect towards production process indicating loweroncentrations to be more suitable for increasing biosurfactantields. The contributions towards total effects were 2.37% and 1.81%,espectively for K2HPO4 and KH2PO4. The F-values for K2HPO4 andH2PO4 were found to be 12.29 and 9.36, respectively whereas the-values were 0.0248 and 0.0377, respectively. These two salts arehe source of K+ and PO4

3− and have a buffering action in the medias well. Low phosphate and a high C/P ratio have been reported

o increase biosurfactant production [17,18]. The negative value offfect coefficient suggests a similar trend in our system. The con-entration level of MgSO4, which acts as a source of Mg2+ ions inhe medium was also found to influence biosurfactant production.

gSO4 was found to possess a negative effect that signifies its effec-

cotbn

ig. 2. Surface-plots showing the relative effects of (A) glucose and NH4NO3, (B) K2HPO4 aroduction.

ering Journal 42 (2008) 254–260

iveness in lower concentrations in the experimental range. Theontribution of MgSO4 towards total effects was 6.48% and wasust next to glucose. The F- and p-values for MgSO4 were 33.57 and.0044, respectively reflecting its high significance in this process.g2+ ion is a cofactor of the Bacillus subtilis Sfp protein that acti-

ates the peptidyl carrier protein domains of surfactin synthetasenzyme [19]. A similar and vital role of magnesium may exist inhe biosurfactant production and other metabolic pathways of thisrganism. The critical character of magnesium may also be an adap-ive measure for this marine microorganism, as magnesium is anmportant constituent of seawater. FeSO4, another critical com-onent required for the growth and biosurfactant synthesis is aource of Fe2+ ions in the medium. The contribution of FeSO4·7H2Oowards total effects was found to be 4.82%. The F- and p-values forhis iron salt were 24.99 and 0.0073, respectively. Fe2+ ions haveeen reported to be vital for biosurfactant synthesis and iron sup-lementation drastically improves the production of surfactin, a

ipopeptide biosurfactant [20,21]. However, excess iron in mediaesulted in acidification, which caused biosurfactant precipitationnd loss in cell viability [22]. In our case however, the higher levelid not inhibit the production process, as the effect was positive.his suggests a higher optimum concentration for use in futureptimization experiments. Fig. 2 shows the surface plots show-ng the relative effect of the significant factors, glucose–NH4NO3,2HPO4–KH2PO4 and MgSO4·7H2O–FeSO4·7H2O on biosurfactantroduction.

.2.2. Non-significant variablesNaCl, a source of Na+ and Cl− ions in the medium was not found

o be statistically significant, contributing only 1.17% to the totalffects and F- and p-value of 6.06 and 0.0695. In spite of beinghe major constituent of seawater NaCl did not show large signifi-ance and effect in biosurfactant production process of this marineicroorganism. This may be due to the important role of K+ ions

n the medium which masks the significance of Na+ ions. CaCl2,he source of calcium in the mineral salts medium, was found to bensignificant in terms of contribution (0.47%) and p-value (>0.1). Thensignificant nature of CaCl2 may be due to the non-involvementf Ca2+ in any important biochemical reaction and the presencef a more vital di-positive ion (Mg2+) in the medium. The threeajor variables taken from the trace-salt component of the stan-

ard medium, that were incorporated in the present study, namelyuSO4·5H2O, ZnSO4·7H2O and MnSO4 did not show any signifi-

ance towards production process with p-value >0.1. Probably, Cu2+

r Zn2+ are not required as enzyme cofactors in any metabolic reac-ion related to the production of biosurfactants. Although Mn2+ haseen reported to enhance the production of surfactin [23], no sig-ificant effect of this component was observed in our studies. This

nd KH2PO4, and (C) FeSO4·7H2O and MgSO4·7H2O concentrations on biosurfactant

S. Mukherjee et al. / Biochemical Engineering Journal 42 (2008) 254–260 259

Table 5Yields of biomass and biosurfactant and the yield coefficients in various combina-tions of the modified medium

Media combinations Biomass (X) (g l−1) Biosurfactant (P) (g l−1) YP/X YP/S

MM-1 5.00 ± 0.10 2.42 ± 0.09 0.48 0.08MM-2 1.51 ± 0.12 0.48 ± 0.08 0.31 0.04MM-3 3.96 ± 0.15 1.58 ± 0.10 0.39 0.07MM-4 5.13 ± 0.20 2.13 ± 0.15 0.41 0.07MM-5 2.23 ± 0.23 0.56 ± 0.05 0.25 0.05S

Bv

siee

3

figth(YSiTmahcpiowlir

Fig. 4. Thin layer chromatography of the biosurfactants obtained from standardrBwv

fcdmutpTeon

M 4.24 ± 0.06 1.31 ± 0.07 0.30 0.06

iosurfactant production in MM-1 was significantly higher compared to MM-3 (p-alue <0.05) and SM (p-value <0.01).

uggests that a minimum basal concentration of these nutrientss sufficient to affect growth and production and their individualffects are negligible compared to the effects of other significantlements.

.3. Improved production medium

The organism showed growth and production in the modi-ed and simpler media containing only the factors essential forrowth and biosurfactant production. The production of biosurfac-ant in the modified medium MM-1 was found to be significantlyigher (Table 5) than the standard medium (SM) and the MM-3modified medium at 0 concentration level). The yield coefficients,P/X and YP/S were significantly higher for MM-1 in comparison toM and MM-3. This justifies a better product formation in mod-fied medium per unit mass of cells or carbon substrate utilized.his substantiates further the validity of Plackett–Burman experi-ents that predict an increase in biosurfactant production in suchmedium. The biosurfactant production was also found to be

igh in MM-4, which contained all factors at high levels. As theontribution of glucose is highest (78.13%) towards biosurfactantroduction, a high level of this promotes biosurfactant production

n spite of the negative effects caused due to high concentration

f K2HPO4, KH2PO4 and MgSO4·7H2O. Biosurfactant concentrationas found to be low in MM-2 and MM-5. Glucose, which is the

imiting factor in biosurfactant production process, was presentn low concentration in these media combination which furtheresults in low yields. From this study it was clear that good biosur-

3

tt

Fig. 3. FTIR spectra of biosurfactant from standard reported medium (BS-S

eported medium (BS-SM) and modified medium (BS-MM). Lane A, BS-SM and Lane, BS-MM viewed under 254 nm UV lamp; Lane C, BS-SM and Lane D, BS-MM sprayedith ninhydrin solution; Lane E, BS-SM and Lane F, BS-MM developed with iodine

apors.

actant yield could be obtained from a simple production mediumontaining the vital components at their concentration levels pre-icted by the Plackett–Burman experiments. A simple productionedium containing least number of essential factors is highly val-

ed in industrial processes. A simple medium not only reduceshe raw materials cost involved in the process but also makes theurification procedure of extracellular metabolites much easier.he statistically designed experiments thus helped to find out thessential media components and formulate a medium containingnly these vital components while excluding the factors that areot essential to the production process of biosurfactants.

.4. Biosurfactant characteristics

FTIR study (Fig. 3) indicated that the quality and the nature ofhe biosurfactant produced in the modified medium were similar tohose produced in the standard medium. Biosurfactants produced

M) compared with biosurfactant from modified medium (BS-MM).

2 ngine

inFftsitptitti

4

ihAosasiiwerthgmbttmsTtLoub

A

KDgAnis

R

[

[

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[24] S.C. Lin, M.A. Minton, M.M. Sharma, G. Georgiou, Structural and immunological

60 S. Mukherjee et al. / Biochemical E

n both of these media showed IR absorption at almost same wave-umber positions thus indicating their similarity at molecular level.urthermore, the IR absorption profile of the biosurfactant obtainedrom both the standard and the modified media showed similarityo surfactin, a lipopeptide biosurfactant and other lipopeptide bio-urfactants like lichenysin [24] and arthrofactin [25] indicating thatt is a lipopeptide type of a biosurfactant. The lipopeptide nature ofhese surfactants was further confirmed by thin layer chromatogra-hy. After chromatographic development, the biosurfactant spotshat were visible under the UV lamp (254 nm) stained yellow withodine indicating the presence of lipid in the molecule. Similarly,hese spots also produced purple spots with ninhydrin indicatinghe presence of peptide moiety in the same molecule confirmingts lipopeptide nature (Fig. 4).

. Conclusions

In the present work the production media components signif-cantly affecting the biosurfactant yields by a marine Bacillus sp.ave been identified using Plackett–Burman experimental design.12-trial Plackett–Burman design was used to evaluate the effect

f 11 major media components selected from a glucose mineralalt medium. Glucose, NH4NO3, K2HPO4, MgSO4·7H2O, KH2PO4nd FeSO4·7H2O were found to be significantly affecting the bio-urfactant production. The effectiveness of these components inncreasing biosurfactant yield was evaluated in terms of their pos-tive or negative effects in the selected experimentation range. Itas found that glucose; NH4NO3 and FeSO4·7H2O had a positive

ffect on biosurfactant production for the selected experimentalange. This suggests an optimum concentration of these variablesowards the higher side of the experimental range. On the otherand K2HPO4, MgSO4·7H2O and KH2PO4 had a negative effect sug-esting an optimum in lower side of the experimental range. Aodified medium was formulated containing only the vital factors

ased on Plackett–Burman screening experiments. Good biosurfac-ant yield obtained in the modified medium further substantiatedhe validity of the Plackett–Burman experiments. The modified

edia combination (MM-1) showed significant increase in the bio-urfactant yield by 84.7% over the reported medium (SM). FTIR andLC studies showed that the quality and property of the biosurfac-ant remains unchanged when produced in the modified medium.ipopeptide biosurfactants were produced by this strain growingn both of these mediums. The results obtained may in prove to beseful in optimization of the simple modified medium to maximizeiosurfactant production.

cknowledgements

S.M. acknowledges CSIR, New Delhi and P.D. acknowledge IIT,haragpur for the financial assistances. R.S. acknowledges theepartment of Biotechnology (DBT), Govt. of India for the project

rant (BT/PR-6827/AAQ/03/263/2005) in marine biotechnology.uthors also gratefully acknowledge members of medical biotech-ology lab, IIT, Kharagpur for their help during the course of

nvestigation. S.M. and P.D. acknowledge Sujoy Sarkar and Subha-ish Das for the photographs.

[

ering Journal 42 (2008) 254–260

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