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Journal of Surfactants andDetergents ISSN 1097-3958 J Surfact DetergDOI 10.1007/s11743-013-1520-y

Achieving the Best Yield in GlycolipidBiosurfactant Preparation by Selecting theProper Carbon/Nitrogen Ratio

Rashmi Rekha Saikia, HemenDeka, Debahuti Goswami, JiumoniLahkar, Siddhartha Narayan Borah,Kaustuvmani Patowary, Plabita Baruah,et al.

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ORIGINAL ARTICLE

Achieving the Best Yield in Glycolipid Biosurfactant Preparationby Selecting the Proper Carbon/Nitrogen Ratio

Rashmi Rekha Saikia • Hemen Deka • Debahuti Goswami •

Jiumoni Lahkar • Siddhartha Narayan Borah •

Kaustuvmani Patowary • Plabita Baruah • Suresh Deka

Received: 28 February 2013 / Accepted: 31 July 2013

� AOCS 2013

Abstract Pseudomonas aeruginosa RS29, the native

biosurfactant-producing strain isolated from the oil fields

of Assam, India was used to investigate the influence of the

carbon nitrogen ratio on production of the biosurfactant.

The biosurfactant producing ability of the strain was

measured based on surface tension (ST) reduction of the

culture medium and the emulsification (E24) index. Pro-

duction was greatly influenced by the sources of nitrogen

and carbon as well as the carbon to nitrogen (C/N) ratio.

Sodium nitrate was the best nitrogen source and the water

miscible carbon source, glycerol was observed as the best

carbon source for maximum biosurfactant production. The

C/N ratio 12.5 allowed the maximum production of bio-

surfactant by the RS29 strain. At this C/N ratio, 55 % ST of

the culture medium was reduced by the produced biosur-

factant. Concentrations of crude and rhamnolipid biosur-

factant obtained at this particular C/N ratio were 5.6 and

0.8 g/l respectively. The RS29 strain was novel as it was

able to produce a sufficient amount of biosurfactant uti-

lizing a much lower amount of the water miscible carbon

source, glycerol. Extraction of the biosurfactant by a

chloroform–methanol (2:1) mixture was the best method to

obtain the highest biosurfactant from the culture medium of

the strain. The biosurfactant was confirmed as a mixture of

mono and di-rhamnolipid congeners, Rha–C10–C10–CH3

being the most abundant one. The biosurfactant was a good

foaming and emulsifying agent.

Keywords Pseudomonas aeruginosa � C/N ratio �Biosurfactant � Rhamnolipid

Introduction

Biosurfactants have received more and more attention in

recent years due to various biological functions and prop-

erties. They have potentials in commercial applications like

in food, in microbiological, pharmaceutical and biomedical

industries, as bio-control agents in agricultural applications

and in health and beauty products for the cosmetic indus-

tries [1–6]. Biosurfactants are environmentally friendly in

nature. When compared to synthetic surfactants, they have

several advantages, including high biodegradability, low

toxicity, low irritancy, and compatibility with human skin

[7, 8] and they are superior to their synthetic counterparts.

The type, quality and quantity of biosurfactants pro-

duced are influenced by the changes in the environmental

conditions [9]. Since the highest activities of microbes can

be attained under suitable environmental conditions; hence

optimization of these conditions is a vital step to achieve

maximum production of biosurfactant. Feeling the need of

these compounds, many studies have been conducted at

present to ensure the production of biosurfactant by

microorganisms [10–12]. But the main challenge with the

production of biosurfactants for commercial applications is

the high production cost. Because of the production cost,

biosurfactants cannot compete economically with the

chemically synthesized ones [13]. Therefore, the present

study was conducted with the aim of obtaining maximum

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11743-013-1520-y) contains supplementarymaterial, which is available to authorized users.

R. R. Saikia � H. Deka � D. Goswami � J. Lahkar �S. N. Borah � K. Patowary � P. Baruah � S. Deka (&)

Environmental Biotechnology Laboratory, Life Sciences

Division, Institute of Advanced Study in Science and

Technology (IASST), Paschim Boragaon,

Guwahati 781035, Assam, India

e-mail: sureshdeka@gmail.com; sureshdeka@yahoo.com

123

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DOI 10.1007/s11743-013-1520-y

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biosurfactant production with the native isolate, Pseudo-

monas aeruginosa RS29 by selecting proper carbon and

nitrogen sources. Detailed investigation was also carried

out to select the best C/N ratio to attain the highest pro-

duction of biosurfactant. The most efficient method was

selected to extract maximum amount of biosurfactant from

the culture media. The biosurfactant produced was further

investigated to characterize it for its composition and

activity.

Experimental Procedures

Materials

Pseudomonas aeruginosa RS29, isolated from crude oil

contaminated soil was used for the present study [14].

Cultures were grown in 500-ml conical flasks containing

100 ml mineral medium with the composition (g/l):

NH4NO3 (2.0), KCl (0.1), KH2PO4 (0.5), K2HPO4 (1.0),

CaCl2 (0.01), MgSO4�7H2O (0.5), FeSO4�7H2O (0.01),

yeast extract (0.1) and 10 ml of trace element solution

containing (g/l): H3BO3 (0.26), CuSO4�5H2O (0.5),

MnSO4�H2O (0.5), (NH4)6Mo7O24�4H2O (0.06) and

ZnSO4�7H2O (0.7). Glycerol (2 % v/v) was used as the sole

carbon source. All chemicals were purchased from Merck

(Mumbai, India). The pH of the medium was adjusted to

7.0 ± 0.5 and the flasks were kept in a shaking incubator

(Orbitek Ljeil) at 37.5 �C and 150 rpm for 4 days. The ST

measurement(s) was carried out in a K11 tensiometer

(Kruss, Germany) using the plate method.

Methods

Investigation of Nitrogen Sources

The nitrogen sources investigated in the study were

ammonium nitrate, ammonium chloride, ammonium sul-

fate, sodium nitrate and potassium nitrate. The nitrogen

salts were added to the mineral medium containing glyc-

erol as the sole carbon source at 0.2 % (w/v) concentration.

A control without any extra nitrogen salt was also studied.

Optical density (OD) at 600 nm, E24 index and critical

micelle dilution (CMD) were measured along with the

measurement of ST. OD was measured with a Shimadzu

UV-1800 UV-spectrophotometer.

The E24 index of the produced biosurfactant was mea-

sured by mixing 3 ml of whole bacterial culture with an

equal volume of n-hexadecane. The mixture was vortexed

at high speed for 2 min and left undisturbed for 24 h. The

E24 index is used to measure the emulsification ability of

biosurfactants and expressed as below [15, 16]

Percentage of height of emulsified layer mmð ÞTotal height of the liquid column mmð Þ

The CMD is defined as the solubility of a surfactant in

an aqueous phase and is commonly used to measure the

efficiency of a surfactant [17]. CMD-1 and CMD-2 were

determined by measuring the ST values of the cell free

culture broth (CFCB) diluted 10- and 100-times with

distilled water [18]. The CFCB was obtained by

centrifuging a 48-h old culture of the strain grown in the

mineral medium with glycerol at 10,0009g for 20 min at

4 �C.

Effect of Carbon Sources

Water miscible and immiscible carbon sources were

investigated for their ability to support growth and bio-

surfactant production of the strain. The carbon sources used

in the study were glucose, glycerol, mannitol, n-hexadec-

ane and olive oil. These were added at the 2 % level to the

mineral medium amended with the best nitrogen source.

The inoculated mineral medium was incubated in a rotary

shaker at 37.5 �C with 150 rpm for 4 days and the ST was

measured every day. OD, E24 index and CMD were

recorded for each of the carbon sources. A control without

any carbon source was also studied in the investigation.

Influence of the C/N Ratio

The C/N ratio is a vital factor influencing the performance

of biosurfactant production [19, 20]. Hence, in the present

study various C/N ratios were investigated to select the

optimum one for maximum biosurfactant production.

Carbon and nitrogen sources used in the study were the

best ones selected from the above studies. The investigated

C/N ratios were 2.5, 7.5, 12.5, 17.5 and 22.5. These were

prepared by keeping the nitrogen source concentration

constant (2 g/l) and changing the concentration of the

carbon source (5, 15, 25, 35, 45 g/l). The cultures were

allowed to grow in a rotary shaker at 150 rpm at 37.5 �C.

Growth of biomass was measured by the UV-spectropho-

tometer. Production of biosurfactant was measured to

select the best C/N ratio along with the measurement of ST.

Quantification of the biosurfactant was performed as

described by Saikia et al. [14]. The produced biosurfactant

was characterized biochemically as well as by LC–MS

analysis which has been described later on.

Extraction and Purification of the Biosurfactant

Three methods were investigated to select the best one for

extraction of the biosurfactant produced by the RS29 strain.

Three different solvent systems were used in these three

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methods. The solvent systems were acetone, chloroform–

methanol and ethyl acetate.

In the first method, the culture broth was centrifuged at

10,0009g for 20 min to remove the cells from the medium.

The clear supernatant served as the source of the crude

biosurfactant. The biosurfactant was recovered from the

CFCB by cold acetone precipitation as described by Pruthi

and Cameotra [21]. The pH of the biosurfactant-containing

CFCB was adjusted to pH 11 and three volumes of chilled

acetone was added to that. It was then allowed to stand for

10 h at 4 �C to eliminate secondary metabolites by pre-

cipitation. The precipitate was collected by centrifugation

and evaporated to dryness to remove residual acetone. In

the second method, biosurfactants from the CFCB was

extracted by a chloroform–methanol (2:1) mixture three

times. The combined extracts were then transferred to a

round-bottom flask connected to a rotary evaporator. The

concentration process was continued at 40 �C until a yel-

lowish-brown colored viscous and consistent extract was

obtained. The crude biosurfactant was then dried and

weighed. In the third method, CFCB was heated at 110 �C

for 10 min to deproteinize it. After cooling, it was acidified

to pH 3.0 by the addition of 2 N HCl. Biosurfactants were

extracted continuously with ethyl acetate at room temper-

ature. The mixture was shaken vigorously and then left

static so that the organic phase got separated from the

aqueous phase. The organic phase was then transferred to a

rotary evaporator and a viscous honey-colored biosurfac-

tant was recovered after solvent evaporation at 40 �C under

reduced pressure. Quantification of the pure biosurfactant

was carried out by a specific method of Chandrasekaran

and Bemiller [22].

Purification of the biosurfactant was performed by liquid

column chromatography according to previous literature

[23] with slight modifications. The crude biosurfactant

(1 g) was dissolved in 5 ml of chloroform and subjected to

a silica gel 60 chromatography column (26 9 3.3 cm)

equilibrated with chloroform. The loaded column was

washed with chloroform and chloroform–methanol mobile

phases were applied as; 50:3 v/v (300 ml), 50:5 v/v

(200 ml) and 50:50 v/v (100 ml) at a flow rate of 1 ml/min.

Fractions of 15 ml were collected and biosurfactant present

in the fractions were detected (ST measurement). Finally,

chloroform–methanol (50:50 v/v) was applied to remove

any remaining biosurfactant from the column. The bio-

surfactant-containing fractions were combined and dried in

a rotary evaporator at 40 �C.

Characterization of Biosurfactant

The purified biosurfactant was analyzed by thin layer

chromatography (TLC). The biosurfactant was dissolved in

a chloroform–methanol (9:1) mixture at a concentration of

10 mg/ml [24]. For analysis, 2 ll of the sample was spotted

onto the silica gel (G60: Merck, Germany) plates. After

drying the sample for 5 min, the plates were developed in

chloroform:methanol:acetic acid:water (25:15:4:2 v/v/v/v)

at room temperature. The chromatogram was sprayed with

a-naphthol reagent followed by concentrated sulfuric acid.

The biosurfactant was separated and identified by LC

coupled to MS using an Agilent 6410 Triple Quad LC–MS

system. The purified sample was dissolved in methanol and

2 ll of the sample was injected into a Zorbax C18 column

(2.1 9 50 mm). The LC flow rate was 0.2 ml/min. An

acetonitrile/water gradient with 0.01 % formic acid was

used (10–90 %) as the mobile phase. ESI–MS was per-

formed in positive mode and analyzed using Agilent

software.

Activity Assay

Foaming

For measuring foaming activity, the flask containing a

2-day old culture of the strain was hand shaken for 2 min

and the stability of the foam was monitored by observing it

for 48 h.

Emulsification

Emulsification activity of the produced biosurfactant was

measured as described previously for the E24 index. Six

immiscible solvents namely, n-hexadecane, hexane, olive

oil, crude oil, diesel and coconut oil were used to observe

the emulsification activity of the biosurfactant.

Oil Displacement

Oil displacement is a method used to determine efficiency

of a given biosurfactant by evaluating the diameter of the

clear zone formed after the addition of surfactant-con-

taining solution on an oil–water interphase. The test is also

used as a preliminary method for identification of biosur-

factant producing strains. Oil displacement activity is

directly proportional to the concentration of the biosur-

factant in the solution [25]. For this assay, 50 ll of crude

oil was added to the surface of 40 ml of distilled water in a

petri dish (15 cm) to form a thin oil layer. Then, 25 ll of

culture broth was gently placed on the centre of the oil

layer. A control plate was also studied where instead of

culture broth only distilled water was placed on the oil

layer. The oil displacement area (ODA) was calculated as-

ODA ¼ 22=7 radius2� �

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Statistical Analysis

Studies were carried out three times with three replications

each and the results represented are the means ± standard

deviations (SD). One way ANOVA was conducted to see

the significance of production of biosurfactant as well as its

extraction from culture media.

Results and Discussion

Investigation of Nitrogen Sources

Application of various nitrogen sources in the culture

media revealed that maximum reduction of ST (55.5 and

55.3 %) was achieved while sodium nitrate and potassium

nitrate were used as nitrogen sources respectively

(Table 1). This might be because of nitrogen salts being

less available from these two salts. Since nitrate first

undergoes dissimilatory nitrate reduction to ammonium

and then assimilation by glutamine-glutamate metabolism;

assimilation of nitrate as a nitrogen source is so slow that it

would simulate a condition of limiting nitrogen [26, 27]. It

had been reported that nitrogen limitation enhances bio-

surfactant production [28]. Ammonium nitrate also showed

significant reduction of ST (54.05 %). ST reduction for

ammonium chloride and ammonium sulfate (13 and

12.71 % respectively) was not prominent as compared to

the other nitrogen sources and the control (40.03 %).

Maximum bacterial growth was observed in an ammo-

nium nitrate-containing mineral medium and for the rest of

the four nitrogen sources almost equal growth was recor-

ded. The lowest bacterial growth was observed in the case

of the control (Table 1). Ammonium nitrate-supplemented

medium supported the growth of the strain more than the

synthesis of biosurfactant by it. Though ammonium sulfate

and ammonium chloride also supported bacterial growth

but synthesis of biosurfactant was very poor in the cases of

these two salts.

The control also showed results. This may be because of

the bacterial utilization of yeast extract present in the

medium in which the strain grew, reduced ST and showed

an E24 index of 50 %. The E24 index was at a maximum

and almost similar for sodium nitrate (80 %) and potassium

nitrate (79 %) followed by ammonium nitrate (72 %). The

minimum was observed for ammonium chloride and

ammonium sulfate (36 % for both) (Table 1).

CMD-1 and CMD-2 were also lowest (27.2 and

40.0 mN/m respectively) for sodium nitrate-supplemented

media followed by potassium nitrate (Table 1). The results

showed little change in efficiency after diluting the sample

100 times. It suggested that a sufficient amount of biosur-

factant was present in the culture medium, and thus its

surface activity was retained even at such a high dilution.

These values are appreciably better than the CMD-1 and

CMD-2 values reported for P. aeruginosa MTCC8165

(29.9 ± 1.0 and 46.5 ± 0.90 respectively), P. aeruginosa

MTCC7815 (36.2 ± 0.35 and 58.1 ± 0.53 respectively),

P. aeruginosa MTCC7812 (48.1 ± 0.46 and 62.6 ± 1.43

respectively) and P. aeruginosa MTCC7814 (38.4 ± 0.64

and 59.8 ± 0.25 respectively) [29].

Carbon Sources

Investigation of water-miscible and immiscible carbon

sources revealed that glycerol was the best carbon source

for maximum biosurfactant production. The strain showed

a 50.46 % reduction in ST when glycerol was used as the

carbon source. In glucose- and mannitol-containing media,

45.16 and 45.31 % ST reductions were achieved respec-

tively. Growth of the strain on olive oil resulted in a

24.86 % decrease in ST and a poor result was recorded for

n-hexadecane (1.9 %) and the control (1.20) (Table 2).

Results of CMD-1 (10- times dilution) and CMD-2 (100-

times dilution) also showed that the best results were

obtained with glycerol as the carbon source followed by

mannitol. The values of CMD-1 and CMD-2 of the bio-

surfactant-containing cell-free medium with glycerol as the

Table 1 Effect of nitrogen source on ST reduction, CMD-1, CMD-2, E24 index and OD600 after 48 h of growth of P. aeruginosa RS29 in

mineral medium

Nitrogen source ST (0 h)

(mN/m)

ST (48 h)

(mN/m)

ST reduction

(%)

CMD-1

(mN/m)

CMD-2

(mN/m)

E24 index

(%)

OD600

Control (without nitrogen source) 60.2 ± 0.23 36.1 ± 0.20 40.03 ± 0.90 49.7 ± 0.16 60.0 ± 0.23 50 ± 0.30 1.39 ± 1.0

Ammonium nitrate 59.2 ± 0.11 27.2 ± 1.0 54.05 ± 1.0 28.7 ± 0.90 42.4 ± 1.0 72 ± 0.25 3.87 ± 1.5

Ammonium chloride 60.0 ± 0.15 52.2 ± 1.20 13.00 ± 0.88 58.8 ± 0.36 59.5 ± 0.49 36 ± 0.35 2.12 ± 0.98

Ammonium sulfate 59.0 ± 0.20 51.5 ± 0.80 12.71 ± 0.89 58.2 ± 0.40 59.7 ± 0.82 36 ± 0.18 2.06 ± 1.2

Sodium nitrate 59.1 ± 0.10 26.3 ± 0.89 55.50 ± 0.20 27.2 ± 0.30 40.0 ± 0.86 80 ± 0.40 2.36 ± 0.86

Potassium nitrate 59.1 ± 0.23 26.4 ± 1.30 55.33 ± 0.12 27.4 ± 0.94 41.2 ± 0.25 79 ± 0.32 2.28 ± 1.15

Results represented are the means ± SD of three independent experiments with three replicates each

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carbon source were 27.2 and 40.0 mN/m respectively,

similar to those mentioned in the case of sodium nitrate.

The E24 index was also maximum for glycerol (80 %).

This was followed by mannitol with E24 index 68 %. The

E24 indexes for olive oil, glucose, n-hexadecane and

control were 67, 60, 55 and 29 % respectively (Table 2).

The maximum OD was observed in the glycerol-containing

medium (Table 2). The results suggest that the RS29 strain

can show its maximum activity in glycerol-containing

media. It was reported that the most of the identified bio-

surfactants are synthesized by microorganisms grown on

water-immiscible hydrocarbons. These compounds are

expensive which in turn raise the overall cost of the process

[30]. Though the literature shows the potentials of n-

hexadecane [31] and vegetable oils [32] as good substrates

for biosurfactant production by Pseudomonas sp., but the

RS29 strain performed better in glycerol rather than the

water-immiscible ones. Similar results have been reported

in the literature [33]. Since costs of water-immiscible

carbon sources are more than water-miscible ones, utili-

zation of glycerol for biosurfactant production is more

economic.

Influence of the C/N Ratio

The C/N ratio is very influential in biosurfactant production

[19, 20] and the optimum value of this ratio can vary

depending on the strain used and the carbon source [34].

Investigation of the influence of the C/N ratio on biosur-

factant production revealed that 12.5 was the optimum

ratio for maximum growth of biomass and biosurfactant

production by the RS29 strain (Fig. 1). An increase in the

C/N ratio from 2.5 to 12.5 resulted in an increase in the

growth of bacterial biomass and production of biosurfac-

tant. Further increases in the C/N ratio reduced the pro-

duction as well as the growth of biomass of the strain. The

highest ST reduction was obtained (55 %) at the C/N ratio

of 12.5 (Table 3). The maximum amount of crude biosur-

factant 5.6 g/l (0.8 g/l rhamnolipid) was obtained at the

C/N ratio of 12.5 (Fig. 1). Differences between crude and

rhamnolipid biosurfactant contents in the culture medium

are in agreement with the literature reporting extraction of

13.35 g/l crude biosurfactant from a 72-h old culture of

Pseudomonas aeruginosa in a palm oil medium with

2.91 g/l rhamnolipid biosurfactant [12]. Growth of biomass

of the strain was also very good at this C/N ratio. It was

reported that a lower C/N ratio was favorable for biosur-

factant production by P. fluorescens Migula 1895-DSMZ

[16]. For P. aeruginosa EM1, Wu et al. [34] observed an

optimum C/N ratio of 26 when glucose was used as a

carbon source and a C/N ratio of 52 when glycerol was

used. They obtained 4.9 g/l biosurfactant using glycerol as

the substrate. Santa Anna et al. [35] demonstrated maxi-

mum production of 0.69 g/l rhamnolipid with the best

tensoactive characteristics (48 % decrease in ST) with

glycerol, a carbon source easily assimilated at a C/N ratio

of 22.8. Rashedi et al. [36] reported that the best biosur-

factant production of 0.69 g/l was obtained with glycerol

and sodium nitrate at a C/N ratio of 55. Therefore, it is

clear that the RS29 strain is better than these strains in

terms of maximum production of biosurfactant at low

amounts of carbon substrate. As it is known that limited

nitrogen enhances production of biosurfactant [28]; keep-

ing the nitrogen content constant if the highest biosurfac-

tant production can be obtained at a low C/N ratio, it shows

that the bacterium is utilizing a lower amount of substrate

for the production of biosurfactant. This is economically

viable.

Extraction and Characterization of the Biosurfactant

Use of a chloroform–methanol (2:1) solvent system was the

best method to extract the maximum amount of biosur-

factant from the culture media. As seen in Table 4, the

maximum biosurfactant amount was obtained from the

culture media when it was extracted with a mixture of

chloroform and methanol at a 2:1 ratio. The biosurfactant

sample produced two purple colored spots on the TLC

plates after spraying with a-naphthol and sulfuric acid.

Spot 1 with an Rf (retardation factor) value equal to

Table 2 Effect of carbon source on ST reduction, CMD-1, CMD-2, E24 index and OD600 after 48 h of growth of P. aeruginosa RS29 in

mineral medium

Carbon source ST (0 h)

(mN/m)

ST(48 h)

(mN/m)

ST reduction

(%)

CMD-1

(mN/m)

CMD-2

(mN/m)

E24 index

(%)

OD600

Control (without carbon source) 51.4 ± 1.0 50.8 ± 0.90 1.20 ± 0.90 68.7 ± 1.1 71.4 ± 1.13 29 ± 1.0 0.785 ± 1.0

Glucose 51.6 ± 0.98 28.3 ± 1.0 45.16 ± 1.0 32.7 ± 0.90 61.4 ± 1.0 60 ± 1.20 2.305 ± 1.5

Mannitol 50.1 ± 1.20 27.4 ± 1.20 45.31 ± 0.88 28.8 ± 0.86 41.5 ± 0.99 68 ± 0.95 2.415 ± 0.98

Glycerol 54.1 ± 0.99 26.8 ± 0.80 50.46 ± 0.89 27.2 ± 1.0 40.0 ± 0.86 80 ± 1.10 3.20 ± 1.20

Olive oil 34.6 ± 1.10 26.0 ± 0.89 24.86 ± 1.20 30.6 ± 1.2 37.5 ± 1.0 67 ± 1.20 2.80 ± 0.86

n-Hexadecane 46.8 ± 0.87 45.9 ± 1.30 1.90 ± 1.12 62.3 ± 0.94 71.5 ± 1.10 55 ± 1.0 1.18 ± 1.15

Values are presented as means ± SD of three independent experiments with three replicates each

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0.42 ± 0.04 and spot 2 with an Rf value equal to

0.78 ± 0.28. This indicated that the biosurfactant sample

was composed of congeners with one and two sugar mol-

ecules influencing the movement of the molecules on the

TLC plate. This is in agreement with Guerra-Santos et al.

[37] who described the production of active compounds by

Pseudomonas aeruginosa on TLC, with the same solvent

system. They obtained Rf values of 0.4 and 0.8 for di- and

mono-rhamnolipid respectively.

As described in the Materials and Methods section, the

MS spectra were acquired in positive ion modes to confirm

the structural assignment of the quasi-molecular ions. The

RS29 strain produced several congeners with M.W. rang-

ing from 334 to 735. Comparison of mass spectra ions with

the available literature [38, 39] revealed the identity of the

congeners which are presented along with their abundance

in Table 5.

Analysis of biochemical as well as spectrometric data of

the biosurfactant revealed the rhamnolipid nature of it. The

rhamnolipid nature of the biosurfactant produced by RS29

is in agreement with previous works reporting production

of rhamnolipid type of biosurfactant by Pseudomonas sp.

[40–42]. Predominance of C10 fatty acids in the rhamn-

olipid biosurfactant was also stated by Thaniyavarn et al.

[12] with the pseudo-molecular ion at m/z 103 as the base

peak of the GC–MS spectrum for Pseudomonas aeruginosa

A41. Arino et al. [43] reported production of C10 fatty acids

as the main component of rhamnolipid biosurfactant by

Pseudomonas aeruginosa GL1 with production of some

amount of C8, C12:1 and C12 fatty acids. LC–MS charac-

terization (Fig. 2) of the produced biosurfactant showed

that the extracted rhamnolipid was a mixture of different

congeners having mono and di-rhamnose groups and with

pseudomolecular ion (m/z) ranging from 334 to 735.

Among the congeners, the predominant one was found to

be Rha–C10–C10–CH3 while the next more abundant con-

gener was Rha–Rha–C8 (Table 5). It was also clear from

the analysis that more mono-rhamnolipid congeners were

present in the biosurfactant than the di-rhamnolipid ones.

Two congeners having unsaturated fatty acids (Rha–C12:2

and Rha–C10–C14:1) were also found in the rhamnolipid

mixture. This is in agreement with results reported earlier

[39]. They have reported both mono- and di-rhamnolipid

congeners from Pseudomonas aeruginosa 47T2 with the

pseudomolecular ion (m/z) ranging from 475 to 703. Rha–

Rha–C8–C10, Rha–C10–C8, Rha–C8–C10, Rha–Rha–C8–

C12:1, Rha–Rha–C10–C10, Rha–Rha–C10–C12:1, Rha–C10–

0

1

2

3

4

5

6

7

2.5 7.5 12.5 17.5 22.5

C/N ratio

Rh

amn

ose

co

nte

nt

(g/l)

C

rud

e b

iosu

rfac

tan

t (g

/l)

0

1

2

3

4

5

6

Bio

mas

s (g

/l)

Rhamnose content Biomass Crude biosurfactant

Fig. 1 Effect of the C/N ratio on the growth of bacteria and

production of biosurfactant in a mineral medium containing glycerol

and sodium nitrate as carbon and nitrogen sources respectively. Bars

indicate standard deviations

Table 3 Rhamnolipid quantification and ST measurement of 48-h

old culture of P. aeruginosa RS29 on glycerol at different C/N

conditions

C/N ratio Rhamnolipid (g/l) ST reduction (%)

2.5 0.49 ± 0.20* 53 ± 0.42

7.5 0.59 ± 0.16* 53 ± 0.38

12.5 0.80 ± 0.18* 55 ± 0.29

17.5 0.60 ± 0.24* 51 ± 0.45

22.5 0.58 ± 0.22* 48 ± 0.32

Values are presented as means ± SD of three independent experi-

ments with three replicates each. Significance of difference:

* p \ 0.05

Table 4 Extraction of biosurfactant using three different methods

Method Biosurfactant extracted (g/l)

Acetone precipitation 4.4 ± 1.9

Extraction by chloroform–methanol 5.6 ± 1.3*

Extraction using ethyl acetate 4.8 ± 1.5

Results are presented as means ± SD of three independent experi-

ments with three replicates each. * p \ 0.05 and * p \ 0.01

Table 5 Identification and characterization of major rhamnolipid

congeners using LC–ESI–MS and their abundance in percent in the

produced biosurfactant

m/z Structural analogue Abundance (%)

476.5 Rha–C10–C8 or Rha–C8–C10 11.9

358.2 Rha–C12:2 10.05

334.41 Rha–C10 5.15

616.7 Rha–C14–C14 5.23

664.82 Rha–Rha–C10–C10–CH3 11.12

678.84 Rha–Rha–C12–C10 3.38

518.7 Rha–C10–C10–CH3 31.86

452.49 Rha–Rha–C8 12.04

558.74 Rha–C10–C14:1 3.12

734.95 Rha–Rha–C14–C12 or Rha–Rha–C12–C14 1.36

594.69 Rha–Rha–C8–C8 4.79

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C10, Rha–Rha–C10–C12, Rha–Rha–C12–C10, Rha–C10–

C12:1, Rha–C12:1–C10, Rha–Rha–C12:1–C12, Rha–Rha–C10–

C14:1, Rha–C10–C12, Rha–C12–C10 were the reported

identified congeners with Rha–Rha–C10–C10 being the

most abundant one.

Activity Assay

The biosurfactant-containing culture supernatant showed

good foaming stability. The foam produced was stable for

48 h indicating prospects of the produced biosurfactant as a

good foaming agent for industrial purposes.

The biosurfactant also showed very good emulsification

activity when tested with various immiscible substrates. As

seen in Table 6, biosurfactant-containing culture media

could emulsify all the tested substrates, namely n-hexa-

decane, hexane, olive oil, crude oil, diesel and coconut oil

which are highly immiscible in water. But the highest E24

index was recorded with crude oil. The recorded E24 with

crude oil was 100 % (Table 6). This was followed by the

E24 index with n-hexadecane of 80 %. Olive oil, diesel and

coconut oil showed approximately equal E24 index.

Lowest E24 index was recorded for hexane (60 %).

The ODA of the biosurfactant was found to be 132.67 cm2

(Fig. 3). According to Thaniyavarn et al. [44], the diameter

of the clearing zone on the oil surface correlates to surfactant

activity. For pure biosurfactant, there is a linear correlation

between the quantity of the surfactant and the clearing zone

diameter. The larger the size, the higher the activity of a

surfactant is. In the control plate where instead of culture

broth only distilled water was used a clear zone did not form.

Tambekar and Gadakh [45] isolated biosurfactant-producing

bacterial strains from petroleum-contaminated soil and

reported two Pseudomonas aeruginosa strains. Investigation

revealed that the strain isolated from garage soil showed

38.47 cm2 and the strain isolated from soil of petrol pump

showed 0.79 cm2 ODA. The isolated Pseudomonas aeru-

ginosa PDKT2 from used engine oil-contaminated soil

reported an oil displacement area of 2.5 cm2 [46]. Techaoei

et al. [47] isolated a Pseudomonas aeruginosa strain

SCMU106 from garage site which showed an oil

Fig. 2 LC chromatogram of the

column purified biosurfactant

extracted with chloroform–

methanol

Table 6 E24 indexes of the

produced biosurfactant with

various substrates

Results are presented as

means ± SD of three

independent experiments with

three replicates each

Solvent Height of the

emulsified layer (cm)

Total height

of the liquid (cm)

E24 index (%)

n-hexadecane 4.16 ± 0.15 5.2 ± 0.10 80 ± 0.14

Hexane 2.52 ± 0.14 4.2 ± 0.12 60 ± 0.13

Olive oil 3.36 ± 0.12 4.8 ± 0.11 70 ± 0.10

Crude oil 5.0 ± 0.0 5.0 ± 0.09 100 ± 0.06

Diesel 3.7 ± 0.11 5.3 ± 0.09 70.5 ± 0.10

Coconut oil 3.5 ± 0.11 5.2 ± 0.10 69 ± 0.11

Fig. 3 Oil displacement activity of the biosurfactant-containing

culture broth

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displacement area of 9.83 and 11.17 cm2 while NH4NO3 and

KNO3 were used as nitrogen sources respectively. The above

discussions revealed that the biosurfactant produced by the

isolated RS29 strain showed excellent activity in terms of the

oil displacement area when sodium nitrate was used as the

nitrogen source.

Conclusion

The yield of biosurfactant is absolutely dependent on the

sources of nitrogen and carbon in the culture medium.

Moreover, the C/N ratio has a great influence on the pro-

duction of biosurfactant. In our study, the RS29 strain was

able to produce the highest amount of biosurfactant at a

very low substrate concentration, demonstrating the effi-

ciency of the strain on glycerol. Obtaining the maximum

amount of biosurfactant from culture media is also

dependent on the extraction procedure used for the pur-

pose. The strain has great practical potential to be used as

an emulsifying and foaming agent.

Acknowledgments We thank the Director of the Institute of

Advanced Study in Science and Technology (IASST), Guwahati, for

providing us with laboratory facilities to carry out the research work.

The corresponding author gratefully acknowledges the Department of

Biotechnology (DBT) from the government of India, for the project

grant (DBT project No. BT/PR 9795/BCE/08/590/2007). We also

thank Mr S Dey, Biotech Park, Assam, India for his assistance in LC–

MS analysis.

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Author Biographies

Rashmi Rekha Saikia is a scientist of the Institute of Advanced

Study in Science and Technology (IASST), Guwahati, India. Her

scientific interests include production of surfactants of microbial

origin and virulence property of the surfactant producing microbes,

application of biosurfactants in petroleum industries such as for the

recovery of hydrocarbons from refinery sludge, bioremediation of

contaminated soil etc.

Hemen Deka is a research associate of IASST, India. His research

interests include biowaste management and currently he is working

on the development of plant–microbe consortia for remediation of

contaminated soil.

Debahuti Goswami is a senior research fellow at IASST, India. She

is presently involved in production enhancement of biosurfactants and

their application for crop disease control.

Jiumoni Lahkar is a research fellow at IASST, India. Currently she

is carrying out research on application of biosurfactants for crop

improvement.

Siddhartha Narayan Borah is a research fellow at IASST, India. He

is pursuing research in the area of plant diseases and the possible

application of biosurfactants for disease control.

Kaustuvmani Patowary is a research fellow at IASST, India.

Presently he is working on high molecular weight polycyclic aromatic

hydrocarbon (PAH) remediation by application of biosurfactant

producing bacteria.

Plabita Baruah is a research fellow at IASST, India. She is presently

working on phyto-remediation of hydrocarbon contaminated soil by

herbs like Cyperus brevifolius.

Suresh Deka is a professor in the Life Sciences Division of IASST,

India. His research interests include bioremediation of contaminated

soil with plants and microbes, degradation of heavy metals, applica-

tion of biosurfactants in agriculture and the petroleum industries.

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