International Journal of Biological Macromolecules 62 (2013) 52–58

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Effect of unconventional carbon sources on biosurfactant production

and its application in bioremediation
Rakeshkumar M. Jain, Kalpana Mody ∗ , Nidhi Joshi, Avinash Mishra, Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar,
Gujarat 364021, India

a r t i c l e i n f o a b s t r a c t

Article history: The potential of an alkaliphilic bacterium Klebsiella sp. strain RJ-03, to utilize different unconventional
Received 29 July 2013 carbon sources for the production of biosurfactant was evaluated. The biosurfactant produced using
Received in revised form 21 August 2013 corn powder, potato peel powder, Madhuca indica and sugarcane bagasse containing medium, exhibited
Accepted 23 August 2013
significantly higher viscosity and maximum reduction in surface tension as compared to other substrates.
Available online 28 August 2013
Among several carbon substrates tested, production of biosurfactant was found to be the highest with corn
powder (15.40 ± 0.21 g/l) as compared to others. The comparative chemical characterization of purified
Keywords:
biosurfactant was done using advance analytical tools such as NMR, FT-IR, SEM, GPC, MALDI TOF–TOF MS,
Alkaliphilic bacteria
Biosurfactant
GC–MS, TG and DSC. Analyses indicated variation in the functional groups, monosaccharide composition,
Bioremediation molecular mass, thermostability. Higher yield with cheaper raw materials, noteworthy stress tolerance
of CP-biosurfactant toward pH and salt as well as compatibility with chemical surfactants and detergents
revealed its potential for commercialization and application in bioremediation.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction contaminated soil, wastewater and cotton cloth under extreme

In recent years interest swayed toward exploration of valuable
biosurfactants for various industrial, agricultural and environmen-
tal applications and hence attentions toward biosurfactant from
new resources have been greatly enhanced [1,2]. Biosurfactants
have diverse chemical structures, compositions and a broad range
of uses in various industries [1–3]. Microbial biosurfactants are
heterogeneous in nature, complex and have structurally different
group of surface active agents. These are produced by a wide vari-
ety of microbes inhabiting different environmental niches which
either adhere to cell surface or excreted extracellularly [1,4–7]. Bio-
surfactants are amphiphilic in nature (with polar and non-polar
moieties) comprised of glycolipids, phospholipids, lipopeptides
and polymeric compounds [8] intend to separate at interfaces
and thus reduce the surface tension [9]. These are ecologically
accepted, non toxic, biodegradable and effective in a wide range of
extreme conditions including temperature, pH, salinity and show
better environmental acceptability [4,8,9] compared to chemical
surfactants. Extremophiles are of interest due to their adaptability
to extreme environment and inherent property for bioremedia-
tion of hydrocarbons, lubricant oil, dyes and toxic metals from
∗ Corresponding author. Tel.: +91 278 2561354; fax: +91 278 2567562.
E-mail address: khmody@csmcri.org (K. Mody).
conditions [1,2,10].
It was reported that millions of tons of hazardous and non-
hazardous wastes are generated each year throughout the world
and therefore there is a global concern for its management and
utilization [11,12]. These wastes are treated chemically and/or
dumped directly in water or soil which again leads to environ-
mental hazards. There are few reports on utilizing these wastes for
the biosurfactant production by micro-organisms [13–19]. Limited
production has been achieved for many biosurfactants at large scale
due to expensive raw materials, low production yield, complex
down stream processing and high purification cost. It is estimated
that the raw material accounts for 30–40% of the total production
cost in most biotechnological processes. Hence, in order to reduce
this cost, it is desirable to use the low cost raw materials [12]. One
of the possibilities explored extensively is the use of organic mat-
ter rich but cheap agro-based raw materials or industrial wastes
as substrates for biosurfactant production. This approach will help
to achieve double benefits of reducing pollution while producing
useful products. A variety of these cheap raw materials supporting
biosurfactant production include plant derived oils like sunflower
oil, oil wastes like soapstock [12] cassava wastewater [13] and oil
refinery wastes [14]. Starch is an abundant by-product in the potato
processing industry [15,16]. Starchy and lignocellulosic materi-
als are potential carbon sources for microbes to produce valuable
products such as extracellular substances (enzyme, polysaccha-
rides, antibiotics, etc.) through fermentation [17]. The low cost

0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2013.08.030
 R.M. Jain et al. / International Journal of Biological Macromolecules 62 (2013) 52–58
agro-industrial wastes include lignocellulosic residues (barley bran
husks, trimming vine shoots, corn cobs and Eucalyptus globulus
chips), jute [17], plant polymer, oil extracts, distillery and whey
wastes, potato process effluent and pea nut cake [18]. Other sources
include sugar plants such as dates syrup, sugar beet, sugar cane or
sugar sorghum [12,18–21]. Sugar and starch processing industries
also produce large amounts of solid residues of starch contain-
ing wastewater [19]. Starch and cellulose are the components of
Madhuca indica, corn, tapioca, wheat bagasse, sugarcane bagasse,
soyabean and potato peel powder. Evaluation of different carbon
substrates may help to overcome the problem of high production
cost.
Previously, an alkaliphilic bacterium, Klebsiella sp. strain RJ-03
has been shown to produce biosurfactant in different conven-
tional carbon substrates [2,22]. In the present study, evaluation
of unconventional low cost carbon sources was undertaken and
compared to conventional sources in terms of biosurfactant yield,
surface activity, viscosity and physicochemical characteristics of
the product. The biosurfactant was evaluated for its compatibility
with detergents to remove lubricant oil from sand and cotton cloth
and compared to chemical surfactants like sodium dodecyl sul-
fate (SDS), Tween80, Triton X-100 and laundry detergent powders
(Aerial, Nirma and Tide).
2. Materials and methods
2.1. Microorganisms and seed culture
An alkaliphilic bacterium, Klebsiella sp. strain RJ-03 (GenBank
accession no. JN398669) isolated from a soil sample of oil contam-
inated site from Gondal, GIDC (N 21◦ 58.95 E 70◦ 47.24 ), Gujarat
(India) was selected for the study [2] The bacterium was maintained
on Horikoshi agar slant at 4 ◦ C for subsequent experiments. A loop-
ful of strain RJ-03 was inoculated into 100 ml Horikoshi medium
(HM; w/v 1% glucose, 0.5% peptone, 0.5% yeast extract, 0.1% K2 HPO4
and 0.02% MgSO4 ·7H2 O; 1% Na2 CO3 after autoclaving) broth of pH
10 and incubated for 24 h at 32 ◦ C and 120 rpm on an orbital rotary
shaker.
2.2. Screening of agro-industrial waste for biosurfactant
production
Different agro-industrial substrates viz. M. indica, sugarcane
bagasse, E. globulus, wheat straw, Jatropha cake, soyabean powder,
corn powder, potato waste powder and seaweed sap used for bio-
surfactant production were subjected to chemical analysis i.e. total
sugars, reducing sugars and total protein. For this, 5 g dry and pow-
dered raw material was suspended in 100 ml distilled water and
autoclaved for 1 h at 100 ◦ C for digestion.
2.3. Production, quantification and purification of biosurfactant
Biosurfactant was produced by inoculating 2% (v/v) seed cul-
ture (OD600 = 2) in 500 ml broth (pH 10) containing 5% of various
unconventional substrates like M. indica (MI), sugarcane bagasse
(SB), wheat straw (WS), rice husk (RH), potato peel powder (PPP),
corn powder (CP), soyabean powder (SP), Jatropha cake (JC) and
seaweed sap (SS). Production media were incubated at 32 ◦ C on
a rotary shaker (120 rpm) for 72 h, centrifuged at 13,000 × g for
20 min at 4 ◦ C to remove the substrate particles, bacterial cells and
debris. Supernatant was acidified to pH 2.0 with 6 M HCl, kept at 4 ◦ C
for 12 h and centrifuged (13,000 × g; 20 min; 4 ◦ C). Biosurfactant
was precipitated by adding 2 fold cold iso-propanol. Products were
dried and yield was compared. Crude products were dialysed for
48 h at 4 ◦ C (12,000 Da cut off dialysis tubing, Sigma) and lyophilized
53
[2]. Purified biosurfactants obtained were stored at −80 ◦ C for fur-
ther studies.
2.4. Scanning electron microscopy
Klebsiella sp. RJ-03 strain was inoculated in CP medium, the most
promising carbon source, and the progress of biosurfactant produc-
tion was observed at different time intervals (0, 24, 48 and 72 h)
using scanning electron microscopy (SEM, LEO 1430 VP) Samples
were mounted on aluminum stub, coated with gold palladium and
observed under SEM at a voltage of 18 kV.
2.5. Viscosity, surface tension emulsification activity and CP
biosurfactant stability studies
Supernatants obtained after 72 h with different unconventional
carbon sources were used to measure viscosity (RS1, HAAKE Instru-
ments, Karlsruhe, Germany) and surface tension (du Nouy ring
tensiometer, Data physics, Germany) at 30 ◦ C. Surface tension of
distilled water and respective production media without inoculum
were taken as control. Emulsification activity of CP-biosurfactant
solution (0.1%, w/v) was determined by measuring the emulsifi-
cation index. Emulsification index was recorded with time and
represented accordingly, i.e. emulsification index after 24 h was
represented as E24 . Stability with respect to pH (2–13), temper-
ature (50–150 ◦ C) and salt stress (2–10%) was determined in the
terms surface tension and viscosity using 1% CP biosurfactant solu-
tion and compared with control. All experiments were carried out
in duplicate under controlled laboratory conditions.
2.6. Chemical composition
Purified biosurfactant samples were analyzed spectrophoto-
metrically for its constituent viz. total sugar, reducing sugar and
protein content. The monosaccharide composition of different
purified biosurfactant samples were analyzed using GC–MS after
alditol-acetate derivatization [2].
2.7. Molecular mass and MALDI TOF–TOF mass spectroscopy
The molecular weight of the purified CP-biosurfactant was
determined by Gel Permeation Chromatography (GPC, 7.8 mm
ID × 300 mm stainless steel, Model Alliance 2695, Waters, USA)
with guard columns. About 80 ␮l (0.1%, w/v in Milli Q water)
of purified biosurfactant was loaded on to a GPC column
Ultrahydrogel-120 and Ultrahydrogel-500 at 30 ◦ C. The column
was calibrated with standard dextran (molecular weight, 5200-
668,000 kDa; PSS, USA) and elution was monitored using a
refractive index detector (2414). MALDI TOF–TOF analysis was car-
ried out on MALDI TOF–TOF analyzer (Applied Biosystem 4800,
USA) with an Nd-YAG laser (355 nm, 200 Hz) operated at 20 kV.
Sample was prepared by dissolving purified CP-biosurfactant
(5 mg ml−1 ) in milli-Q water and mixed with equal volume of
matrix ␣-Cyano-4-hydroxycinnamic acid (10 mg ml−1 ). Each spec-
trum was collected in positive ion reflector mode with an average of
1400 laser shots per spectrum. Reproducibility of the spectrum was
checked and spectra were analyzed after centroid and de-isotoping
using Data explorer software (Applied Biosystem, USA) [2,22,23].
2.8. FT-IR, 1 H and 13 C NMR analysis
Selected purified biosurfactants produced by utilizing different
unconventional carbon sources were subjected to Fourier trans-
form infrared spectroscopy (FT-IR) to elucidate the chemical nature
by identifying the types of functional groups and chemical bonds.
The FT-IR spectrum was recorded in 4000–400 cm−1 region on a
54 R.M. Jain et al. / International Journal of Biological Macromolecules 62 (2013) 52–58

GX-FTIR system (Perkin Elmer, USA) where a KBr pellet was used Table 1
Chemical composition of unconventional substrates used in this study.
as a background reference. For NMR (500 MHz, Bruker Avance-II
spectrometer, Switzerland) analysis, lyophilized CP-biosurfactant Substrate Total sugars (%) Reducing sugars (%) Protein (%)
was dissolved in D2 O (50 mg ml−1 ) and 1 H & 13 C NMR spectra Madhuca indica 73.00 62.70 8.60
were recorded at 70 ◦ C with 5000–5200 accumulations, 5.9 ␮s pulse Corn powder 76.00 66.00 4.70
duration, 1.2 s acquisition time and 6 ␮s relaxation delay. Sugarcane bagasse 62.70 15.15 3.30
Potato peel powder 60.00 23.00 1.534
Rice husk 6.30 3.90 4.42
2.9. Thermal gravimetric (TG) and differential scanning Jathropa cake 6.50 0.254 3.70
calorimetric (DSC) analysis Seaweed sap 0.68 0.03 0.04
Wheat straw 6.6 4.42 3.60

TG and DSC analyses of CP-biosurfactant were carried out with

Mettler Toledo TGA/SDTA System (Greifensee, Switzerland). About
5–8 mg of lyophilized CP-biosurfactant sample was loaded on a
platinum pan and its energy level was scanned in the range of
50–500 ◦ C under a nitrogen atmosphere with a temperature gra-
dient of 10 ◦ C/min. The analyses were performed under gradual
increase in temperature by plotting the weight percentage and heat
flow against temperature [1].
2.10. Application of the biosurfactant in lubricant oil removal
from contaminated sand and cotton cloth
The physico-chemical characteristics like pH, particle size, mois-
ture content, porosity, bulk density, cation exchange capacities,
total carbon, total nitrogen, elements, surface area and average pore
diameter of soil were analyzed however density, specific gravity,
physical state and color were determined for lubricant oil, using
standard methods [2]. About 400 g sandy soil was impregnated
with 200 ml of lubricant oil for the oil removal study. A measure
of 35 g of contaminated sandy soil was transferred into 100 ml of
different surfactant solutions (1%, w/v) like CP-biosurfactant, SDS,
Tween80, Triton-X100 along with a control flask containing 100 ml
distilled water and incubated on a rotary incubator shaker at 30 ◦ C,
100 rpm for 24 h. After incubation, the solution was centrifuged at
7000 rpm for 10 min to separate the aqueous phase from oil con-
taining soil which was further extracted with hexane. The hexane
was recovered using a rotary evaporator and the residual lubricant
oil was measured gravimetrically [2]. Compatibility, stability and
efficiency of the biosurfactant to remove oil were studied and com-
pared to commercially available detergents to explore its potential
as a detergent additive. Detergents and biosurfactant were indi-
vidually dissolved in water (1 and 0.5%, w/v) and their efficiency
to remove oil from an oil-contaminated cotton cloth was checked
individually as well as in combination with the biosurfactant (1:1
ratio). An amount of 6 g lubricant oil was poured on a 25 cm × 25 cm
cotton cloth and allowed to dry at 40 ◦ C for 24 h. Each piece of
cloth impregnated with oil was soaked in flasks containing 100 ml
tap water (control), biosurfactant, detergent solutions and biosur-
factant:detergent solution (1:1 v/v) respectively. Flasks were kept
on a shaker at 30 ◦ C, 100 rpm for 60 min. Post-wash water was
used to measure the amount of oil removed from cotton cloth.
The extraction process was repeated thrice, hexane was recovered
using rotary evaporator and the residual lubricant oil was measured
gravimetrically. All experiments were carried out in duplicate.
Protein content was found to be maximum (8%) in M. indica
while it was approximately 4% in all the other raw materials used
[24]. High reducing sugar contents in M. indica, corn powder, potato
peel powder and sugarcane bagasse proving them to be the most
promising unconventional carbon and nitrogen source for biosur-
factant production. It was observed that unconventional substrates
having high sugar content, such as corn powder, M. indica, potato
peel powder, soybean powder and sugarcane bagasse, promoted
biosurfactant production. It was observed that bacterium grow vig-
orously in wheat straw, rice husk and E. globulus, Jatropha cake
and seaweed sap with no or minimal biosurfactant production.
Among all the different unconventional carbon substrates used in
the study, corn powder was found to be best in the terms of yield
and other properties (Table 2).
3.2. Viscosity, surface tension, emulsification activity and
stability of CP-biosurfactant
The cell-free culture supernatant of strain RJ-03 which was
grown in liquid CP medium showed the highest biosurfactant
production (15.4 g/l), reduction in surface tension (from 69.03 to
44.037 mN/m) and viscosity (62.17 cP) compared to other pro-
duction media (Table 2). CP was found to be the promising
substrate with respect to yield of biosurfactant. A positive corre-
lation between yield and viscosity was observed, however, surface
tension was found inversely correlated with yield as previously
[25,26].
The emulsifying activity determines the strength of biosurfac-
tant in retaining the emulsion of hydrocarbons or oils in water [27].
The extracted CP-biosurfactant efficiently emulsified hydrocarbons
and oils (Table 3). Formation of stable emulsion was observed with
xylene, toluene, hexadecane and petrol with 0.2% biosurfactant
concentration, reflected by high emulsification indices found in the
range of 100 to 65% up to 24 h. Some commercial emulsifiers, par-
ticularly those having fatty acid components, tend to form clumpy
solution and thus limit their applications [28]. The carbohydrate
and proteinaceous component of bacterial biosurfactant played an
important role in the emulsification of hydrocarbons and oils, apart
from functional groups [2,9]. High and stable emulsification of CP-
biosurfactant was attributed by high viscosity, which enhances
Table 2
Comparative effect of different unconventional carbon substrates on biosurfactant
production and its properties.

3. Result and discussion Carbon Biosurfactant Viscosity (cP) Surface tension

substrates yield (g/l) (mN/m)
3.1. Screening of agro-industrial wastes, production and CP 15.40 62.17 44.03
quantification of biosurfactant production PPP 9.60 57.60 45.46
MI 7.56 50.61 46.63
SB 6.20 48.19 47.09
High content of total sugar (34–76% dry weight) and reducing SP 3.40 38.12 52.03
sugars were detected in corn powder, M. indica, potato peel powder JC 1.58 27.08 60.36
and sugarcane bagasse compared to other substrates like wheat SS – 10.79 68.17
straw, soyabean powder, Jatropha cake, rice husk and seaweed sap WS 1.03 26.08 54.01
RH 0.94 26.01 59.03
(Table 1).
 R.M. Jain et al. / International Journal of Biological Macromolecules 62 (2013) 52–58 55

Table 3 3.4. Chemical compositions

Emulsification activity of CP biosurfactant with hydrocarbons and oil.

Hydrocarbons/oils Emulsification Emulsification Total sugar, reducing sugar and protein contents, ranged
index (E0 ) index (E24 ) from 50–72%, 2.8–4.8% and 2.7–6.9% respectively in biosurfac-
Carbon tetrachloride 78 65 tant products. Heteropolysaccharide nature of the biosurfactant
Dichloromethane 90 65 was revealed by its monosaccharide composition analysis which
Ground nut oil 80 62 showed presence of both, hexose and pentose sugars in vary-
Petrol 90 72
ing proportions. Monosaccharide composition of SPY-biosurfactant
Toluene 80 80
Hexadecane 80 75 is quite different from the monosaccharide composition of CP-
Xylene 78 70 biosurfactant. In the present study, varying monosaccharide com-
position was observed with different carbon sources (Table 5). Here,
mannose was the major sugar followed by glucose and galactose,
Table 4
Stability CP biosurfactant solution (1%, w/v) under different conditions. especially in CP-biosurfactant. Surprisingly, biosurfactant pro-
duced in the presence of PPP was found to be a homopolysaccharide
Parameter Surface tension (mN/m) Viscosity (cP)
composed of mannose. Biosurfactants produced by Marinobacter
Control 44.09 62.87 sp., Vibrio sp., Acetobacter sp., Halomonas sp., Bacillus sp., Pseu-
3.5% NaCl 61.08 44.62 domonas sp., Corynebacterium sp. Klebsiella sp. and Halobacter sp.
5% NaCl 56.03 27.08
were comprised of carbohydrate, uronic acid, protein and sulfate
10% NaCl 58.07 17.09
80 ◦ C 44.12 60.09 [2,9]. Biosurfactant reported in the present study can be classified
100 ◦ C 44.03 60.12 as a polysaccharide–protein complex (polymeric microbial surfac-
150 ◦ C 59.04 10.6 tant). Proteins present in biosurfactant play an important role in
emulsification with hydrocarbons and oils.
the emulsification abilities of hydrocarbons [29,30]. Low molecular
weight biosurfactants are used as flocculants and does not support 3.5. Molecular mass and MALDI TOF–TOF mass spectroscopy
stable emulsions, however high molecular weight biosurfactants
act as emulsion stabilizers. It was reported that uronic acid and pro- The gel permiation chromatogram generated a single peak cor-
teinaceous components of biosurfactant play an important role in responding to 2366 kDa approximately; with 4.0702 polydispersity
the emulsification in addition to acetyl functional group which pro- and 10.65 min retention time (Supplementary S2). Biosurfactants
vides hydrophobicity and imparting enhanced emulsifying activity are divided in low and high molecular weight, having differ-
[2,9]. ent chemical structures and surface properties. High molecular
The diverse application of biosurfactants in different fields weight biosurfactants, produced from Bacillus sp., Acinetobacter sp.
depends on its stability at different temperatures, pH and salinity. and Pseudomonas sp, are reported to be used as emulsifiers [8].
The CP-biosurfactant was thermostable and showed no significant Matrix assisted laser desorption- ionization mass spectroscopy is
effect of temperature up to 100 ◦ C, thereafter at 150 ◦ C substan- an advanced analytical tool and recently used for biosurfactant [2].
tial changes were observed in its surface active characteristics with MALDI TOF–TOF mass spectroscopy of CP-biosurfactant represents
decrease in viscosity and no reduction in surface tension. In con- mass peaks (m/z 193.84–1003.23) corresponding to pentose, hex-
trast, chemical surfactants such as SDS exhibit a significant loss ose sugars moieties, disaccharides, trisaccharides and revealed the
of surface active properties above temperature 70 ◦ C [31]. The presence of oligosaccharide chains consisting of different ratios of
CP-biosurfactant exhibited its ability to retain its surface-active pentose and hexose sugars (Supplementary S3).
properties in a wide range of pH (2, 7 and 12) and at salt concen-
tration up to 3.5% NaCl (Table 4). High thermo stability, tolerance 3.6. FT-IR, 1 H and 13 C NMR analysis
to varying pH and high salt concentration make CP-biosurfactant a
potential candidate for bioremediation and its possible exploration FT-IR spectra showed (Fig. 1) a broad stretched intense
in food, pharmaceutical and cosmetics industries [31]. peak around 3391–3413 cm−1 characteristic of hydroxyl groups
assigned to the carboxylic acid group of uronic acids. An
3.3. Scanning electron microscopy asymmetrical C H stretching vibration of aliphatic CH2 group
attributed around 2927–28 cm−1 , 2356–64 cm−1 and the peak
A progressive production of CP-biosurfactant was studied. SEM around 2107 cm−1 designated aliphatic C H bonds. It showed
monograph showed single bacterial cell without biosurfactant at asymmetric vibrations (for the CH aliphatic stretching and C H
0 h and as time progresses, production of biopolymeric substances bending bonds of the CH3 , CH2 and CH groups) which con-
was observed around the bacterial cells from 24 h to 48 h, thereafter firmed the presence of alkanes and inter & intramolecular hydrogen
bacterial cells were aggregated at 72 h because of overproduction bonding. The peak around 1630–51 cm−1 and the peaks near
of biosurfactant (Supplementary S1). 1417–1446 cm−1 suggested the presence of C O group present in

Table 5
Chemical composition of biosurfactant produced in media containing different carbon sources.

Carbon source Chemical compositiona Monosaccharide constituentsb

Total Reducing Total Arabinose (%) Rhamnose (%) Fucose (%) Mannose (%) Galactose (%) Glucose (%) Ribose (%)
sugar (%) sugar (%) protein (%)

CP 72 4.70 3.0 – 1.60 2.62 51.45 17.24 27.09 –

PPP 67 2.87 2.70 – – – 100 – – –
MI 50 3.9 6.90 1.72 8.39 1.53 31.41 26.39 22.57 7.99
SB 65 3.0 3.30 1.13 7.53 2.33 28.91 13.76 25.22 21.12
a
Chemical composition analyzed by spectrophotometer.
b
Monosaccharide constituents of total sugar analyzed by GC–MS.
56 R.M. Jain et al. / International Journal of Biological Macromolecules 62 (2013) 52–58

Fig. 1. FT-IR spectra of the purified biosurfactant produced from media containing different unconventional substrate.

carboxylate and amide moieties of protein, respectively. The broad
stretch of C O C, C O at 1016–1245 cm−1 showed the presence
of carbohydrates. Peaks at 1016–35 cm−1 (PII band: polysaccha-
rides), 760–763 (of CH2 group) and 657–675 cm−1 (of NH group)
evidenced the presence of glycopeptide moieties. A symmetrical
methyl group ( CH3 ) corresponding to the sugar moiety. The ı
value at 2.125 ppm showed the CH-proton of C5/C6 carbon of de-
oxy sugar. The range of chemical shift (ı) signals from 3.43 to 5.47
possibly indicated a glycosidic linkage of pentose/hexose sugars.
13 C signals of five anomeric carbon at ı = 77.145, 73.087,

stretched peak near 1384 cm−1 indicated the presence of carboxyl
groups. Absorption peaks in the range around 760–571 cm−1 cor-
respond toward stretch of alkyl-halides. The presence of acetyl and
carboxyl functional groups in the biopolymer proved its anionic
nature which could be useful for binding and thus remediating
divalent cations. The FT-IR confirmed the glycopeptide nature of the
biosurfactants which was similar to the earlier reports on Klebsiella
sp. strain RJ-03 [2,31,32]. The carboxyl groups provided overall neg-
ative charge to the biopolymer, thereby supporting binding and
adsorptive properties for divalent cations by electrostatic interac-
tions [9].
1 H NMR spectra of purified CP-biosurfactant (Fig. 2) revealed
71.399, 71.342, 69.342 and 99.432 of repeating monosaccha-
rides units were observed in 13 CNMR spectrum analysis of the
CP-biosurfactant (Fig. 3) while signal in the range of ı = 60.452 con-
firms the presence of acetyl group. The C1 signals for ␤-glucose
(99.432) & ␣-glucose (80.020); C2 signals for ␤-glucose (77.026),
␣-glucose (73.087) & ␤-galactose (71.399) were also observed.
NMR characteristic spectral peaks of CP-biosurfactant showed close
resemblance with previous report of SPY-biosurfactant [2].
3.7. Thermal gravimetric (TG) and differential scanning
calorimetric (DSC) analysis

a number of peaks (signals) corresponding to different functional Thermal stability of any biosurfactant is an important character-
groups assigned to sugars moieties and protein content. The pres- istic for its commercial application. Degradation of biosurfactants
ence of chemical shift (ı) value at 0.976–1.678 ppm indicated the took place by two or three well differentiated steps as observed

1 13
Fig. 2. H spectrum of purified CP-biosurfactant. Fig. 3. C NMR spectrum of purified CP-biosurfactant.
 R.M. Jain et al. / International Journal of Biological Macromolecules 62 (2013) 52–58
Fig. 4. Thermal gravimetric analysis of biosurfactants obtained from different
unconventional substrate.
in TGA graph and depends upon the properties of biosurfactants
(Fig. 4). Biosurfactants showed a weight loss in first step due to
presence of moisture (water and alcohol) during temperature range
of 50–210 ◦ C followed by second step 100–380 ◦ C and thereafter
third step took place above 350 ◦ C to complete degradation. An
initial weight loss of approximately 9.83, 5.81, 4.9 and 1.58% for
CP, PPP, MI and SB respectively, were observed between 50 and
210 ◦ C followed by second phase of degradation in the range of
100–380 ◦ C, where about 50.77, 65.40 48.42 and 44.09% weight loss
was detected for CP, PPP, MI and SB respectively. In the PPP biosur-
factant, maximum degradation was found at 360 ◦ C as compared
to other biosurfactants, while complete weight loss was observed
at 350–400 ◦ C. CP-biosurfactant showed maximum degradation
above 380 ◦ C temperature and its thermograph was distinct from
previous TG analyses. Microbial glucan, produced by Geobacillus
tepidamans V264 was stable up to 250 ◦ C with maximum degra-
dation at 280 ◦ C [10] while SPY biosurfactant showed maximum
degradation at 300 ◦ C [2].
Fig. 5. Differential scanning calorimetric analysis of biosurfactants obtained from
different unconventional substrate.
57
The DSC thermogram (Fig. 5) of CP, PPP, MI and SB biosurfac-
tant showed crystallization temperature (Tc) at midpoint 82.29,
82.70, 75.15 and 85.61 (onset temperature 49.81, 56.37, 49.21
and 83.89 ◦ C) and melting point for Tm1 peaks at 244.02, 260.33,
151.61 and 175.77 ◦ C (onset temperature 236.83, 241.75, 156.76
and 173.40 ◦ C), respectively (Fig. 5). Melting point for Tm2 peaks at
334.42, 337.10, 235.46 and 256.29 ◦ C (onset temperature 312.05,
316.48, 242.89 and 256.80 ◦ C) was observed for CP, PPP, MI and
SB biosurfactant respectively while, Tm3 peaks at and 334.19 ◦ C
(onset temperature and 313.38 ◦ C) was detected in SB biosurfactant
(Fig. 5).
3.8. Application of the biosurfactant in lubricant oil removal from
contaminated sand and cotton cloth
The physicochemical characteristics of soil and lubricant oil
used in the present study were analyzed previously [2]. In the
present bioremediation study, higher concentration of lubricant oil
was used to impregnate cotton cloth (25 cm × 25 cm) and soil (35 g),
(about 2 and five times respectively) compared to previous reports
[2]. Performance of CP-biosurfactant was found significantly high
for removal of lubricant oil from soil compared to chemical sur-
factants (Supplementary S4). About >96% of oil was recovered in
the presence of CP-biosurfactant however 57–65% recovery was
found by chemical surfactants. Performance of biosurfactant was
excellent and it alone could remove >90% oil compared to 51–66%
oil removal by commercially available detergents from cotton cloth
(Supplementary S5). Additionally, biosurfactant exhibited compat-
ibility with commercially available detergents. Biosurfactants are
more efficient, effective and eco-friendly and thus have advantages
over chemical surfactants. Bioremediation by using biosurfactants
are considered green as it removes oil contaminants by reduc-
ing surface and interfacial tension and thus supports mobilization
[33–36]. Enhanced oil removal was further observed from the cot-
ton cloth during washing by detergents supplemented with the
biosurfactant as compared to detergent solutions alone. It is known
that commercially available detergents contain anionic surfac-
tants, bleaching agents, water softening builders and enzymes [37].
Results evidenced that CP-biosurfactant showed excellent stability
and compatibility with commercially available detergents, the pre-
requisite to be used as an additive. Previously, different aqueous
biosurfactant solutions (aescin, lecithin, rhamnolipid, saponin and
tannin) showed its possible application in washing of crude oil con-
taminated soil [33]. Rhamnolipid and SDS removed 80% oil while
lecithin could remove about 42% oil from the contaminated soil.
Previously 57–90% oil removal was reported from contaminated
soil by using biosurfactant produced by Pseudomonas aeruginosa
SP4, Bacillus subtilis PT2, Rhodococcus ruber and Klebsiella sp. RJ-03
[2,33–35]. Inability of water to remove hydrophobic compounds
from sandy soil enhances need of surfactant as they are ampho-
teric in nature (having both hydrophobic and hydrophilic moieties).
Biosurfactants display excellent surface activity in comparison to
chemical surfactant due to their bulky molecular weight and com-
plex biodegradable structure. The use of biosurfactants for removal
of hydrocarbons from contaminated soil and cotton cloth are very
limited compared to physical and/or chemical methods and need
to be addressed [2,4,37].
4. Conclusions
A very few attempts have been made to exploit unconven-
tional carbon sources for biosurfactant production. In the present
study, enhanced production of biosurfactant was achieved by an
alkaliphilic bacterium Klebsiella sp. using corn powder with better
stability at high temperature, in a wide range of pH and salt stress.
58 R.M. Jain et al. / International Journal of Biological Macromolecules 62 (2013) 52–58
CP-biosurfactant showed excellent ability of oil removal from soil
and cloths (Supplementary S6). Further, it showed efficiency, com-
patibility and stability with various commercial detergents and
its additive interaction with chemical surfactants improved the
washing performance. This led to conclude its economic indus-
trial production as well as application as a laundry detergent
additive.
Acknowledgments
RMJ and NJ are thankful to Council of Scientific and Industrial
Research, New Delhi (CSIR-SRF) and Ministry of New and Renew-
able Energy for financial support respectively. The authors are
grateful to Dr. Vinod Boricha, Mr. Harshad Brahmbhatt, Mr. Jayesh
Chaudhary, Mr. Vinod Agarwal and Mr. Gaurav Vyas of the Analyt-
ical Discipline for analytical assistance.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.
2013.08.030.
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