1 s2.0 S0141813013004546 Main
1 s2.0 S0141813013004546 Main
1 s2.0 S0141813013004546 Main
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.
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 53
agro-industrial wastes include lignocellulosic residues (barley bran [2]. Purified biosurfactants obtained were stored at −80 ◦ C for fur-
husks, trimming vine shoots, corn cobs and Eucalyptus globulus ther studies.
chips), jute [17], plant polymer, oil extracts, distillery and whey
wastes, potato process effluent and pea nut cake [18]. Other sources 2.4. Scanning electron microscopy
include sugar plants such as dates syrup, sugar beet, sugar cane or
sugar sorghum [12,18–21]. Sugar and starch processing industries Klebsiella sp. RJ-03 strain was inoculated in CP medium, the most
also produce large amounts of solid residues of starch contain- promising carbon source, and the progress of biosurfactant produc-
ing wastewater [19]. Starch and cellulose are the components of tion was observed at different time intervals (0, 24, 48 and 72 h)
Madhuca indica, corn, tapioca, wheat bagasse, sugarcane bagasse, using scanning electron microscopy (SEM, LEO 1430 VP) Samples
soyabean and potato peel powder. Evaluation of different carbon were mounted on aluminum stub, coated with gold palladium and
substrates may help to overcome the problem of high production observed under SEM at a voltage of 18 kV.
cost.
Previously, an alkaliphilic bacterium, Klebsiella sp. strain RJ-03 2.5. Viscosity, surface tension emulsification activity and CP
has been shown to produce biosurfactant in different conven- biosurfactant stability studies
tional carbon substrates [2,22]. In the present study, evaluation
of unconventional low cost carbon sources was undertaken and Supernatants obtained after 72 h with different unconventional
compared to conventional sources in terms of biosurfactant yield, carbon sources were used to measure viscosity (RS1, HAAKE Instru-
surface activity, viscosity and physicochemical characteristics of ments, Karlsruhe, Germany) and surface tension (du Nouy ring
the product. The biosurfactant was evaluated for its compatibility tensiometer, Data physics, Germany) at 30 ◦ C. Surface tension of
with detergents to remove lubricant oil from sand and cotton cloth distilled water and respective production media without inoculum
and compared to chemical surfactants like sodium dodecyl sul- were taken as control. Emulsification activity of CP-biosurfactant
fate (SDS), Tween80, Triton X-100 and laundry detergent powders solution (0.1%, w/v) was determined by measuring the emulsifi-
(Aerial, Nirma and Tide). cation index. Emulsification index was recorded with time and
represented accordingly, i.e. emulsification index after 24 h was
2. Materials and methods represented as E24 . Stability with respect to pH (2–13), temper-
ature (50–150 ◦ C) and salt stress (2–10%) was determined in the
2.1. Microorganisms and seed culture terms surface tension and viscosity using 1% CP biosurfactant solu-
tion and compared with control. All experiments were carried out
An alkaliphilic bacterium, Klebsiella sp. strain RJ-03 (GenBank in duplicate under controlled laboratory conditions.
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 2.6. Chemical composition
(India) was selected for the study [2] The bacterium was maintained
on Horikoshi agar slant at 4 ◦ C for subsequent experiments. A loop- Purified biosurfactant samples were analyzed spectrophoto-
ful of strain RJ-03 was inoculated into 100 ml Horikoshi medium metrically for its constituent viz. total sugar, reducing sugar and
(HM; w/v 1% glucose, 0.5% peptone, 0.5% yeast extract, 0.1% K2 HPO4 protein content. The monosaccharide composition of different
and 0.02% MgSO4 ·7H2 O; 1% Na2 CO3 after autoclaving) broth of pH purified biosurfactant samples were analyzed using GC–MS after
10 and incubated for 24 h at 32 ◦ C and 120 rpm on an orbital rotary alditol-acetate derivatization [2].
shaker.
2.7. Molecular mass and MALDI TOF–TOF mass spectroscopy
2.2. Screening of agro-industrial waste for biosurfactant The molecular weight of the purified CP-biosurfactant was
production determined by Gel Permeation Chromatography (GPC, 7.8 mm
ID × 300 mm stainless steel, Model Alliance 2695, Waters, USA)
Different agro-industrial substrates viz. M. indica, sugarcane with guard columns. About 80 l (0.1%, w/v in Milli Q water)
bagasse, E. globulus, wheat straw, Jatropha cake, soyabean powder, of purified biosurfactant was loaded on to a GPC column
corn powder, potato waste powder and seaweed sap used for bio- Ultrahydrogel-120 and Ultrahydrogel-500 at 30 ◦ C. The column
surfactant production were subjected to chemical analysis i.e. total was calibrated with standard dextran (molecular weight, 5200-
sugars, reducing sugars and total protein. For this, 5 g dry and pow- 668,000 kDa; PSS, USA) and elution was monitored using a
dered raw material was suspended in 100 ml distilled water and refractive index detector (2414). MALDI TOF–TOF analysis was car-
autoclaved for 1 h at 100 ◦ C for digestion. ried out on MALDI TOF–TOF analyzer (Applied Biosystem 4800,
USA) with an Nd-YAG laser (355 nm, 200 Hz) operated at 20 kV.
2.3. Production, quantification and purification of biosurfactant Sample was prepared by dissolving purified CP-biosurfactant
(5 mg ml−1 ) in milli-Q water and mixed with equal volume of
Biosurfactant was produced by inoculating 2% (v/v) seed cul- matrix ␣-Cyano-4-hydroxycinnamic acid (10 mg ml−1 ). Each spec-
ture (OD600 = 2) in 500 ml broth (pH 10) containing 5% of various trum was collected in positive ion reflector mode with an average of
unconventional substrates like M. indica (MI), sugarcane bagasse 1400 laser shots per spectrum. Reproducibility of the spectrum was
(SB), wheat straw (WS), rice husk (RH), potato peel powder (PPP), checked and spectra were analyzed after centroid and de-isotoping
corn powder (CP), soyabean powder (SP), Jatropha cake (JC) and using Data explorer software (Applied Biosystem, USA) [2,22,23].
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 2.8. FT-IR, 1 H and 13 C NMR analysis
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 Selected purified biosurfactants produced by utilizing different
for 12 h and centrifuged (13,000 × g; 20 min; 4 ◦ C). Biosurfactant unconventional carbon sources were subjected to Fourier trans-
was precipitated by adding 2 fold cold iso-propanol. Products were form infrared spectroscopy (FT-IR) to elucidate the chemical nature
dried and yield was compared. Crude products were dialysed for by identifying the types of functional groups and chemical bonds.
48 h at 4 ◦ C (12,000 Da cut off dialysis tubing, Sigma) and lyophilized 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
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.
Total Reducing Total Arabinose (%) Rhamnose (%) Fucose (%) Mannose (%) Galactose (%) Glucose (%) Ribose (%)
sugar (%) sugar (%) protein (%)
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 methyl group ( CH3 ) corresponding to the sugar moiety. The ı
stretch of C O C, C O at 1016–1245 cm−1 showed the presence value at 2.125 ppm showed the CH-proton of C5/C6 carbon of de-
of carbohydrates. Peaks at 1016–35 cm−1 (PII band: polysaccha- oxy sugar. The range of chemical shift (ı) signals from 3.43 to 5.47
rides), 760–763 (of CH2 group) and 657–675 cm−1 (of NH group) possibly indicated a glycosidic linkage of pentose/hexose sugars.
evidenced the presence of glycopeptide moieties. A symmetrical 13 C signals of five anomeric carbon at ı = 77.145, 73.087,
stretched peak near 1384 cm−1 indicated the presence of carboxyl 71.399, 71.342, 69.342 and 99.432 of repeating monosaccha-
groups. Absorption peaks in the range around 760–571 cm−1 cor- rides units were observed in 13 CNMR spectrum analysis of the
respond toward stretch of alkyl-halides. The presence of acetyl and CP-biosurfactant (Fig. 3) while signal in the range of ı = 60.452 con-
carboxyl functional groups in the biopolymer proved its anionic firms the presence of acetyl group. The C1 signals for -glucose
nature which could be useful for binding and thus remediating (99.432) & ␣-glucose (80.020); C2 signals for -glucose (77.026),
divalent cations. The FT-IR confirmed the glycopeptide nature of the ␣-glucose (73.087) & -galactose (71.399) were also observed.
biosurfactants which was similar to the earlier reports on Klebsiella NMR characteristic spectral peaks of CP-biosurfactant showed close
sp. strain RJ-03 [2,31,32]. The carboxyl groups provided overall neg- resemblance with previous report of SPY-biosurfactant [2].
ative charge to the biopolymer, thereby supporting binding and
adsorptive properties for divalent cations by electrostatic interac- 3.7. Thermal gravimetric (TG) and differential scanning
tions [9]. calorimetric (DSC) analysis
1 H NMR spectra of purified CP-biosurfactant (Fig. 2) revealed
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 57
4. Conclusions
CP-biosurfactant showed excellent ability of oil removal from soil [10] M. Kambourova, R. Mandeva, D. Dimova, A. Poli, B. Nicolaus, Carbohydrate
and cloths (Supplementary S6). Further, it showed efficiency, com- Polymer 77 (2009) 338–343.
[11] B. Nicolaus, M. Kambourova, E.T. Oner, Environmental Technology 31 (2010)
patibility and stability with various commercial detergents and 1145–1158.
its additive interaction with chemical surfactants improved the [12] R.S. Makkar, S.S. Cameotra, I.M. Banat, AMB Express 1 (5) (2011) 1–19.
washing performance. This led to conclude its economic indus- [13] M. Nitschke, G.M. Pastore, Bioresource Technology 97 (2006) 336–341.
[14] M. Nitschke, S.G. Costa, R. Haddad, L.A. Goncalves, M.N. Eberlin, J. Contiero,
trial production as well as application as a laundry detergent Biotechnology Progress 21 (2005) 1562–1566.
additive. [15] W. Bednarski, M. Adamczak, J. Tomasik, M. Plaszczyk, Bioresource Technology
95 (2004) 15–18.
[16] K. Das, A.K. Mukherjee, Process Biochemistry 42 (2007) 1191–1199.
Acknowledgments [17] Q. Wang, S. Chen, J. Zhang, M. Sun, Z. Liu, Z. Yu, Bioresource Technology 99
(2008) 3318–3323.
RMJ and NJ are thankful to Council of Scientific and Industrial [18] S.R. Chowdhury, R.K. Basak, R. Sen, B. Adhikari, Bioresource Technology 102
(2011) 6629–6632.
Research, New Delhi (CSIR-SRF) and Ministry of New and Renew-
[19] R. Thavasi, S. Jayalakshmi, I.M. Banat, Bioresource Technology 102 (2011)
able Energy for financial support respectively. The authors are 3366–3372.
grateful to Dr. Vinod Boricha, Mr. Harshad Brahmbhatt, Mr. Jayesh [20] D. Onbasli, B.J. Aslim, Journal of Environmental Biology 30 (2009) 161–163.
[21] F. Freitas, V.D. Alves, M.A.M. Reis, Trends in Biotechnology 29 (2011) 388–398.
Chaudhary, Mr. Vinod Agarwal and Mr. Gaurav Vyas of the Analyt-
[22] R.M. Jain, K. Mody, N. Joshi, A. Mishra, B. Jha, Colloids and Surfaces B: Biointer-
ical Discipline for analytical assistance. faces 108 (2013) 199–204.
[23] A. Mishra, K. Kavita, B. Jha, Carbohydrate Polymer 83 (2011) 852–857.
[24] R.M. Jain, K.H. Mody, J. Keshri, B. Jha, Marine Pollution Bulletin 62 (2011)
Appendix A. Supplementary data
2377–2383.
[25] J.M. Luna, L. Sarubbo, G.M. Campos-Takaki, Brazilian Archives of Biology and
Supplementary data associated with this article can be found, Technology 52 (2009) 785–793.
in the online version, at http://dx.doi.org/10.1016/j.ijbiomac. [26] R. Fusconi, R.M.N. Assuncao, R.M. Guimaraes, G.R. Fiho, A.E.H. Machado, Carbo-
hydrate Polymer 79 (2010) 403–408.
2013.08.030. [27] D.G. Cooper, B.G. Goldenberg, Applied Microbiology and Biotechnology 53
(1987) 224–229.
References [28] S. Mukherjee, P. Das, R. Sen, Trends in Biotechnology 24 (2006) 509–515.
[29] F. Freitas, V.D. Alves, M. Carvalheira, N. Costa, R. Oliveira, M.A.M. Reis, Carbo-
hydrate Polymer 78 (2009) 549–556.
[1] R.M. Jain, K. Mody, A. Mishra, B. Jha, Carbohydrate Polymer 87 (2012) [30] W.M.F.W. Nawawi, P. Jamal, M.Z. Alam, Bioresource Technology 101 (2010)
2320–2326. 9241–9247.
[2] R.M. Jain, K. Mody, A. Mishra, B. Jha, Carbohydrate Polymer 89 (2012) [31] S. Joshi, C. Bharucha, S. Jha, S. Yadav, A. Nerurkar, A.J. Desai, Bioresource Tech-
1110–1116. nology 99 (2008) 195–199.
[3] S.S. Cameotra, R.S. Makker, Current Opinion in Microbiology 7 (2004) 262–266. [32] P. Das, S. Mukherjee, R. Sen, Journal of Applied Microbiology 104 (2008)
[4] E. Rosenberg, E.Z. Ron, Applied Microbiology and Biotechnology 52 (1999) 1675–1684.
154–162. [33] K. Urum, T. Pekdemir, Chemosphere 57 (2004) 1139–1150.
[5] A. Mishra, B. Jha, Bioresource Technology 100 (2009) 3382–3386. [34] L.M. Whang, P.W.G. Liu, C.C. Ma, S.S. Cheng, Journal of Hazardous Materials 151
[6] K. Kavita, A. Mishra, B. Jha, Biofouling 27 (2011) 309–317. (2008) 155–163.
[7] R.P. Singh, M.K. Shukla, A. Mishra, P. Kumari, C.R.K. Reddy, B. Jha, Carbohydrate [35] C.C. Lai, Y.C. Huang, Y.H. Wei, J.S. Changa, Journal of Hazardous Materials 167
Polymers 84 (2011) 1019–1026. (2009) 609–614.
[8] M. Banat, R.S. Makkar, S.S. Cameotra, Applied Microbiology and Biotechnology [36] C. Calvo, M. Manzanera, G.A. Silva-Castro, L. Uad, J. Gonzalez-Lopez, Science of
53 (2000) 495–508. the Total Environment 407 (2009) 3634–3640.
[9] P.V. Bramhachari, P.B. Kishor, R. Ramadevi, R. Kumar, B.R. Rao, S.K. Dubey, [37] A.K. Mukherjee, Letters in Applied Microbiology 45 (2007) 330–335.
Journal of Microbiology and Biotechnology 17 (2007) 44–51.