Bioremediation & Biodegradation

Research
Article
Research
Article

Qazi et al., J Bioremed Biodeg 2013, 4:7

http://dx.doi.org/10.4172/2155-6199.1000204

Open
OpenAccess
Access

Yeast Extract as the Most Preferable Substrate for Optimized

Biosurfactant Production by rhlB Gene Positive Pseudomonas putida SOL10 Isolate
Muneer Ahmed Qazi1, Zulfiqar Ali Malik1,2, Ghazi Dino Qureshi1, Abdul Hameed1 and Safia Ahmed1
1
2

Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
Department of Microbiology, Faculty of Natural Sciences, Shah Abdul Latif University, 66111 Khairpur Mirs, Sindh-Pakistan

Abstract
Oil contaminated sites are enriched source of microorganisms that produce a variety of surface active
amphiphilic compounds known as biosurfactants. Pseudomonas putida SOL-10 strain isolated from oil contaminated
soil of Fimkassar oil field, Chakwal, Pakistan, was identified by standard morphological, biochemical and 16S rRNA
sequence analysis methods. SOL-10 strain was initially screened for biosurfactant production using oil spreading
test and then manifestation of rhlB (rhamnolipid) gene was confirmed by PCR using gene-specific primers. Maximum
biosurfactant production in terms of surface tension (29.9 mN m-1) and emulsification index (E24, 73.45%), was
achieved when the strain was grown in MSM supplemented with yeast extract (1.5-2 %, w/v) and urea (0.1 %, w/v)
as carbon and nitrogen sources, respectively, and the physical parameters were adjusted at pH 7.0, temperature
30C, 150 rpm agitation speed. The biosurfactant emulsified various hydrocarbons tested, being more effective
against xylene and kerosene (85.19% and 70.59%, respectively). The crude biosurfactant also showed stability at
a wide range of temperature (25-80C), pH (1-9) and salt concentration (1-5%, w/v). The stability and hydrocarbon
emulsifying potential of the biosurfactant indicated its possible use as decent contender for future environmental
applications like biodegradation and bioremediation of organic pollutants.

Keywords: Yeast extract; rhlB gene; Pseudomonas putida;

biosurfactant; Hydrocarbon emulsification
Introduction
Biosurfactants are surface active amphiphilic compounds
produced mainly by various microorganisms [1]. Generally, surface
active compounds are of two types, i.e. chemically synthesized
(surfactants) and biologically synthesized (biosurfactants). The
chemical surfactants are produced from petrochemicals and are
considered to be environmentally unsafe because they can lead to an
ecological imbalance due to toxicity and less biodegradability [2,3]. On
the contrary, biosurfactants are environment friendly because these are
less toxic, biodegradable. The other advantages of the biosurfactants
over chemical surfactants include their specific activity even at extreme
environmental conditions and production from renewable resources
[4-6]. The biosurfactants have widely been applied in various fields like
food and agriculture, detergents and cosmetics, environmental cleanup
and oil recovery, biomedical and therapeutics [6-8] during recent years.
Due to imminent petroleum crisis worldwide, the current market
demand for industrially sustainable biosurfactants has grabbed the
attention of many companies for commercial production of costeffective and environment friendly substitutes to synthetic surfactants
[3,9,10]. Therefore, the global market of biosurfactants, growing at
a CAGR of 3.5%, is expected to reach USD 2,210.5 million in 2018
[11]. This in turn, has also encouraged a tremendous progress in the
field of biosurfactant research and has attracted the attention of many
researchers towards relatively unexplored natural resources. There is a
constant search for new and novel microbial strains having capability
for the production of value added green chemicals, new energy sources
and new drugs of therapeutic potential.
A large variety of surface active compounds have been isolated from
hundreds of microorganisms including glycolipids, phospholipids,
lipopeptides, lipoproteins, glycoproteins, polymeric and particulate
structures etc. [10,12]. Among microorganisms, Pseudomonas
aeruginosa for rhamnolipids, Bacillus subtilis for surfactin and
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

Candida spp. for sophorolipids and mannosylerythritol lipids are most

extensively studied [1,13]. The largest group of rhamnolipid producing
bacteria belongs to the genus Pseudomonas [14]. Apart from a constant
debate among the scientists about the prevalence of rhamnoilipd
genes in bacteria other than Pseudomonas species, some novel strains
of bacteria having unique ability to produce rhamnolipidshave
recentlybeen reported [15,16]. Rhamnolipid production in few
pathogens including Burkholderia mallei and B. pseudomallei and
the non-pathogenic B. thailandensis,has also been reported [17]. In
bacteria, the primary biosynthetic and regulatory genes are present
in an rhlABRI gene cluster which is involved in the production of
rhamnolipids [18]. Theres a continuous debate among the scientific
community over prevalence of rhamnolipid genes in bacteria other
than P. aeruginosa. In the current study, a bacterial strain Pseudomonas
putida, isolated from one of the oil fields of Pakistan, was screened and
optimized for rhamnolipidproduction. P. putida, considered to be a
non-pathogenic member of Pseudomonads, is found mostly in moist
environments like soil and water (especially in rhizosphere). It is found
to be involved in plant protection from pests, plant growth promotion,
and environmental clean-up of persistent organic pollutants. The
bacterium has also been considered to have enormous potential for
biotechnological applications [19].

*Corresponding author: Safia Ahmed, Department of Microbiology, Faculty of

Biological Sciences, Quaid-i-Azam University Islamabad 45320, Pakistan, Tel:
+9251-90643009; E-mail: safiamrl@yahoo.com
Received June 09, 2013; Accepted September 21, 2013; Published September
27, 2013
Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast
Extract as the Most Preferable Substrate for Optimized Biosurfactant Production
by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4:
204. doi:10.4172/2155-6199.1000204
Copyright: 2013 Qazi MA, et al. This is an open-a ccess article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.

Volume 4 Issue 7 1000204

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

Page 2 of 10

Although, biosurfactants are advantageous over chemical

surfactants available in the market, the limited yields, foaming issues,
poor recoveries and higher production- as well as purification-costs
are still major hurdles in their production at commercial scale. Apart
from that, all microbial processes are influenced by environmental and
culture conditions that also play a major role in type and productivity
of the biosurfactants [4,20]. Therefore, the major goal of this study
was to evaluate the effect of cultural and environmental conditions on
optimal biosurfactant production by P. putida SOL-10 isolate.

Materials and Methods

Microorganism and culture maintenance
The bacterial isolate SOL-10, isolated from Fimkessar oil
field, Chakwal, Pakistan was obtained from culture collection of
Microbiology Research Labs., Quaid-i-Azam University, Islamabad.
The strain was already known as a potential crude oil-biodegrading
agent [21]. The bacterium was routinely cultured on nutrient agar and
preserved on nutrient agar slants at 4C in refrigerator for routine use.
Culture stocks of pure strain were prepared in nutrient broth with
glycerol (20%) for long term preservation at -70C. The isolate SOL-10
was screened for biosurfactant producing ability using agar plate-based
oil spreading test [22].

Physiological and biochemical characterization of SOL-10

isolate
The bacterial strain was primarily characterized on the basis
of Gram reaction, motility, spore and capsule formation. The
physiological and biochemical characterization of SOL-10 strain that
included: hydrolysis of starch, lipids and casein; catalase, oxidase,
indole production citrate and carbohydrate (lactose) utilization tests,
was performed as per standard methods according to Bergeys Manual
of Determinative Bacteriology [23]. The Analytical Profile Index (API)
was also obtained to further confirm biochemical characteristics of
SOL-10 strain with API 20E Kit and API identification software using
online APIweb services (BioMrieux, France).

16S rRNA based identification

The bacterial genomic DNA was extracted from overnight pure
culture of SOL-10 strain using ethanol precipitation method [24].
Briefly, 5 to 6 pure bacterial colonies were picked and suspended in
240 L distilled water. To this, 20-30 L of sodium dodecylsulfate
(SDS), 80 L of PK(Proteinase K) buffer and 40 L of PK were mixed
and incubated at 55C for 1 h for disintegration of cells. 100 L of 6M
NaCl was added. Centrifugation was done at 14,000 rpm for 5 min.
Supernatant was taken and 1 mL of chilled 100% ethanol was added
in order to precipitate DNA. Tubes were then mixed well, centrifuged
at 12,000 rpm and supernatant was discarded. Washing of the pelleted
DNA was carried out twice with 1 mL of 70% ethanol, centrifuged and
supernatant was discarded. The pellet was air dried and 100 L of TE
buffer was added. The partial sequencing of the 16S rRNA gene was
commercially carried out at the Genomic Division, Macrogen Inc.,
Seoul, Korea,using universal amplification and sequencing primers
(Table 1) and ABI PRISM Big DyeTM Terminator Cycle Sequencing
Ready Reaction Kit (PE Biosystem, USA). Electrophoresis of sequencing
reaction was completed using the automated ABI PRISM 3730l DNA
Sequencer (Applied Biosystems, USA).

Phylogenetic correlation analysis

The partial sequence of the 16S rRNA gene obtained in this study
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

was analyzed and compared with nucleotide sequence databases in the

National Center for Biotechnology Information (NCBI) website using
Basic Local Alignment Search Tool (BLAST) program (http://www.
ncbi.nlm.nih.gov/BLAST), in order to confer percentage sequence
similarities. The evolutionary history of SOL-10 strain was inferred
using the Neighbor-Joining (NJ) method [25]. The evolutionary
distances were computed using the Maximum Composite Likelihood
(MCL) method [26]. Phylogenetic analyses were conducted in
Molecular Evolutionary Genetics Analysis (MEGA) software (Version
4.0) [27].

Molecular screening for rhlB gene

PCR for rhlB gene amplification was carried out was in
Thermocycler 2700 (Applied Biosystems, USA) using gene-specific
primers (kpd-F 5-GCCCACGACCAGTTCGAC-3 and kpd-R
5-CATCCCCCTCCCTATGAC-3) according to Danashekar et al.
[28]. The PCR amplification cycle consisted of an initial denaturation
step at 94C for 5 min, followed by 34 cycles of 25 sec at 94C and
annealing at 54C for 40 sec. Afterwards, an elongation cycle (72C
for 50 sec) was followed by a final extension step (72C for 6 min).
The final PCR product (10 L) was resolved with gel electrophoresis
using 1 % agarose gel in 0.5 TBE buffer against 1 kb DNA marker
(Fermentas) and ethidum bromide-stained bands were visualized
under UV-transilluminator. The gel images were taken with Bio-Rad
gel documentation system (Bio-Rad Laboratories, Inc., USA).

Culture conditions for growth and biosurfactant production

Mineral salts medium (MSM) as described by Abouseoud et al.
[29], with slight modification, was used throughout the fermentation
experiments. The medium contained (g L-1 of deionized water):
Na2HPO4, 2.2; KH2PO4, 1.4; MgSO4.7H2O, 0.6; FeSO4.7H2O, 0.01; NaCl,
0.05; CaCl2, 0.02; and 0.1 mL of trace elements solution containing g
L-1 of deionized water: ZnSO4.7H2O, 2.32; MnSO4.4H2O, 1.78; H3BO3,
0.56; CuSO4.5H2O, 1.0; NH4MoO4.2H2O, 0.39; KI, 0.66.The pH of
medium was adjusted to 7.0 0.2 using 1 M HCl and 1M NaOH and
was autoclaved at 121C and 15 lb pressure for 20 min. The carbon and
nitrogen sources were separately sterilized and added to the production
medium at 2% and 0.1% concentrations respectively. All the chemicals
were purchased from Sigma (Sigma-Aldrich, USA).

Preparation of the inoculum

A 5% of the inoculum, 16-18 h grown culture in nutrient broth
was separated by centrifugation at 5,000rpm and washed twice with
saline. After second centrifugation, the biomass pellet was aseptically
re-suspended into MSM and maintained with an optical density of 1.0.
Test

Result

Gram reaction

Motility

Sporulation

Capsulation

Starch hydrolysis

Lipid hydrolysis

Casein hydrolysis

Catalase

Oxidase

Indole

Citrate utilization

Lactose fermentation

+ (with Acid & Gas)

Table 1: Morphological and biochemical features of SOL-10 strain.

Volume 4 Issue 7 1000204

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

Page 3 of 10

This cell suspension was then used as inoculum in production medium

at 5% (v/v) concentration.

Optimization of cultural conditions for biosurfactant

production
Different environmental as well as nutritional factors were optimized
for the production of biosurfactant and the cultivation was performed
in 250 mL shake flasks within shaking incubator at certain range of pH
(2.0 to 10.0), temperature (25, 30, 37 and 45C)and agitation speed (0,
120, 150 and 200 rpm). However, the effect of nutritional factors like
carbon source(yeast extract, glucose, glycerol, sucrose, -hexadecane,
xylene, kerosene and olive oil) and nitrogen source (KNO3, NH4NO3,
NaNO2, NaNO3, urea, yeast extract, vitamin B2 (Riboflavin), L-Leucin
and L-Arginine) on the biosurfactant production by SOL-10 strain was
evaluated. For that purpose, shake flask experiments were performed
in 250 mL flasks containing 100 mL MSM plus 2% and 0.1% of each
carbon and nitrogen source, respectively, at 30C and 150 rpm for 5
days. In another experiment, cultivation was also performed with
different concentrations of the most suitable carbon source at 0.5, 1.0,
1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5% (w/v), in order to determine the effect
of varying carbon source concentration on biosurfactants producing
ability of the isolatein order to optimize the yield.

Analytical methods
The qualitative and quantitative analysis of biosurfactantscontaining cell-free supernatant (CFS) of culture broth was performed.
The samples were collected after every 24 h, centrifuged at 12,000 rpm
for 15 min at 4C prior to analysis. All the analyses were performed
in triplicates and the averages of the three were taken. The Oil
Displacement Activity (ODA) test was performed according to
Rodrigues et al. [30]. The CFS (20 L) was dropped on crude oil-film
over surface of 40 mL distilled water in 90 mm petri dish. The zone of
oil repellence was visualized and measured in millimeter (mm), quickly
after performing the assay. The percentage emulsification index (E24) of
the CFS and crude extract was determined according to the method of
Cooper and Goldenberg using Eq. 1 [31].
eHT
(1)
E24 (=
%)
100
tHT
Where eHT denotes the emulsion height and tHT indicates
total height of the solution. The heights were measured in terms of
centimeters (cm) in triplicates and the means were calculated.
The surface tension (SFT) of the cell-free broth, and crude
biosurfactant solution was measured with a digital tensiometer
(EasyDyne K20, KRUSS GmbH, Germany) using standard Whilmay
plate method at room temperature according to the manufacturers
instructions. All the measurements were taken as the mean of five
measurements in mN m-1 SD.

Isolation and stability of crude biosurfactant

The 72 h old culture broth was centrifuged at 12,000 rpm (15 min,
4C) and filtered (0.45 m syringe filters). The cell free supernatant
(CFS) was acidified with 6N HCl to pH 2 and left overnight at 4C.
After centrifugation, the precipitates were collected and re-dissolved
in phosphate buffer pH 7. The biosurfactants were then extracted
three times with equal volume of chloroform-methanol (2:1) and
pooled. The solvent was evaporated by using rotary evaporator at
45C. The remaining brown extract served as crude biosurfactant.
The effect of a wide range of pH (1-11), temperature (25-121C),
and salt concentrations (1-7%) on activity and stability of the crude
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

biosurfactant was tested. Briefly, the crude biosurfactant (100 mg/50

ml, w/v) was dissolved in range of pH buffers (1, 3, 5, 7, 9, 11) and
incubated for 1 h. Subsequently, the crude biosurfactant (100 mg/50
ml, w/v), dissolved in most suitable buffer, was exposed to different
temperature treatments (at 25, 40, 60, 80, 90, 100C) in water bath
for 1 h and also autoclaved at 121C under 15 lb pressure. Different
concentrations of NaCl (1, 2, 3, 4, 5, 6 and 7%) were added to the crude
biosurfactant solution (100 mg/50 ml, w/v) prepared in distilled H2O
and incubated for 1 h at room temperature. The activity and stability
of the biosurfactant was determined after each treatment in terms of
surface tension, emulsification index and oil displacement activity.

Hydrocarbon emulsifying potential of biosurfactant

The hydrocarbon emulsifying capability of the biosurfactant against
different hydrophobic sources was evaluated. For that purpose, equal
volumes of each hydrocarbon (i.e., n-hexane, n-hexadecane, kerosene,
xylene, glycerol and olive oil) and CFS were mixed at high speed using
vortex mixer for 2 min and the emulsifying indices were measured after
24 h using Eq. 1.

Statistical data analysis

For statistical analyses, all the experiments were run in triplicates.
The averages, standard deviations and analysis of variance (ANOVA),
at 95% confidence level, were computed by using Microsoft Excel
2010 version. The standard errors for mean values, at 95% confidence
level, were calculated and represented as error bars in all the graphical
representations. The least significant difference (LSD) among the
hydrocarbons tested was calculated by using Statistix software
(Version, 8.1).

Results and Discussion

The most common property of all the biosurfactants is that they
reduce surface and intra-molecular forces of heterogeneous systems
by partitioning at air-liquid, liquid-liquid or solid-liquid interface.
Such a property of biosurfactants helps them to reduce the surface
tension and convert highly immiscible hydrocarbons into small
droplets or micelles, and increase their bioavailability. These micelles
are then easily taken up and utilized for growth and metabolism
by microorganisms. Therefore, it is experienced that hydrocarbon
contaminated sites are ideal sources for the isolation of biosurfactant
producing microorganisms. Many scientists have recently reported
variety of microorganisms having capability to produce biosurfactants
from oil contaminated environments [2,32-37]. The bacterial strains
isolated from oil contaminated sites are more prone to biosurfactant
production.
Consequently in current study, the bacterial isolate P. putida
SOL-10, from oil contaminated soil, was screened and optimized for
biosurfactant producing ability. The physiological and biochemical
characteristics of SOL-10 are given in Table 2. Based on negative starch,
lipids and casein hydrolysis tests and positive catalase, oxidase, indole
and citrate utilization, and lactose fermentation tests, the strain was
Primer
name

Primer sequence

Amplification

27F

5-AGAGTTTGATCMTGGCTCAG-3

1492R

5-TACGGYTACCTTGTTACGACTT-3

518F

5-CCAGCAGCCGCGGTAATACG-3

800R

5-TACCAGGGTATCTAATCC-3

Sequencing

Table 2: List of primers used in this study for amplification and sequencing of 16S
rRNA gene.

Volume 4 Issue 7 1000204

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

Page 4 of 10

tentatively identified as Pseudomonas putida. The API results obtained

later on by using API software also confirmed that the isolate has 99%
homology with P. putida strains. To further confirm the identity of
SOL-10 strain, 16S rRNA sequencing was performed. The nucleotide
sequence obtained after necessary trimming was 901 bp in size. The
similarity search by BLAST program revealed maximum similarity
(~98%) was with P. putida strains (Figure 1). Therefore the strain was
named as P. putida SOL-10 strain. The partial nucleotide sequence has
been submitted into online NCBI GeneBank repository and is available
under accession number (JX534510).
A positive screening test (5.5 0.2 cm, oil repellence zone) indicated
that the isolate SOL-10 could be a suitable candidate for biosurfactant
production. Figure 2 shows the gel image of the amplified product of
rhamnosyltransferase l (rhlB) gene (723 bp) and compared with 1kb
molecular weight DNA marker and another rhamnolipid positive isolate
of P. aeruginosa isolated during current study (unpublished data).Most
of the research documented on the production of rhamnolipids has
been focused mainly on rhamnolipids of P. aeruginosa strains [38, 39],
whereas, very little studies have been conducted on P. putida so far [4042]. Different studies reported the presence of unidentified rhamnolipid
congeners in P. putida [40,43]. Similarly, in current study, we identified
arhlB gene positive P. Putida isolate from oil contaminated soil.
On the basis of primary screening results, SOL-10 isolate was
further investigated for the effect of culture conditions like temperature,
pH, agitation, and carbon and nitrogen sources that usually effect

growth and biosurfactants production [44]. Figure 3 shows the effect

of different culture conditions on the biosurfactant-producing abilities
of SOL-10, wherein the maximum biosurfactant production was
achieved at 30C (SFT, 30.89 0.76 mN m-1; E24, 72.73 3.5%) after 48 h
of incubation. Although, the strain SOL-10 did not show significant
biosurfactant production at 25, 37, and 45C represented by 35.76 2.1,
55.0 4.4 and 49.09 5.2%emulsifying activities, respectively (Figure
3A). However, significant reduction in surface tension of the cellfree culture broth was also achieved at 25, 37, and 45C [SFT (mN
m-1)=33.57 0.44, 34.45 1.1, 40.26 0.93, respectively]. In general, the
isolate revealed significant growth and biosurfactant production in
the range of mesophilic temperature, i.e. 30 to 40C, while a notable
decrease in biosurfactant production was observed at higher and
lower temperature ranges. This could be due to depressing effects of
temperature on the biomass, E24, interfacial tension (IFT) and critical
micelle dilutions [45]. The change in the pH of production medium
also affected the biosurfactant production by P. putida SOL-10 strain.
The strain did not show growth as well as biosurfactant production at
pH 2.0, however, the maximum biosurfactant production was observed
in the pH range 6-8 (E24, 69%; SFT, 30 to 32 mN m-1) after 72 h of
incubation and only slightly declined (E24, 62.5%; SFT, 38.44 0.9 mN
m-1) during 120 h, while considerable activities were also detected in
samples with pH range of 3.0 to 10.0 (Figure 3B). The pH tolerance at
wide range may be attributed to the ability of the bacterium to produce
alkaline byproducts, which in turn shifted the pH of the medium

Figure 1: Phylogenetic tree of Pseudomonas putida SOL-10 strain showing evolutionary relationship inferred by NJ method with 22 taxa.

J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

Volume 4 Issue 7 1000204

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

Page 5 of 10

Figure 2: PCR amplified product of rhlB gene. Lane-1: 1 kb DNA marker, Lane-2: positive control, Lane-3: amplified product of rhlB gene of rhamnolipid positive isolate
P. putida SOL-10.

Figure 3: Effect of culture conditions on emulsifying activity and surface tension of culture broth in relation to biosurfactant production by P. putida SOL-10; A.
temperature, B. pH, C. agitation, D. carbon, E.nitrogen source, and F. yeast extract conc.

towards alkaline side (Figure 4). This is one of the reasons for growth
and biosurfactant production by P. putida SOL-10 strain when grown
at lower pH limits. The characteristic might in future be helpful in

J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

application of the strain and the product in leather, paper, textile and
other industries working on variable pH ranges.
The cultivation broth showed maximum biosurfactant production

Volume 4 Issue 7 1000204

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

Page 6 of 10

mN m-1), it was found that supplementation of urea or L-leucin into

the production medium maximized the productivity (E24, 73.45 1.6%
and 73.15 1.0%; SFT, 29.91 0.95 and 30.24 0.87 mN m-1, respectively)
as compared to nitrates of sodium, ammonium and potassium (Figure
3E).

Figure 4: Growth and biosurfactant production kinetics ofP. putida SOL-10

isolate grown in MSM under optimized culture conditions. Emulsification index
(E24,
); surface tension (SFT,
); oil displacement activity (ODA,
); cell dry weight (CDW,
); pH ( ); and crude biosurfactant weight (BS
yield,
).

at 150 rpm (E24, 72.73 1.5%; SFT, 30.42 0.2 mN m-1), while it remained
comparatively lower at 120 rpm and 200 rpm (E24, 50 2.4%; SFT, 32.66
0.5 mN m-1and E24, 55.23 2.2%; SFT, 33.2 0.9 mN m-1, respectively)
during 48 h. Similarly, fermentation experiments for biosurfactant
production by P. putida strains at vigorous shaking speed have also
been reported [40,46]. In the current study, the bacterial strain was
also found capable of producing biosurfactants under static condition
(0 rpm), giving an E24 of 40% after 48 h (Figure 3C). The absence of
agitation resulted in extended lag phase, short exponential and long
stationary phases (data not shown), which may be due the insufficient
oxygen level than normally required during the initial acclimatization
of the bacterium in growth medium. The higher agitation rate also
negatively influenced the biosurfactant production possibly due
to shear stress that would have resulted in detrimental effects on
biosurfactant yield and growth kinetics of the bacterium. Our findings
of optimum temperature, pH and agitation are somehow concurrent
with previous works on the production of biosurfactants by different
strains of P.putida that showed maximum production on 28C, pH 7.0
and 150 rpm [40,41,46]. The best results for rhamnolipids production
from Pseudomonas species have been reported with the addition of
either oils or residues rich in oils to the culture medium [29,47-49].
Subsequently, the effect of different carbon and nitrogen sources
was investigated (Figure3D-3F). Maximum biosurfactant production
was observed in the medium containing 1.5-2.5% (w/v) yeast extract
as carbon source (E24, ~72%; SFT, 29.95 mN m-1), however, varying
concentration of yeast extract was found to have little effect on the
biosurfactant production (Figure 3D and 3F). The strain demonstrated
biosurfactant production with all the nitrogen sources tested. Figure
3E is showing the effect of organic and inorganic nitrogen sources on
biosurfactant producing ability of P. putida SOL-10 isolate. The best
emulsifying activities (E24, 73%) were detected when urea and L-leucin
were used as nitrogen sources at a conc. of 0.1% (w/v) along with
yeast extract (conc. 1.5%, w/v), as compared to inorganic nitrogen
sources such as sodium nitrate, ammonium nitrate and potassium
nitrate at 30C, 150 rpm and pH 7.0, showing maximum biosurfactant
production. Likewise, none of the tested nitrogen sources displayed
detrimental effects on growth as well as the biosurfactant production,
which was really interesting. Although, the use of yeast extract resulted
in significant biosurfactant production (E24, 72.33 1.3%; SFT, 30.54 0.25
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

The introduction of compounds containing amine groups like

yeast extract and urea usually triggers the biosynthesis of either directly
the peptide-containing biosurfactants or the enzymes regulating the
biosynthesis of other biosurfactant types. Yeast extract is obviously the
whole cell extract of yeast cells. It majorly contains proteins, short chain
peptides, carbohydrates, free amino acids and nucleic acids. Therefore,
it is supposed to be a rich source of not only carbon and nitrogen but also
some essential trace elements as well. It was also therefore concluded
that yeast extract alone can be utilized as a good source of nitrogen
as well as carbon, which provided sufficient carbon and nitrogen
content required by the isolate for biosynthesis of metabolic products.
The production of biosurfactant by using amino acids L-Leucin and
L-Arginine as nitrogen sources may be due to the direct uptake of
amino acids as precursors for the surfactant biosynthesis, improving
the biosurfactant yield [50,51]. The ammonium salts and amino acids
might have further been incorporated into the biosurfactants primary
structure resulting in positively charged functional groups making
them cationic in nature, which shifted pH of the medium to alkaline
side [52]. This might have enabled the P. putida SOL-10 to tolerate a
wide range of pH for growth and metabolite production. Similar results
were obtained with P. putida ML2 when nitrogen was supplied at the
oxidation level of ammonia (urea) than with nitrogen provided as
nitrate, although the strain was also able to assimilate nitrates [41].
Previous work on biosurfactant production emphasized on the
optimization of cultural conditions, because such factors could affect
both the amount and type of biosurfactant produced [41,45,51].
In another study [53], biosurfactant production from gamma-ray
induced P. putida 300-B mutant was carried out using distant carbon
sources (viz. hydrocarbons, waste frying oils WFOs, vegetable oil
refinery wastes and molasses). This was also one of the reasons to use
olive oil, glycerol, kerosene, xylene and hexadecane as possible carbon
sources for the production of biosurfactants from P. putida SOL-10 in
current study. But in case of P. putida SOL-10, it was found that these
hydrocarbons (except yeast extract) did not play a significant role in
the production of biosurfactants. The only emulsification activity (E24,
25%) was found when olive oil was used. This may be due to their high
hydrophobic lipid contents [51]. Although, we did not check the effects
of different concentrations of those hydrocarbons, further studies
are under the way for other factors that could affect growth as well
as biosurfactant production. However, the maximum emulsification
activity in samples with olive oil (E24, 25.37%) after 48 h have illustrated
that it could be used as an additional medium supplement for the
growth and as inducer for biosurfactant production by the strain. It was
observed that olive oil and glucose could be used as inducers of growth
and biosurfactants production by P. putida SOL-10 along with yeast
extract (Figure 3D). Similar results were also reported with soybean
WFO (waste frying oil) as carbon source and glucose as growth initiator
under fed-batch cultivation [49]. The production of biosurfactants by
P. fluorescens strain using substrates such as n-hexadecane, olive oil
and glucose with an emulsifying activity of 49% when olive oil was used
as the carbon source has also been reported [29].
Figure 4 shows the growth and biosurfactant production
kinetics of SOL-10 isolate under optimized conditions in 2L shaking
flak fermentation setup, where a growth associated production of

Volume 4 Issue 7 1000204

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

Page 7 of 10

biosurfactants was observed that declined during late stationary and

death phase. It might be due to the enzymatic hydrolysis and uptake
of these molecules because of substrate scarcity in the medium, or
the inhibitory effect of biosurfactants on cell growth above a certain
concentration, which induce subsequent degradation of these molecules
[54]. The partial characterization of the biosurfactant produced under
optimized conditions was performed by measuring the surface tension,
pH, oil displacement zone and emulsification index (E24, %) in relation
to biosurfactant yield and cell-dry weight. The surface tension was
determined against a standard of de-ionized water (72 mN m-1). The
surface tension of the cultivation broth started decreasing from the
very first day (40.03 mN m-1) and reduced to 29.9 mN m-1 during 96
h. Similarly, the emulsifying activity of CFS reached to 73% after 72
h of incubation, which also confirmed the biosurfactant production
by the strain. Thereafter, the surface tension slightly increased and
emulsification index of the CFS declined that illustrated the depletion
of biosurfactant from the cultivation broth after 120 h. The decline in
biosurfactant level might be due to the inhibitory effect of biosurfactant
on cell growth above certain concentration, which induced subsequent
degradation of the molecules [54]. Another explanation could be the
enzymatic hydrolysis and uptake of these molecules by the cells due
to substrate scarcity in late growth phases of bacteria. The pH of the
medium shifted to alkaline side with increasing concentration of the
biosurfactant, decreasing surface tension and increasing emulsifying
activity that indicated the cationic nature of the biosurfactant (Figure
4). The ability of biosurfactant to dissolve in water and alkalinity also
indicated the presence of hydrophilic portion (i.e. carbohydrate, amino
acid, phosphate or cyclic peptide) in it [55].
The cell-free broth was then analyzed for critical micelle dilution
(CMD) in order to quantify the biosurfactant concentration and to
identify the optimum harvesting time for product recovery. Table 3
shows the time dependent relationship of surface tension and CMD
of the cell-free broth at 3-fold dilutions (i.e. 10x, 100x, 1000x). It was
observed that the surface tension of the CFS abruptly changed with
dilution during initial 48 h of the incubation time, however, it was
stable later on. This indicated enrichment of the production medium
with significant amount of biosurfactant after 48 h of the incubation.
The lowest surface tension representing most significant biosurfactant
concentration was thus achieved during 72 to 96 h of incubation (Table
3). The critical micelle concentration (CMC) of the crude biosurfactant
after recovery from fermentation broth was found to be in the range
of 230 mg L-1 (w/v). The rhamnolipids from different bacteria have
been reported to have their CMC values in the range of 10-600 mg L-1
[56,57].
After recovery from fermentation broth, the biosurfactant was
investigated for its stability over a broad range of pH, temperature and
salinity (Figure 6A-6C). Figure 5A shows that the biosurfactant was
stable at broad pH range having maximum activity at pH 7 (E24, 75%,
SFT, 34.9 mN m-1, ODA, 2.6 cm). At the lower pH range maximum
activity was found at pH 5, while at the alkaline pH range activity was
high at pH 9. In general, there was no significant loss of activity at a
pH range of 1-11. The biosurfactant produced by P. putida SOL-10
was also found to be thermostable, where the surface and emulsifying
activities were maintained even at the higher temperature of 100C
and 121C, while the maximum activity (E24, 73%, SFT, 33.1 mN m-1,
ODA, 1.7 cm) was observed at the moderate temperature range,
i.e. 40C (Figure 6B). Little changes were observed in the activity of
biosurfactant between1-7% salt concentrations (Figure 6C). The results
of this study were concurrent with previous reports about the stability
of biosurfactants produced by P. fluorecens, P. aeruginosa MA01 and B.
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

cereus NK1 [2,29,58]. The results were also found in accordance with the
biosurfactant isolated from AlcanivoraxdieseloleiB-5 [59]. The stability
of biosurfactant in the present work over a wide range of environmental
conditions suggests that it maintains its activity at extreme conditions. A
variety of hydrophobic sources i.e., n-hexane, n-hexadecane, kerosene,
xylene, olive oil and mustard oil were evaluated in order to determine
the emulsifying capability of the biosurfactant produced by P. putida
SOL-10. Table 4 shows the statistical analysis of variance (ANNOVA)
of emulsifying ability for all the hydrophobic sources tested. The
results revealed that the biosurfactant has significantly higher (p<0.05)
emulsifying capability against xylene and kerosene (>85% and ~70%
respectively), as compared to other hydrophobic sources used (Figure
7).
The P. putida SOL-10 is a potential soil-borne, non-pathogenic
biosurfactant producing strain. The optimization results concluded
the selective behavior of the strain for biosurfactant production under
favorable conditions. The preference of selective carbon and nitrogen
sources at certain concentrations, as well as at specific environmental
conditions demonstrated the importance of optimization studies in
such cases. Although, yeast extract is mostly considered as nitrogen
source, it also has sufficient amount of carbon that served as sole
carbon and energy source for SOL-10 isolate during current study.
However, due to expensiveness of yeast extract, further investigations
Time

SFT SD1

CMD-1 SD2

CMD-2 SD

(h)

(mNm )

(mN m )

(mN m )

(mN m-1)

NC3

71.34 0.02

71.33 0.02

71.36 0.02

71.36 0.02

-1

-1

-1

CMD-3 SD

43.6 0.45

48.27 0.025

68.33 0.015

69.58 0.06

24

40.3 0.36

44.78 0.09

57.24 0.04

69.12 0.01

48

34.6 0.06

34.65 0.11

36.72 0.01

45.43 0.06

72

31 0.02

31.36 0.023

31.28 0.05

38.54 0.07

96

29.9 0.03

30.68 0.05

30.02 0.02

32.62 0.055

120

31.2 0.06

31.56 0.026

31.98 0.23

32.54 0.03

Average surface tension standard deviation (mN m ) of triplicate experiments.

Average critical micelle dilution (CMD) standard deviation (mN m-1) of triplicate
experiments. CMD-1=10x dilution; CMD-2=100x dilution; CMD-3=1000x dilution.
3
Negative control=Un-inoculated mineral salts medium (MSM)
1

-1

Table 3: Relationship between surface tension and critical micelle dilution (CMD)
of the cell free broth of P. putida SOL-10 strain grown in MSM under optimized
culture conditions.

Figure 5: Critical micelle concentration (CMC) of the crude biosurfactant

extract of P. putida SOL-10 strain in relation to surface tension.

Volume 4 Issue 7 1000204

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

Page 8 of 10

Figure 7: Hydrocarbon emulsifying capability of P. putida SOL-10

biosurfactant produced after specific time intervals during cultivation under
optimized conditions.

are needed to replace yeast extract with alternate cheaper substrates

for the production of surface active metabolites by the isolate, in
order to make the process cost-effective as well. The current study
would certainly aid some important knowledge to the existing data
about biosurfactant production by different microorganisms, and
the hydrocarbon emulsifying property of the biosurfactant can be
helpful for future applications like bioremediation of contaminated
environments.
Acknowledgments
The authors are highly grateful to Higher Education Commission (HEC),
Government of Pakistan, for funding this study under HEC-Project No. 1366,
entitled Production and characterization of biosurfactants produced by indigenous
microorganisms. The authors have no conflict of interest to declare.

References
1. Banat IM, Franzetti A, Gandolfi I, Bestetti G, Martinotti MG, et al. (2010)
Microbial biosurfactants production, applications and future potential. Appl
Microbiol Biotechnol 87: 427-444.
2. Sriram MI, Kalishwaralal K, Deepak V, Gracerosepat R, Srisakthi K, et al.
(2011) Biofilm inhibition and antimicrobial action of lipopeptide biosurfactant
produced by heavy metal tolerant strain Bacillus cereus NK1. Colloids Surf B
Biointerfaces 85: 174-181.
3. Marchant R, Banat IM (2012) Biosurfactants: a sustainable replacement for
chemical surfactants? Biotechnol Lett 34: 1597-1605.
4. Saikia RR, Deka S, Deka M, Sarma H (2012) Optimization of environmental
factors for improved production of rhamnolipid biosurfactant by Pseudomonas
aeruginosa RS29 on glycerol. J Basic Microbiol 52: 446-457.
5. Makkar RS, Cameotra SS, Banat IM (2011) Advances in utilization of renewable
substrates for biosurfactant production. AMB Express 1: 5.
Figure 6: Stability of crude biosurfactant at varying pH (A), temperature (B)
and salt concentrations (C).

Source of Variation

SS

df

Between Hydrocarbons

5,665.88

Within Replicates

2,063.48

24

Total

7,729.36

29

MS

P-value

1,133.18 13.18 0.000003

F crit
2.62

85.98

SS=Sum of square
5
Df=degree of freedom
6
MS=mean square
4

Table 4: Single factor (one-way) ANNOVA for hydrocarbon-emulsifying ability of

the biosurfactant produced by SOL-10 isolate.

J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

6. Khopade A, Biao R, Liu X, Mahadik K, Zhang L, Kokare C (2012) Production

and stability studies of the biosurfactant isolated from marine Nocardiopsis sp.
B4. Desalination 285: 198204.
7. Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial applications
of microbial surfactants. Appl Microbiol Biotechnol 53: 495-508.
8. Lima TMS, Fonseca AF, Leo BA, Mounteer AH, Ttola MR (2011) Oil recovery
from fuel oil storage tank sludge using biosurfactants. J BioremedBiodegrad 2:
125. DOI:10.4172/2155-6199.1000125.
9. Marchant R, Banat IM (2012) Microbial biosurfactants: challenges and
opportunities for future exploitation. Trends Biotechnol 30: 558-565.
10. Mukherjee S, Das P, Sen R (2006) Towards commercial production of microbial
surfactants. Trends Biotechnol 24: 509-515.
11. Transparency Market Research (2012) Biosurfactants market - global scenario,

Volume 4 Issue 7 1000204

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

Page 9 of 10
raw material and consumption trends, industry analysis, size, share and
forecasts, 2011 - 2018".
12. Ron EZ, Rosenberg E (2001) Natural roles of biosurfactants. Environ Microbiol
3: 229-236.
13. Muthusamy K, Gopalakrishnan S, Ravi K, Sivachidambaram P (2008)
Biosurfactants: properties, commercial production and application. CurrSci 94:
736-774.
14. Das P, Mukherjee S, Sen R (2008) Genetic regulations of the biosynthesis of
microbial surfactants: an overview. Biotechnol Genet Eng Rev 25: 165-185.
15. Rezanka T, Siristova L, Sigler K (2011) Rhamnolipid-producing thermophilic
bacteria of species Thermus and Meiothermus. Extremophiles 15: 697-709.
16. Rooney AP, Price NP, Ray KJ, Kuo TM (2009) Isolation and characterization
of rhamnolipid-producing bacterial strains from a biodiesel facility. FEMS
Microbiol Lett 295: 82-87.
17. Toribio J, Escalante AE, Sobern-Chvez G (2010) Rhamnolipids: Production
in bacteria other than Pseudomonas aeruginosa. Eur J Lipid SciTechnol 12:
1082-1087.

35. Darvishi P, Ayatollahi S, Mowla D, Niazi A (2011) Biosurfactant production

under extreme environmental conditions by an efficient microbial consortium,
ERCPPI-2. Colloids Surf B Biointerfaces 84: 292-300.
36. Roldn-Carrillo T, Martnez-Garca X, Zapata-Peasco I, Castorena-Corts
G, Reyes-Avila J, et al. (2011) Evaluation of the effect of nutrient ratios on
biosurfactant production by Serratia marcescens using a Box-Behnken design.
Colloids Surf B Biointerfaces 86: 384-389.
37. Pradeep NV, Anupama, Anitha G, Renukamma, Avvaru S, et al. (2012)
Bioremediation of oil contaminated soil using biosurfactant produced by
Pseudomonas aeruginosa. J Res Biol 2: 281-286.
38. Raza ZA, Khan MS, Khalid ZM, Rehman A (2006) Production kinetics and
tensioactive characteristics of biosurfactant from a Pseudomonas aeruginosa
mutant grown on waste frying oils. Biotechnol Lett 28: 1623-1631.
39. Nitschke M, Costa SG, Haddad R, Gonalves LA, Eberlin MN, et al. (2005) Oil
wastes as unconventional substrates for rhamnolipid biosurfactant production
by Pseudomonas aeruginosa LBI. Biotechnol Prog 21: 1562-1566.
40. Tuleva BK, Ivanov GR, Christova NE (2002) Biosurfactant production by a new
Pseudomonas putida strain. Z Naturforsch C 57: 356-360.

18. Abdel-Mawgoud AM, Lpine F, Dziel E (2010) Rhamnolipids: diversity of

structures, microbial origins and roles. Appl Microbiol Biotechnol 86: 13231336.

41. Bonilla M, Olivaro C, Corona M, Vazquez A, Soubes M (2005) Production and

characterization of a new bioemulsifier from Pseudomonas putida ML2. J Appl
Microbiol 98: 456-463.

19. Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, et al. (2002) Complete
genome sequence and comparative analysis of the metabolically versatile
Pseudomonas putida KT2440. Environ Microbiol 4: 799-808.

42. Raza ZA, Khan MS, Khalid ZM (2007a) Evaluation of distant carbon sources
in biosurfactants production by a gamma ray-induced Pseudomonas putida
mutant. Process Biochem 42: 686-692.

20. Saharan B, Rahu R, Sharma D (2011) A review on biosurfactants: fermentation,

current developments and perspectives. Genet EngBiotechnol J 2011: GEBJ29.

43. Martnez-Toledo A, Ros-Leal E, Vzquez-Duhalt R, Gonzlez-Chvez Mdel

C, Esparza-Garca JF, et al. (2006) Role of phenanthrene in rhamnolipid
production by P. putida in different media. Environ Technol 27: 137-142.

21. Malik ZA (2009) Biodegradation of crude oil by soil bacteria. Ph.D dissertation,
Quaid-i-Azam University Islamabad, Pakistan.

44. Desai JD, Banat IM (1997) Microbial production of surfactants and their
commercial potential. Microbiol Mol Biol Rev 61: 47-64.

22. Sakurai S, Manabe Y, Imanaka T, Morikawa M (1993) Method for screening

microorganism capable of producing biosurfactant and measuring activity of
biosurfactant. NIKKO BIO GIKEN KK.

45. Pruthi V, Cameotra SS (2003) Effect of nutrients on optimal production

of biosurfactants by Pseudomonas putidaA Gujarat oil field isolate. J
SurfactDeterg 6: 65-68.

23. Holt JG (1994) Bergey's Manual of Determinative Bacteriology. (9th edn),

Lippincott Williams & Wilkins, Baltimore. ISBN: 0-683-00603-7.

46. Kuiper I, Lagendijk EL, Pickford R, Derrick JP, Lamers GE, et al. (2004)
Characterization of two Pseudomonas putida lipopeptide biosurfactants,
putisolvin I and II, which inhibit biofilm formation and break down existing
biofilms. Mol Microbiol 51: 97-113.

24. Rasheed F, Ahmad T, Ali M, Ali S, Ahmed S, et al. (2011) High frequency of
cagA and vacA s1a/m2 genotype among Helicobacter pylori infected gastric
biopsies of Pakistani children. Malays J Microbiol 7: 167-170.
25. Saitou N, Nei M (1987) The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425.
26. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies
by using the neighbor-joining method. Proc Natl Acad Sci U S A 101: 1103011035.

47. Costa SG, Nitschke M, Haddad R, Eberlin M, Contiero J (2006) Production

of Pseudomonas aeruginosa LBI rhamnolipids following growth on Brazilian
native oils. Process Biochem 41: 483-488.
48. Benincasa M, Accorsini FR (2008) Pseudomonas aeruginosa LBI production
as an integrated process using the wastes from sunflower-oil refining as a
substrate. Bioresour Technol 99: 3843-3849.

27. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary

Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596-1599.

49. Solomon JRD, Legge RL (2008) The effects of culture condition on the
degradation of PCE and production of biosurfactant by Pseudomonas
aeruginosa. Int J IntegBiol 4: 45-49.

28. Danashekar K, Natarajan D, Mathiyazhagan N (2011) Amplification of

biosurfactant producing gene (rhlB) from Pseudomonas aeruginosa isolated
from oil contaminated soil. Int J Pharma Bio Sci 2(1): 497-504.

50. Lawrence JF (1984) Liquid chromatography in environmental analysis. In:

HPLC determination of surfactants and related compounds. PavelJandera
(Eds.), The Humana Press Inc., p. 157, ISBN 0-89603-045-8.

29. Abouseoud M, Maachi R, Amrane A, Boudergua S, Nabi A (2008) Evaluation

of different carbon and nitrogen sources in production of biosurfactants by
Pseudomonas fluorescens. Desalination 223: 143-151.

51. Lotfabad TB, Shourian M, Roostaazad R, Najafabadi AR, Adelzadeh MR,

et al. (2009) An efficient biosurfactant-producing bacterium Pseudomonas
aeruginosa MR01, isolated from oil excavation areas in south of Iran. Colloids
Surf B Biointerfaces 69: 183-193.

30. Rodrigues L, Banat IM, Teixeira J, Oliveira R (2006) Biosurfactants: potential

applications in medicine. J Antimicrob Chemother 57: 609-618.
31. Cooper DG, Goldenberg BG (1987) Surface-active agents from two bacillus
species. Appl Environ Microbiol 53: 224-229.
32. Arutchelvi J, Doble M (2010) Characterization of glycolipid biosurfactant from
Pseudomonas aeruginosa CPCL isolated from petroleum-contaminated soil.
Lett Appl Microbiol 51: 75-82.
33. Arutchelvi J, Doble M (2011) Mannosylerythritol Lipids: Microbial Production
and Their Applications. In: Biosurfactants: From Genes to Applications,
Sobern-Chvez G (Ed), pp. 145-177, Springer, Mnster, Germany.
34. Bharali P, Das S, Konwar BK, Thakur AJ (2011) Crude biosurfactant from
thermophilicAlcaligenesfaecalis: feasibility in petro-spill bioremediation.
IntBiodeterBiodegr 65: 682-690.

J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

52. Sandrin C, Peypoux F, Michel G (1990) Coproduction of surfactin and iturin

A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis.
Biotechnol Appl Biochem 12: 370-375.
53. Raza ZA, Rehman A, Khan MS, Khalid ZM (2007) Improved production of
biosurfactant by a Pseudomonas aeruginosa mutant using vegetable oil
refinery wastes. Biodegradation 18: 115-121.
54. Mukherjee S, Das P, Sivapathasekaran C, Sen R (2009) Antimicrobial
biosurfactants from marine Bacillus circulans: extracellular synthesis and
purification. Lett Appl Microbiol 48: 281-288.
55. Nitschke M, Costa SG (2007) Biosurfactants in food industry. Trends Food
SciTechnol 18: 252-259.
56. Dubeau D, Dziel E, Woods DE, Lpine F (2009) Burkholderia thailandensis

Volume 4 Issue 7 1000204

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013) Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

Page 10 of 10
harbors two identical rhl gene clusters responsible for the biosynthesis of
rhamnolipids. BMC Microbiol 9: 263.

surface optimization of biosurfactant produced by Pseudomonas aeruginosa

MA01 isolated from spoiled apples. Prep Biochem Biotechnol 43: 398-414.

57. Abdel-Mawgoud AM, Lpine F, Dziel E (2010) Rhamnolipids: diversity of

structures, microbial origins and roles. Appl Microbiol Biotechnol 86: 13231336.

59. Qiao N, Shao Z (2010) Isolation and characterization of a novel biosurfactant

produced by hydrocarbon-degrading bacterium Alcanivorax dieselolei B-5. J
Appl Microbiol 108: 1207-1216.

58. Abbasi H, Sharafi H, Alidost L, Bodagh A, Zahiri HS, et al. (2013) Response

Submit your next manuscript and get advantages of OMICS

Group submissions
Unique features:

User friendly/feasible website-translation of your paper to 50 worlds leading languages

Audio Version of published paper
Digital articles to share and explore

Special features:

Citation: Qazi MA, Malik ZA, Qureshi GD, Hameed A, Ahmed S (2013)
Yeast Extract as the Most Preferable Substrate for Optimized Biosurfactant
Production by rhlB Gene Positive Pseudomonas putida SOL-10 Isolate. J
Bioremed Biodeg 4: 204. doi:10.4172/2155-6199.1000204

J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal

250 Open Access Journals

20,000 editorial team
21 days rapid review process
Quality and quick editorial, review and publication processing
Indexing at PubMed (partial), Scopus, EBSCO, Index Copernicus and Google Scholar etc
Sharing Option: Social Networking Enabled
Authors, Reviewers and Editors rewarded with online Scientific Credits
Better discount for your subsequent articles

Submit your manuscript at: http://www.omicsonline.org/submission

Volume 4 Issue 7 1000204