Volume 1 Number 2 (Summer 2009) 54 - 61

Characterization of novel biosurfactant producing strains of Bacillus

spp. isolated from petroleum contaminated soil
Nasr S 1, Soudi M R 1*, Mehrnia M R 2 , Sarrafzadeh M H 2
1
Department of Microbiology, Faculty of Sciences, Alzahra University,Tehran, Iran.
2
Biotechnology and Environment Group, Department of Chemical Engineering, College of Engineering,
Tehran University,Tehran, Iran.

Received: October 2008. Accepted: February 2009.

ABSTRACT

Background and objectives: Biosurfactants are surface active agents with broad range commercial applications in
various industries and have considerable advantages over their chemical counterparts.

Materials and Methods: In this study, bacteria were isolated from contaminated and uncontaminated soil and
selected during preliminary screening using hemolytic activity, oil spreading and oil collapsed techniques. Isolates
with at least more than one positive response to these three methods were subjected to complementary screening by
measuring surface tension reduction as well as emulsification capacity. The criteria for selection of potent isolates were
surface tension reduction below 40 mN/m and emulsification capacity of more than fifty percent.

Results: Using these stepwise screening procedures, two biosurfactant/bioemulsifier producing isolates have been suc-
cessfully selected that were able to reduce surface tension effectively and one of which formed a stable emulsion.

Conclusion: Phylogenic relationships of the two potential candidates were determined comparing the 16Sr DNA gene
sequences, revealing them as two isolates of Bacillus subtilis and Bacillus cereus that can be used in pilot scale for industrial
production of new biosurfactant/bioemulsifier.

Keywords: Bacillus, biosurfactant, emulsification, petroleum, soil.

INTRODUCTION liquids or at solid- liquids interface (Bioemulsifier).

Biosurfactants usually exhibit emulsifying capacity
Surfactants of both biological and chemical
but bioemulsifiers do not necessarily reduce
origin are amphipathic molecules that accumulate
surface tension (4). Many microorganisms produce
at the interfaces and reduce surface and interfacial
extracellular or membrane-associated surface active
tension at the interfaces between liquids, solids,
compounds that play essential roles in the survival
and gases thereby allowing them to mix or disperse
of the producing microorganisms either through
readily as emulsions in water or other liquids (1-3).
facilitating nutrient transport or providing microbe-
Surface active compounds are divided into two
host interaction or playing role as biocides (5, 6).
main types: those that reduce surface tension at the
air-water interface (Biosurfactant) and those that Biosurfactants are categorized mainly by their
reduce the interfacial tension between immiscible chemical composition and microbial origin.
Generally, their structures include a hydrophilic
* Corresponding author: Mohammad Reza Soudi. Ph.D. moiety consisting of amino acids or peptides, mono-,
Address: Department of Microbiology, Faculty of Sciences, Alzahra
di- or polysaccharides and hydrophobic moiety
University, Vanak Ave, Tehran, Iran.
comprising unsaturated or saturated fatty acids.
Tel: +982188044051-9 (2733)
Fax: +982188058912 Accordingly, the major classes of biosurfactants
E-mail: msoudi@alzahra.ac.ir, msoudi@yahoo.com include glycolipids, lipopeptides, lipoprotein,

54
 NOVEL BIOSURFACTANT PRODUCING BACILLI 55

phospholipids, fatty acids, polymeric biosurfactant colonies were isolated and purified by replicating
and particulate biosurfactants (7, 8). This wide range on the same solid medium to obtain pure cultures.
of structural diversity results in broad spectrum of
Preliminary screening of biosurfactant/
potential industrial applications including production
bioemulsifier producing bacteria. Pure isolates
of food, cosmetics, and pharmaceuticals, agriculture,
were cultured in E-medium at 30°C and 200 rpm
mining, enhanced oil recovery, transportation of
for 9 days (14).The broth cultures were centrifuged
crude oil, cleaning oil storage tanks and pipelines
at 22000×g for 45 min (15). The supernatant was
and soil remediation (9-11). Biosurfactant producing
subsequently subjected to the preliminary screening
microorganisms belong to different genera including:
methods using oil spreading and oil collapse methods
Arthrobacter spp., Bacillus spp., Candida spp.,
as below.
Clostridium spp., Corynebacterium spp., Nocardia
spp., Pseudomonas spp., Rhodococcus spp. and more Oil spreading method. Oil spreading technique
other genera have been reviewed (2-12). was carried out according to the method described
previously by Youssef et al. (14) and Plaza et al. (6).
The enormous market demands for surfactants
Briefly, fifty milliliter of distilled water was added
are currently met by numerous synthetic mainly
to the petri dishes followed by addition of 100 µl of
petroleum-based, chemical surfactants. These
crude oil to the surface of water. Then 10 µl of cell
compounds are usually toxic to the environment and
free culture broth was dropped on to the crude oil
non-degradable. Tightening environmental regulations
surface. The diameter of clear zone on the oil surface
and increasing awareness for the need to protect the
was measured and compared to 10 µl of distilled
ecosystem have effectively resulted in an increasing
water as negative control.
interest in biosurfactants as possible alternative to
chemical surfactants (2) . They have advantages over Oil collapse method. A modified oil collapse
their chemical rivals in bioavailability, biodegradability, method was carried out using 96 well microtiter-
activity under extreme condition, lower toxicity, plates containing 100 µl mineral oil which was
ecological acceptability, structural diversity, equilibrated for an hour at room temperature. Ten
productivity on cheap and renewable substrates, microliter of supernatant of culture broth was added
capacity for modification and mass production through to the surface of a well and the picture captured after
biotechnology and genetic engineering (2, 5, 12). 1 minute using 10 × objective lens of microscope.
At present few biosurfactants have been used on Biosurfactant production was considered positive
an industrial scale due to the lack of cost effective when the drop diameter was at least 0.5 mm larger
production processes. Therefore the search for than those produced by distilled water and also by
biosurfactant producing microorganisms that can be culture medium as negative controls (6- 14).
grown economically on industrial scale continues (4). Hemolytic activity. Isolates were screened on
Thus, the main goal of this study was to introduce blood agar plates containing 5% (v/v) sheep blood
novel microorganisms with potential for biosurfactant/ and incubated at 37°C for 48 h. Hemolytic activity
bioemulsifier production. was detected as the presence of a clear zone around
MATERIALS AND METHODS bacterial colonies (6- 14).

Soilsamples.Soil samples included oil contaminated Complementary screening. Isolates which were
soil and an active farm soil. The physical and positive for at least more than one of preliminary
chemical characteristics including temperature, methods were subjected to the complementary
pH, moisture and electrical conductivity were screening to verify their ability to produce
determined using a portable analytical instrument biosurfactant / bioemulsifier.
(HACH Company) (13). Direct isolation of the Surface tension measurements. Surface tension
microorganisms was carried out using serial dilution reduction was measured using Krüss Hamburg
on (up to 10-7) of soil samples in 0.85 % sterile saline. Nr2215 Tensiometer and by submerging the platinum
Using the spreading method on nutrient agar plates, ring in the cell free culture broth and recording the
total bacterial count was measured after incubation force required to pull it through the air–liquid interface
at 30°C for 24 hours. Morphologically distinct (16). The results were compared to distilled water
56 SOUDI ET AL . IRAN. J. MICROBIOL. 1 (2 ) : 54 - 61

Table 1. Microbial and physiochemical characteristics of soil samples.

Total count Moisture Electrical Temperature

Soil samples pH conductivity
)CFU g-1) (%) (mS) (°C)

Oil contaminated 104×3.1 7.35 9.25 0.60 28

Newly harvested farmland 105×1.1 7.67 16.27 0.29 29

and medium composition (as negative control) and response to hemolytic activity; 32 positive results were
Tween 20 (as a positive control). The criterion used obtained for oil spreading and 16 positive responses
for selecting biosurfactant-producing agents was the were obtained when oil collapse method was used; and
ability of the isolates to reduce surface tension below in total 14 isolates or 13.72% putative biosurfactant
40 mN/m (4- 17). producer were obtained with positive responses to more
than one of the above preliminary screening methods.
Emulsification capacity (E24). Emulsification
activity test was performed according to Krepsky et al. The putative biosurfactant producing isolates were
(18). The results were compared with water and pure screened in complementary stage using two methods.
E-medium as negatives controls and 1% solution of SDS Results from these experiments indicate that the
(a common chemical bioemulsifier) as positive control. surface tension varies from 23.3 mN/m to 57.6 mN/m
A criterion cited for emulsion stabilizing capacity is the and the emulsion activity ranging from 0 to 100% for
ability to maintain at least 50% of original emulsion different hydrocarbon sources (Table 2).
volume 24 hours after formation (4). Two-way ANOVA It is interesting that a given isolate showed
analysis (p <0.05) was performed on emulsion activity different and distinct emulsifying response
data to determine significant differences in emulsion when exposed to each of hydrocarbon sources.
activity in the presence of different hydrocarbon sources Following complementary screening, five potential
while the difference between isolates were a blocked biosurfactant producing strains were isolated,
factor in this analysis. Statistical analysis was performed of which 2 were gram positive and were further
using Minitab (Version 15) software. characterized (Table 3). Sequencing of 16S rDNA
Biochemical and genetic analysis of superior produced by polymerase chain reaction of bacterial
isolates. Partial sequencing of 16S rRNA of two superior DNA using universal primers revealed that superior
isolates was carried out in Pasteur Institute Laboratories, gram positive biosurfactant-producing isolates were
France. Morphological and biochemical identification closely related to two Bacillus spp., including Bacillus
tests were also performed following directions in subtilis, and Bacillus cereus (16) . Determination of
Bergey’s Manual of Systematic Bacteriology (19). the morphological and biochemical traits of these
isolates confirmed the results of phylogenetic studies.
RESULTS
DISCUSSION
Two soil samples were screened for biosurfactant-
producing microorganisms. To depict the environment Biosurfactants are amphiphilic molecules with
from which the bacteria were isolated, physical great diversity, environmental acceptability and broad
and chemical characteristics of soil samples were spectrum of functions and industrial applications which
determined (Table 1). make them interesting bio-products. Soil is a known
habitat and source of versatile microorganisms and
The grown population of the oil-contaminated since the microorganisms capable of emulsifying
and farmland soil were 3.1 ×104 CFU/g and 1.1×105 and solubilizing hydrophobic agents have an
CFU/g, respectively. The initial isolation yielded apparent advantage over their competitors,
a total of 102 pure isolates which were grown on sampling of this nature provides a source rich
nutrient agar. Among them, 16 isolates gave positive in microorganisms with desired characteristics.
 NOVEL BIOSURFACTANT PRODUCING BACILLI 57

Table 2. Detection of biosurfactant producing isolates by preliminary and complementary screening methods.

Preliminary methods Complementary methods

E24 E24 E24

Surface
Hemolytic Oil Oil In presence In presence
Isolate tension In presence of
activity collapse spreading of of
(mN/m) Crud oil
Kerosene n-Hexadecane

SN1 - + ++ 23.3±0.05 53.59±0.56 63.18±2.06 100±0

SN12 + - + 26.2±0.05 2.17±0.23 2.22±0.25 67.53±3.98

Cw20 - + + 44.6±0.82 52.59±3.42 57.93±3.93 100±0

CY7 - + + 51.1±1.23 0±0 0±0 72.85±1.01

FY1 - + + 51.6±1.69 2.35±0.30 0.81±0.58 94.04±4.86

Fy4 + + + 43.1±0.80 2.90±0.46 2.61±0.37 82.9±6.48

Fw1 ++ + - 57.6±2.29 2.27±1.23 3.32±0.91 100±0

FY13 + + + 37.8±0 0±0 1.47±1.18 72.62±5.24

Fw13 + + + 52.0±2.35 0±0 0±0 67.33±6.22

Cw29 - + + 48.4±3.48 0±0 0±0 61.73±0.54

NP1 - + +++ 57.5±1.01 3.12±1.62 2.13±0.21 80.6±8.11

FY11 + - ++ 31.9±0.04 0±0 0±0 69.98±9.06

Cw26 + + - 53.3±2.43 58.73±1.62 65.93±0.89 61.51±3.20

51.54±0.83
To1
+ - + 35.08±0.91 0±0 0±0
Controls
Culture
- - - 65.8±1.32 0±0 0±0 77.23±0
medium

water - - - 70.1±0.07 3.91±1.90 2.21±0.27 100±0

Tween20 - - - 35.2±0 - - -

1% SDS - - - - 68.50±1.78 65.87±3.24 66.50±0.62

Sea water
- - - - 0±0 0±0 100±0
4% NaCl
58 SOUDI ET AL . IRAN. J. MICROBIOL. 1 (2 ) : 54 - 61

Table 3. Biochemical characterization of superior biosurfactant producing isolates

Characteristics SN1 SN12 Characteristics SN1 SN12

Cell morphology Bacilli Bacilli Utilization of

Gram reaction + + Citrate + -

Spore forming + + Egg-yolk lecithinase - +

Catalase + + Formation of Indole - -

Growth at pH 6.8, nutrient

Oxidase - - + +
broth

Growth at pH 5.7, nutrient

Motility + - + +
broth

Voges-Proskaur test + - Growth in NaCl

Acid from 2% + +

D-Glucose + + 5% + +

L-Arabinose + - 7% + +

D-Xylose + - 10% + -

D- Manitol + - Growth at

Gas from glucose - - 5°C - -

Hydrolysis of 10°C - -

Casein + + 30°C + +

Gelatin + - 40°C + -

Starch + + 50°C + -

Esculin + + 55°C - -

Urea - - 65°C - -

Tween80 - + Nitrate reduction to Nitrite + +

- : negative results , +: positive results
 NOVEL BIOSURFACTANT PRODUCING BACILLI 59

Pure isolates were cultured in production medium and (22, 23). As observed in Table 2, some of these
following centrifugation, supernatants were used for results are quite impressive compared to the most
preliminary screening since excretion type bacteria potent biosurfactant producing microorganism
that release biosurfactants to the culture medium are among Bacillus spp. known to reduce the surface
more interesting from the industrial point of view tension of water to 27 mN/m (12, 24). The second
than bacteria with adherent biosurfactants due to the factor that has been studied in complementary
simplicity and economical aspects of the recovery stage was emulsion activity using three different
process (4- 20). hydrocarbons including crude oil, kerosene and
n-hexadecane. About 13% positive isolates in
The primary screening of biosurfactant
preliminary stage showed emulsion activity. It is
producing bacteria was carried out using hemolytic
interesting that a given isolate showed different
activity, oil collapse and oil spreading techniques.
and distinct responses when it was exposed to each
Selection of these methods was due to their strong
of hydrocarbon sources suggesting appropriate
advantages including simplicity, low cost, quick
selection of hydrocarbon source is necessary
implementation and use of relatively common
for determination of emulsification capacity.
equipment that is accessible in almost every
Amiriyan et al. 2004 suggested that emulsifier
microbiological laboratory; however, as expected,
activity depends on the affinity of bioemulsifier
these methods are not perfect or flawless. As
for hydrocarbon substrates which involves a direct
previously mentioned by Youssef et al. (6) and
interaction with hydrocarbon itself rather than
Plaza et al. (14) in the hemolytic method, there are
an affect on surface tension of the medium (25).
many bio-products that can cause red blood cell
We used water and artificial sea water (4%NaCl)
lysis which do not necessarily have to be surface
as negative controls which suggest that crude oil
actives molecules.
may not be an appropriate hydrocarbon source
The drop collapse method depends on the for the study of emulsion activity and statistical
principle that a drop of liquid containing a analysis has verified a significant difference
biosurfactant collapses and spreads over the oily between the response of a given isolate to the
surface. There is a direct relationship between hydrocarbon source (Table 4).
the diameter of the sample and concentration
In the present study, two superior isolates SN1
of the biosurfactant and in contrast, the drop
(Bacillus subtilis), SN12 (Bacillus cereus) with
lacking biosurfactant remains beaded due
biosurfactant-producing ability and the former
to the hydrophobicity of the oil surface that
with emulsion capacity were isolated from
cause aggregation of droplets (9- 21) but this
petroleum-contaminated soil. Their ability to reduce
method is not sensitive in detecting low levels
surface tension and emulsion capacity makes
of biosurfactant production. The results of our
them new potential candidates for biosurfactant
experiments indicate, 15% of total isolates were
and bioemulsion production. Further studies have
positive for hemolytic activity or oil collapse
been initiated to identify their properties and
and 31% were considered positive based on
consequently determine the potential of their
oil spreading and since these methods have different industrial applications in particular
shown differences, the isolates with more enhanced oil recovery application.
than one positive response were exposed to
complementary screening. The latter screening ACKNOWLEDGEMENTS
stage included surface tension and emulsion
This work was financially supported by the
activity measurements.
National Iranian Oil Company (NIOC). The authors
Forty percent of positive isolates in preliminary would like to thank colleagues at the National
screening have shown reduction of surface tension Laboratory of Industrial Microbiology at Alzahra
below the standard index (40 mN/m). The criterion University and the Chemistry-Physics laboratory at
used for selecting biosurfactant production is the University of Tehran. We also extend our gratitude
ability to reduce surface tension below 40 mN/m (4 to Anne Le Flèche in France Pasture Institute for 16S
- 17) while some authors have suggested 30 mN/m rDNA sequencing.
60 SOUDI ET AL . IRAN. J. MICROBIOL. 1 (2 ) : 54 - 61

Table 4. Two-way ANOVA analysis for determining significant difference of emulsion activity in presence of different
hydrocarbon sources using Minitab software.

Source DF SS MS F P

hydrocarbon 2 44717.1 22358.6 108.01 0.000

block 14 13406.3 957.6 4.63 0.000

Error 28 5796.2 207.0

Total 44 63919.6

S = 14.39 R-Sq = 90.93% R-Sq(adj) = 85.75%

Individual 95% CIs For Mean Based on

Level N Mean StDev

crude oil 14 79.59 15.18

n- hexadecane 14 13.46 25.37

kerosene 14 12.01 22.30

Pooled StDev

+---------+---------+---------+---------
(---- * ---)
(--- * ----)
(---- * ---)
+---------+---------+---------+---------
0 25 50 75

Pooled StDev = 21.38

DF:Degree of freedom, SS:Sum of square, MS:Mean of square, F:Fisher test.

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