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comparative study for biosurfactant production by using Bacillus subtilis and Pseudomonas aeruginosa

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Botany Research International 2 (4): 284-287, 2009
ISSN 2221-3635
© IDOSI Publications, 2009
Correspoding Author: T. Priya Department of Microbiology, Annamalai University,
Annamalai Nagar-608 002 Tamil Nadu, India
284
Comparative Study for Biosurfactant Production by
Using Bacillus subtilis and Pseudomonas aeruginosa
T. Priya and G. Usharani
Department of Microbiology, Annamalai University, Annamalai Nagar-608 002 Tamil Nadu, India
Abstract: Biosurfactants are amphiphilic compounds produced by various bacteria and fungi which reduce
surface and interfacial tension. Oil contaminated soil were collected from four different automobile shop in
Mayiladuthurai, Tamilnadu, India. Bacillus subtilis and Pseudomonas aeruginosa were isolated and identified
from these four samples and these were screened for biosurfactants production using vegetable oil, kerosene,
petrol and diesel by oil spreading technique and emulsification stability test and the best one of Bacillus
subtilis and Pseudomonas aeruginosa were used for biosurfactants production using vegetable oil, kerosene,
petrol and diesel as source. The isolated biosurfactants were identified using TLC method. Among four different
oils; diesel is the best source for the production of biosurfatant in both Bacillus subtilis and Pseudomonas
aeruginosa and Pseudomonas aeruginosa have higher activity than Bacillus subtilis.
Key words: Biosurfactant Bacillus subtilis Pseudomonas aeruginosa and TLC
INTRODUCTION biosurfactants. Rhamnolipid is one of the type of
Biosurfactants are amphiphilic compounds are linked to one or two molecules of -hydroxydecanoic
produced on living surfaces, mostly microbial cell acid while the OH group of one of the acids is involved
surfaces or excreted extracellularly and contain in glycosidic linkage with the reducing end of the
hydrophobic and hydrophilic moieties that reduce rhamnose disaccharide, the OH group of the second
surface tension and interfacial tension between individual acid is involved in ester formation [1]. Rhamolipid is
molecules at the surface and interface respectively [1]. produced by Pseudomonas aeruginosa, a gram-negative,
Biosurfactants are produced by different microorganisms motile, non-spore forming bacteria. Surfactin is a cyclic
such as bacteria, fungi and yeast. Biosurfactants have lipopeptide commonly used as an antibiotic [4].
several advantages, including low toxicity, high Surfactin’s structure consists of a peptide loop of seven
biodegradability, low irritancy and compatibility with aminoacids (L-asparagine, glycine, two L-leucine, L-valine
human skin [2]. Biosurfactants have gained importance and two D-Leucines) and anhydrophobic fatty acid
in the fields of enhanced oil recovery, environment chain thirteen to fifteen carbon long [5]. In the various
bioremediation, food processing and pharmaceuticals [3]. course of studies of its properties, surface was found
Biosurfactant producing microorganisms were to exhibit effective characteristics like antibacterial,
naturally present in the oil contaminated soil. Oil antiviral, antifungal, antimycoplasma and hemolytic
contaminated environment contain large amount of activities [6]. Surfactin is produced by Bacillus subtilis,
hydrocarbons. Hydrocarbons are composed of a gram-positive, motile, spore forming bacteria.
complex chemical structure i.e., aliphatic and aromatic The present study focused on the biosurfactant
hydrocarbons. Microorganisms exhibit emulsifying production by Bacillus subtilis and Pseudomonas
activity by producing biosurfactants and utilize the aeruginosa isolated from oil contaminated soil using
hydrocarbons as substrate often mineralizing them or four different oils as substrate and the biosurfactant
converting them into harmless products. production was screened by oil spreading technique and
Among the different classes of biosurfactants, emulsification stability test. Isolated biosurfactants were
rhamnolipid and surfactin are best studied identified by using TLC method.
glycolipids, in which one or two molecules of rhamnose
Bot. Res. Intl., 2 (4): 284-287, 2009
285
MATERIALS AND METHODS lipopeptide biosurfactant as red spots and anthrone
Isolation and Identification of Bacillus Subtilis and 95 mL ethanol)-to detect glycolipid biosurfactant as
Pseudomonas Aeruginosa: Oil contaminated soils were
collected from four automobile shops in Mayiladuthurai.
Serial dilution technique using nutrient agar was
performed to isolate bacteria from soil. Staining
techniques and biochemical tests [7] were performed to
identify Bacillus subtilis and Pseudomonas aeruginosa.
Screening for Biosurfactant Activity: Biosurfactant different oil contaminated soil. Bacillus subtilis is a
activity of isolated bacteria was detected by using oil gram positive, motile, endospore forming bacteria and
spreading technique and emulsification stability test in it hydrolyses starch, casein and gelatin (Table1).
four different oils namely vegetable oil, Kerosene, petrol, Pseudomonas aeruginosa is a gram negative, rod
diesel. shaped, motile bacteria and its biochemical characters
Oil Spreading Technique: The 50 ml of distilled water was
added to a large Petri dish (15 cm diameter) followed by Screening for Biosurfactant Activity
the addition of 20µl of oil to the surface of water, 10µl of Oil Spreading Technique: In oil spreading technique, all
supernatant of culture broth [8]. four isolates of Bacillus subtilis (BS1, BS2, BS3 and BS4)
Emulsification Stability (E24) Test: E24 of culture produces biosurfactants activity in vegetable oil,
samples was determined by adding 2 ml of oil to the same kerosene, petrol and diesel. Among four Bacillus subtilis
amount of culture, mixing with a vortex for 2 min and isolates BS3 produces the higher zone formation of 6mm,
leaving to stand for 24 hours. The E24 index is given as 17 mm, 16mm and 20 mm in vegetable oil, kerosene, petrol
percentage of height of emulsified layer (mm) divided by and diesel respectively. Similarly PS3 produces higher
total height of the liquid column (mm) [9]. zone formation of 8mm, 15mm, 18mm and 22mm in
Optimization of Growth: Bacterial growth was optimized Both BS3 and PS3 shows higher biosurfactant activity in
using different parameters like pH, temperature, potassium diesel and PS3 have more activity than BS3 (Table 3).
di-hydrogen phosphate and Magnesium sulphate.
Biosurfactant Production: Bacteria were inoculated on
the mineral salt agar medium [10]. Colonies grown on this
media were inoculated in mineral broth which contain 2%
of oil (vegetable oil, kerosene, petrol, diesel ) and it was
incubated in an in an optimized condition for 24 to 48
hours in a shaker operating at 120 rpm / min.
biosurfactants was isolated using acid precipitation
method at pH 2 using HCl [11].
Analytical Method
Thin Layer Chromatography: Preliminary characterization
of the biosurfactant was done by TLC method. A portion
of the crude biosurfactant was separated on a silica gel
plate using CHCl :CH OH:H O (70:10:0.5, v/v/v) as
33 2
developing solvent system with different color
developing reagents. Ninhydrin reagent (0.5 g ninhydrin
in 100 mL anhydrous acetone) was used to detect
reagent (1 g anthrone in 5 mL sulfuric acid mixed with
yellow spots [12].
RESULT AND DISCUSSION
Isolation and Identification of Bacillus Subtilis and
Pseudomonas Aeruginosa:Bacillus subtilis and
Pseudomonas aeruginosa were isolated from four
were shown in Table 2.
and Pseudomonas aeruginosa (PS1, PS2, PS3 and PS4)
vegetable oil, kerosene, petrol and diesel respectively.
Table 1: Characteristics of Bacillus subtilis
S. No Test Result
1 Gram Staining Grampositive rod
2 Endospore staining Positive
3 Starch Hydrolysis Positive
4 Casein Hydrolysis Positive
5 Gelatin Hydrolysis positive
Table 2: Characteristics of Pseudomonas aeruginosa
S. No Test Result
1 Gram staining Negative
2 Indole Negative
3 Methyl red Negative
4 Voges proskauer Negative
5 Citrate Positive
6 TSI K/K
7 Glucose Positive
8 Maltose Positive
9 Lactose Negative
10 Sucrose Negative
Optimization of pH
0
0.2
0.4
0.6
0.8
1
1.2
6 6.5 7 7.5 8 8.5
pH Val ue
OD value
BS
PS
Optimization of Temperature
0
0.2
0.4
0.6
0.8
1
1.2
1.4
35 36 37 38 39 40
Temperature ( °C)
OD Value
BS
PS
Optimization of Potassium dihydrogen
Phosphate concentration
0
0.2
0.4
0.6
0.8
1
1.2
0.1 0.2 0.3 0.4 0.5 0.6
Potassium dihydrogen phosphate
concentration (% )
OD Value
BS
PS
Bot. Res. Intl., 2 (4): 284-287, 2009
286
Table 3: Oil Spreading Technique for Bacillus subtils (BS) and
Pseudomonas aeruginosa (PS)
E24 Value (%)
-------------------------------------------------------------------------------
Sample Vegetable oil Kerosene Petrol Diesel
BS PS BS PS BS PS BS PS
1 34 40 43 42 45 48 50 56
2 32 41 43 45 48 52 51 59
3 45 48 52 55 55 58 60 68
4 40 42 49 43 50 50 53 56
Table 4: Emulsification stability test for Bacillus subtils (BS) and
Pseudomonas aeruginosa (PS)
Zone Formation (mm)
----------------------------------------------------------------------------
Sample Vegetableoil Kerosene Petrol Diesel
BS PS BS PS BS PS BS PS
1 5 4 12 11 14 15 15 17
2 4 5 11 13 13 16 16 19
3 6 8 17 15 16 18 20 22
4 5 4 12 14 14 17 17 18
Table 5: Analysis of biosurfactants using Thin Layer Chromatography
Rf Value
Carbon source BS PS
Vegetable Oil 0.48 0.64
Kerosene 0.51 0.63
Petrol 0.52 0.68
Diesel 0.58 0.72
BS-Bacillus subtilis
PS-Pseudomonas aeruginosa
Emulsification Stability (E24) Test: All the four isolates
of Bacillus subtilis and Pseudomonas aeruginosa have
the ability of emulsifying oils. The highest E24 value was
observed in BS3 are 45, 52, 55 and 60 mm in vegetable oil,
kerosene, petrol and diesel respectively. Similarly the
highest E24 value was observed in PS3 are 48, 55, 58 and
68 in vegetable oil, kerosene, petrol and diesel
respectively. Among the four oil the higher E24 value was
observed in diesel and PS3 shows the better E24 value
than the BS3 (Table 4).
As BS3 and PS3 shown higher surfactant activity,
they have taken for Biosurfactant production using four
different oils (Table 5).
Process Optimization for Biosurfactant Production
Optimization of pH: The pH ranges from 6, 6.5, 7, 7.5, 8
and 8.5 were used for the optimization of pH for the
growth of BS3 and PS3. Both the organism have higher
growth rate at pH 7 and PS3 shows more growth than BS3
(Fig. 1).
Fig. 1: BS-Bacillus subtilis
PS-Pseudomonas aeruginosa
Fig. 2: BS-Bacillus subtilis
PS-Pseudomonas aeruginosa
Fig 3: BS-Bacillus subtilis
PS-Pseudomonas aeruginosa
Optimization of Temperature: The temperature
ranges from 35°C to 40°C for the optimization.
At 37°C BS3 and PS3 shows higher growth rate
and the PS3 shows better growth rate than BS3 at 37°C
(Fig. 2).
Optimization of Potassium Di-hydrogen Phosphate:
Potassium di-hydrogen phosphate is used in the
concentration of 0.15% to 0.6% is used for
optimization. The maximum growth was observed at
0.4% concentration in both BS3 and PS3. PS3 shows
better growth than BS3 (Fig. 3).
Optimization of Magnesium sulphate
concentration
0
0.2
0.4
0.6
0.8
1
1.2
0.1 0 .2 0.3 0.4 0.5 0.6
Magnesium sulphate
(%)
OD Val ue
BS
PS
Bot. Res. Intl., 2 (4): 284-287, 2009
287
Fig. 4: BS-Bacillus subtilis 6. Pooja Singh, and S.S. Cameotra 2004. Potential
PS-Pseudomonas aeruginosa applications of microbial surfactants in biomedical
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acid precipitation method at pH2 was identified by Lactococcus lactis 53, Colloids and Surfaces B:
TLC method. Surfactin from Bacillus subtilis was Biointerfaces., 49: 79-86.
identified by red color spot where as rhamnolipid from 9. Sarubbo, L.A., 2006. Production and Stability
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Biosurfactants or microbial surfactants are surface-active biomolecules that are produced by a variety of microorganisms. Biosurfactants have gained importance in the fields of enhanced oil recovery, environmental bioremediation, food processing and pharmaceuticals owing to their unique properties such as higher biodegradability and lower toxicity. Interest in the production of biosurfactants has steadily increased during the past decade. However, large-scale production of these molecules has not been realized because of low yields in production processes and high recovery and purification costs. This article describes some practical approaches that have been adopted to make the biosurfactant production process economically attractive. These include the use of cheaper raw materials, optimized and efficient bioprocesses and overproducing mutant and recombinant strains for obtaining maximum productivity. Here, we discuss the role and applications of biosurfactants focusing mainly on medicinal and therapeutic perspectives. With these specialized and cost-effective applications in biomedicine, we can look forward to biosurfactants as the molecules of the future.
enhanced oil recovery. Biochemical Engineering, J., Microbial production of biosurfactants and their 42: 172-179. importance
  • N G K Karanth
  • P G Deo
  • N K Veenanadig
Karanth, N.G.K., P.G. Deo and N.K. Veenanadig, 1999. enhanced oil recovery. Biochemical Engineering, J., Microbial production of biosurfactants and their 42: 172-179. importance. Curr. Sci., 77: 116-123.
Characteristics of applications of biosurfactants as biological and biosurfactant produced by Pseudomonas aeruginosa immunological molecules
  • H Yin
  • J Qiang
  • Y Jia
  • J Ye
  • H Peng
  • H Qin
Yin H., J. Qiang, Y. Jia, J. Ye, H. Peng, H. Qin, 2. Cameotra, S.S. and R.S. Makkar, 2004. Recent N. Zhang and B. He, 2008. Characteristics of applications of biosurfactants as biological and biosurfactant produced by Pseudomonas aeruginosa immunological molecules, Curr. Opin. Microbiol, S6 isolated from oil-containing wastewater. Process 7: 262-266. Biochemistry., 44: 302-308.
Isolation and comparison of Pseudomonas aeruginosa SP4 for microbial surfactant
R. 2006. Oliveira Physicochemical and Functional S. Chavadej, 2008. Isolation and comparison of Pseudomonas aeruginosa SP4 for microbial surfactant