Optimization of Biosurfactant Production
by Bacillus licheniformis DW3
Muneer Ahmed Qazi, Maria Abid, Abdul Hameed and Safia Ahmed
Department of Microbiology Quaid i Azam University,
Islamabad.
Title:
Author(s):
Affiliation:
Table of Contents
Materials and Methods
Screening
Results
Screening
Merits
BiodegradabilityGenerally low toxicityBiocompatibility and digestibilityAvailability of raw materials Acceptable production economicsUse in environmental controlSpecificity Vast application fields
Challenges
Expensiveness at large scaleHigh grade purityLow productivityUse of expensive mediaPoor understanding of synthesis
regulationFoam formation
Two classification systems:
On the basis of Molecular mass
• Low-molecular-mass molecules
• High-molecular-mass molecules
On the basis of Polar nature
• Anionic• Cationic• Neutral• Amphoteric
Biosurfactants are biological surface active agents that are:
Amphiphilic
Biodegradable
Less toxic
Environmentally compatible
Highly selective
Specifically active
Enhanced Oil Recovery (EOR) Bioremediation and
Biodegradation of hydrocarbons
Pharmaceuticals Cosmetics Food industry Textile industry Detergents and cleaners Herbicide and Pesticide
formulations Leather and Paper industries Agriculture Bioleaching of Metals Immunological molecules Biomedical field
Introduction
Reduce:
• Surface tension• Critical Micelle Concentration
(CMC)
• Interfacial tensions
Improve:
• Bioavailability of Hydrocarbons
Form:
• Conditioning film at interface
Remove:
• Lipopolysaccharide layer of microbes
To screen bacteria for biosurfactant production
To maximize biosurfactant’s yield from B. licheniformis DW3 by optimizing different cultural and environmental conditions, such as: Inoculum size Temperature pH Carbon sources Carbon source concentration Nitrogen source Agitation speed Oil as additional Carbon Source
Objectives of the study
Study Plan
Materials and Methods
Mat
eria
ls &
Met
hods
Methodology
Screening
Production &
Optimization
Microorganism: Best Bioemulsifier producer strain was further studied for Production and Optimization experiments.
Inoculum Preparation: A 5% of seed culture of the bacterial strain grown in nutrient broth at 30 ºC, 150rpm, for 18-24hours was used as inoculum.
Production Medium: The Mineral Salts Medium (MSM) in addition with Carbon and Nitrogen sources separately sterilized was used as production medium.
Optimization of culture conditions was carried out.
• Microorganism Bacillus endophyticus MD1, Bacillus
subtilis SNW3, Bacillus licheniformis DW3, Psychrobacter sp. DW6, Pseudomonas putida SOL-10 and Bacillus sp. SS1
• Primary ScreeningOil Spread Method (JP05211892)Oil Displacement Area (ODA)Luria Bertani (LB) Agar
• Secondary ScreeningEmulsification Activity (E24) %Drop-Collapse TestMineral Salts Medium (MSM)
Media and their composition
Luria Bertani (LB) Agar
Tryptone 1%Sodium chloride 0.5%Yeast extract 0.5%Agar 1.5% pH 7.0±0.2
Mineral Salts Medium (MSM) (g/L) Na2HPO4 2.2KH2PO4 1.4MgSO4.7H2O 0.6
FeSO4.7H2O 0.01NaCl 0.05CaCl2 0.02Yeast Extract 0.02
and 0.1ml of trace element solution containing (g/L):
ZnSO4.7H2O 2.32MnSO4.4H2O 1.78H3BO3 0.56CuSO4.5H2O 1.0
NH4MoO4.2H2O 0.39KI 0.66EDTA 1.0
pH 7.0±0.2
+ 1.5-2 % carbon and 0.1 % nitrogen sourcesseparately sterilized.
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Optimization parameters for Biosurfactant Production
Inoculum's size (1, 2, 3, 4, 5, 7, and 10%).
Temperature (25, 30,37,45 and 50°C).
Agitation speed (0, 100, 150 and 200rpm).
pH (3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5 and 10).
Mat
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Continued…
Carbon sources Peptone, malt extract, corn oil, glucose, yeast extract,
olive oil, used oil, soyabean oil
Carbon source concentration 0.5, 1, 1.5 and 2%
Nitrogen sources NaNO3, NaNO2, NH4NO3, and Urea
Oil as additional Carbon Source 0.5,1% soyabean and 0.5,1% used oil
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Analytical Methods
Oil Spread Method (JP05211892)
1. A few drops of crude oil were dropped onto the solid surface of LB agar and uniformly spread and left for 24h.
2. After 24h the plates were centrally inoculated with cultures to be screened, and incubated at 37 ºC for 24h.
3. Halos of oil repellence were observed and halo size was then measured.
4. Halo size was measured in cm.
Oil Displacement Activity (ODA) test
(Rodrigues et al., 2006)
1. The 50ml of distilled water was added
to a large Petri dish (15 cm diameter).
2. 20μl of crude oil is then added to the
surface of water.
3. 10μl of culture supernatant broth is
then poured in center of the oil film.
4. Zone of displacement is visualized
and measured.
5. ODA = 22/7 (radius)2 cm2
Emulsification Activity (E24) %
(Techaoei et al., 2007)
1. Equal volumes of kerosene and cell-free supernatant in test tube were vortexed at high speed for 2 min and allowed to stand for 24h.
2. The E24 index is given as percentage of the height of emulsified layer (cm) divided by the total height of the liquid column (cm).
Drop Collapse Method (Krepsky et al., 2007)
1. The drop of cell culture or culture
supernatant is dropped onto a
hydrophobic oil coated surface.
2. The size and shape of the drop is
anlyzed for biosurfactant production.
3. If the drop contains surfactant it is
collapsed and the size of drop is
increased.
4. Size of drop is measured in mm/μm.
RESULTS
Primary Screening by oil spread technique
S. No. Bacterial sp. Halo size (cm)
1 Bacillus subtilis SNW3 2.5 ± 0.2
2 Pseudomonas putida SOL-10 5.5 ± 0.2
3 Bacillus endophyticus MD1 2.0 ± 0.2
4 Psychrobacter sp. DW6 1.5 ± 0.2
5 Bacillus licheniformis DW3 4.5 ± 0.2
6 Bacillus sp. SS1 0.5 ± 0.2
Res
ult
s
Secondary Screening by Emulsification Index (E24) %
B. subtilis SNW3 P. putida SOL-10 B. licheniformis DW30
10
20
30
40
50
60
70
Em
uls
ific
ati
on
In
dex
(E
24
) %
Bacterial strains
24 hrs 48 hrs 72 hrs 96 hrs 120 hrs
Res
ult
s
Res
ult
s
Effect of Inoculum Size
0
10
20
30
40
50
60
0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96
1 2 3 4 5 7 10
Time (hours)
E24
(%
)
0
1
2
3
4
5
6
7
8
Gro
wth
O.D
(60
0nm
)
E24 (%) OD
Effect of Temperature
0
10
20
30
40
50
60
70
0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96
25°C 30°C 37°c 45° 50°C
Time(hours)
E24
(%
)
Res
ult
s
Effect of Agitation
0
10
20
30
40
50
60
0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96
No Aggitation 100 rpm 150rpm 200rpm
Time (hours)
E24
(%)
0123456789
Gro
wth
O.D
(600
nm)
E24 (%) OD
Res
ult
s
Effect of pH
0
10
20
30
40
50
60
70
0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96
3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
Time (hours)
E24 (
%)
0
1
2
3
4
5
6
Grow
th O.
D (60
0nm)
E24 (%) O.D
Res
ult
s
Effect of Carbon Sources
0
10
20
30
40
50
60
70
0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96
peptone malt extract corn oil glucose yeastextract
olive oil used oil soyabeanoil
Time(hours)
E24(
%)
00.511.522.533.544.55
Gro
wth
OD(
600n
m)
E24 OD
Res
ult
s
Effect of Carbon Source Concentration
0
10
20
30
40
50
60
70
0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96
0.50% 1% 1.50% 2% 2.50%
Time(hours)
E24
(%)
0
1
2
3
4
5
6
7
Gro
wth
O.D
(60
0nm
)
E24 (%) OD
Res
ult
s
Effect of Nitrogen Source
05
1015202530354045
0 24
48
72
96 0 24
48
72
96 0 24
48
72
96 0 24
48
72
96
Sodium Nitrate Sodium Nitrite AmmoniumNitrate
Urea
Time(hours)
E24(%
)
012345678910
Gro
wth
.OD
(600n
m)
E24 (%) OD
Res
ult
s
Effect of oil as an additional carbon source
05
101520253035404550
0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96
0.5% SoyabeanOil
1% SoyabeanOil
0.5% Used Oil 1% Used Oil
Time(hours)
E24
(%)
0
2
4
6
8
10
12
Gro
wth
OD
(600
nm)
E24(%) OD
Res
ult
s
Production of Biosurfactants under optimized conditions
0
10
20
30
40
50
60
70
0 24 48 72 96
Time(hours)
E24
(%)
0
1
2
3
4
5
Gro
wth
OD
(600
nm
)
E24(%) OD
Res
ult
s
Conclusion B. licheniformis DW3 is a potent biosurfactant producer
Optimum parameters for maximum production of biosurfactant were found as:
Inoculum's size 5%
Temperature 30ºC
pH 8.0
Continued.....
Agitation 150rpm
2% yeast extract as carbon source
Highest emulsification index (E24%=62.43) was attained at optimized conditions
Although the experiments with different nitrogen sources and oil as an additional carbon source revealed some negative effects on biosurfactant production, they had positively supported heavy growth.
ThanksAny question??