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

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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).

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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??