Physico-Chemical Characterization of a Biosurfactant Produced by

Pseudomonas Fluorescens

Surfactants
Surfactants are amphipathic molecules that reduce the surface tension between phases of differing polarity, such as oil and water.
Surfactants are used as emulsifiyng , dispersing, solubilizing, foaming agents. Nowadays surfactants are one of the most important substances for many fields of industry pharmacy, food industry, design of washing agents, petroleum industry, agriculture, environmental protection and remediation An excessive use of chemical surfactants leads to technogenic load on environment, flora and fauna, affects on food products

Biosurfactants

Naturally occurring surface-active compounds derived from microorganisms

Advantages over chemical surfactants

High efficiency in broad range of pH and salt concentrations, thermostability low toxicity, inherent good biodegradability and ecological acceptability.

Types
Most microbial surfactants are complex molecules, comprising different structures that include peptides, glycolipids, glycopeptides, fatty acids and phospholipids.

Rhamnolipid Rhamnolipid

Surfactin lipopeptide

Problems with biosurfactant production

Limited yield (productivity) Separation problems Batch process Substrate inhibtion could occur

SOLUTION?
Various techniques are used for cell or biomass immobilized, such as flocculation, adsorption on surfaces, covalent bonding to carriers, cross linking of cells, encapsulation in a polymer gel, and entrapment in a polymeric matrix.
IMMOBILIZATION METHODS ADSORPTION ENCAPSULATION ADHESION ENTRAPMENT IN GEL OR POLYMERMATRIX

CELL IMMOBILIZATION!

Advantages and limits of immobilized cells process

Advantages
Higher cell densities and cell loads Increased volumetric productivity eliminates the costly processes of cell recovery and cell recycle. Possibility of continuous reactor design Limited effect of substrate inhibition

Limits
Diffusional limitations Support plugging Product inhibition

Supports
Either natural biopolymers (polysaccharides, alginate, carrageenan, and agar) or synthetic polymer (polyacrylates, polyurethanes, and polyether's) can be used as gel-forming agents.

Calcium alginate
Calcium Alginate gel is widely employed for immobilization of whole cells since it is less toxic than synthetic polymers and easily gelled under mild conditions.
Entrapment in insoluble Ca alginate gel is recognized as a rapid, nontoxic, inexpensive, versalite and the most often used method for immobilization of cells.

Objectives
Study the feasibility and effect of immobilization of Pseudomonas fluorescens on the kinetics of biosurfactant production compared to free cells process. Biosurfactant Separation and characterization

MATERIAL & METHODS

Microorganism:Pseudomonas fluorescens Migula 1895-DSMZ Fermentation:
Mineral salts medium+ olive oil(2%), as a carbon source; ammonium nitrate, (C/N=10); Ambient temperature; agitation on orbital shaker 200 tr/min;

Assays:
Surface tension (ST) (De Nouy Tensiometer Kruss K6), emulsification index (E24%), Biomass (dry weight) and pH

Immobilization Technique
Entrapment in calcium alginate
Optimum Conditions for immobilization were obtained by Response Surface Method with three factors : Sodium alginate concentration
Calcium chloride concentration Biomass loading To obtain maximum Responses of surface tension and E24
washing Sodium alginate
Cell suspension

Syringe(20G)

Calcium alginate beads

RESULTS

Biosurfactant production kinetics by free [A] and immobilized [B] cells of Pseudomonas fluorescens
75 60
)

10 8
) ) ) )

75 60 45

10

8
)

ST(dyne/cm) ( E24 (%) (

45 30 15 0 0 20 40 60

6 4

ST(dyne/cm) ( E24 (%) (

pH ( Biomass(

6 30 15 0 0
pH (

[A]

2 0 80 100 120

[B]

2 20 40 60 80 100 120 140

Time(hours)

Time(hours)

Biosurfactant production kinetics by free [A] and immobilized [B] cells of Pseudomonas fluorescens
Minimum ST (dyne/cm) Maximum E24 (%)

Optimum time (hours)

pH variations

Observations

Free cells

30

67

40-50

6.8-7.0

ST re-increase and E24 decrease after 48 hours

Immobilized cells

35

62

72

6.8 down to 5 (0-72hours) 5.0 up to 7.0 (72-96hours)

No significant changes of optimum ST or E24 after 72 hours

 Biosurfactant separation

Easy recovery with acetone precipitation Yield (6g/l) Purer product with immobilized cell technique

Biosurfactant characterization

Positive rhamnose test Good foaming and emulsifying activity

Effect of temperature [A], pH [B] and salinity (NaCl)[C] on biosurfactant activity

High thermal stability

Surface activity increases with pH No significant effect of salinity

80
Surface tension (dyne cm )
-1

Critical micellar concentration (CMC)

Low CMC value : CMC = 290mg/l

70 60 50 40 30 20 10

0 1 2 3 4 5 6 7 8 -1 Biosurfactant concentration(g L )

Wetting capacity

Polystyrene Polystyrene with adsorbed biosurfactant

98,7

ability effect on hydrophobic surfaces Contact angle () (polystyrene Eau CH2I2 Formamide surface)coated with biosurfactant:
30,8 74,8

Good wetting

43

58

63,2

Naphthalene solubility enhancement

Naphthalene solubility increase from 30mg/L (distilled water) up to 200 mg/L (6-7 folds) in biosurfactant aqueous solution (0.5 g/L). Naphthalene solubility in water containing biosurfactant was also affected by pH and salinity.
250 250

Solubilized Naphthalene(mg/l)

Solubilized Naphthalene(mg/l)

200 150 100 50 pH 5 pH 7 pH 11

200 150 100 50

5% 10% 20% 0%

0 1 2 3 4 5 Boiosurfactant concentration(g/l)

1 2 3 4 5 Boiosurfactant concentration(g/l)

Fig.4: Effect of pH and salinity on naphthalene solubility in aqueous biosurfactant solutions

Conclusions
Diffusional limitations in alginate beads affected the

kinetic of biosurfactant production. Stable emulsions were obtained with immobilized cells. Easy separation Thermal and chemical stability increasing with pH Interesting characteristics for use in environmental and industrial applications at normal and extreme conditions.