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TITLE PAGE

ISOLATION AND CHARACTERIZATION OF BIOSURFACTANT PRODUCTION BY A DICULTURE OF Pseudomonas spp AND Azotobacter

vinelandii.

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CERTIFICATION Umeji, Ann Adaora is a postgraduate student in the Department of Biochemistry with the registration number PG/M.Sc/07/42539 has satisfactorily completed the requirements of course and research for the award of the Master of Science degree in Industrial Biochemistry and Biotechnology. The work in this report is original and has not been submitted in part or full for any other degree, diploma or certificate of this or any other university. −−−−−−−−−−−−−−−−−−−− −−−−−−−−−−−−−−−−−−−−−− Prof. I.N.E. Onwurah Prof. I.N.E. Onwurah (Supervisor) (Head of Department) −−−−−−−−−−−−−−−−−−−−−−− (External Examiner)

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DEDICATION This research work is dedicated to the Almighty God for His unfailing love, mercy and faithfulness and to my parents and siblings for their love, support and encouragement.

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ABSTRACT Biosurfactants are surface active agents produced extracellularly or as part of the cell membrane by bacteria, yeast and fungi, and they lower the surface and interfacial tensions of immiscible fluids. A total of nine bacterial isolates were obtained from deliberately crude oil – contaminated soil beside the biochemistry postgraduate laboratory. These isolates were studied for their biosurfactant producing potentials. Three isolates designated C1P, NC11P and C1A were selected for this study on the basis of their characteristics and good growth. Isolates C1P and NC11P were identified as Pseudomonas spp while C1A was identified as Azotobacter vinelandii. Biosurfactant production was assessed by such parameters as drop collapse, haemolysis, emulsification and oil spreading test. During the production of biosurfactants their growth in the culture medium was assessed by optical density at the wavelength of 600nm. The isolates showed an exponential growth in the medium. Isolates C1P and NC11P showed a strong and positive correlation of 0.959. NC11P was used for the amendment culture with inadequate nitrogen source. The diculture of NC11P* and C1A showed significant growth and nitrogen fixation exemplified with turning of moistened red litmus paper to blue. The diculture broth supplemented with crude oil gave the highest growth (OD600nm= 2.408) and also a precipitate of 3.830g and a cell biomass of 0.066g when compared to the sole culture broth of NC11P* whose precipitate was 1.180g and a cell biomass of 0.031g. After extraction and purification of the biosurfactant produced by the diculture, it was found that the biosurfactant contained 61% carbohydrate, 30% lipid and 9% protein and was classified as a carbohydrate- protein- lipid complex.

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ACKNOWLEDGEMENT My sincere appreciation goes to my supervisor Prof. I.N.E. Onwurah for his fatherly supervision and bringing out the best in me. My heartfelt gratitude goes to Dr. C.U. Anyanwu for his constant patience, fatherly and professional guidance, without which this work could not be completed. I am grateful to Prof. O.U. Njoku and Prof. F.C. Chilaka for their concern and words of encouragement during the research work. I acknowledge the academic and non-academic staff of Biochemistry, I will not fail to mention people like Prof. L.U.S. Ezeanyika, Dr. H.A. Onwubiko, Dr. O.F.C. Nwodo, Dr. V. Ogugua, Mr. P.A.C. Egbuna, Mr Ikwuagu, and Mr. O.C. Enechi and also Microbiology department for different form of assistance during this program, especially Dr. E.A. Eze, and Mr O. Nwokoro. I recognize the constant support and contributions of my senior friend Arc. Ifeanyi Odedo, and the contributions and support from Dr. Chima Nwanguma, Mr. C.I. Nnamchi, and Mr. Babagana Wakil, I cannot quantify the blessings I have derived from this friendship, not a day goes by that I am not enriched by you. I equally appreciate the concern of my siblings; Nky, Ije, Obii and Uju, friends and colleagues both in Biochemistry, Microbiology, PG Hall and Innovation centre among whom are Obinna Oje, Boniface, Attai, Gloria Okpala, Uche Nwodo, Alfred, Samson, Ruth, Adaeze, Nwanneka, Mrs Chigozie Oseke, Nebechi, Paul, Nuru, Lanre, Precious, Michael, Parker and Oga Uche, as well as the staff of records office, Personnel Services especially Mr. B.E. Orji, Mrs. F.O. Nnadi and Mrs. C. Timothy, I am highly indebted to you all and I pray God will bless you all until you cannot stand it. My gratitude goes to all the members of the Graduate Student Fellowship (GSF) and Bishop David Abioye of Living Faith Church whose words during WOFBI classes motivated me to embark on this degree and Pastor Peace Efemena. I appreciate Mr. G.I. Owunne for his love and support and Engr. N.R. Ukaigwe for giving me a shoulder to lean on. I also acknowledge various authors, whom I made references to their work, I am indeed grateful. Finally, I am deeply grateful to Nigeria - Sao tome and Principe under Addax Petroleum for the grant given to me for this research work. Umeji, Ann Adaora. October, 2009.

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TABLE OF CONTENTS Pages TITLE PAGE…………………………………………………………………….i CERTIFICATION PAGE……………………………………………………….ii DEDICATION…………………………………………………………………..iii ABSTRACT……………………………………………………………………...iv ACKNOWLEDGEMENT……………………………………………………….v TABLE OF CONTENTS…………………………………………………………vi LIST OF TABLES………………………………………………………………..ix LIST OF FIGURES………………………………………………………………x CHAPTER ONE: INTRODUCTION 1.1 Introduction...…..………..…………………………………………………...1 1.2 Biosurfactants…………………………………………………………………2 1.2.1Biosurfactant producing bacteria….…………………………………………..2 1.3 Types of biosurfactants………..………………………………………………..3 1.3.1 Low- molecular mass biosurfactants………………………………………..3 1.3.1.1 Glycolipids………..…………………………………………………………4 1.3.1.2 Phospholipids………………………………………………………………..7 1.3.1.3 Lipoproteins and Lipopeptides………………………………………………8 1.3.2 High- molecular mass biosurfactants………………………………………...9 1.3.2.1 Polymeric biosurfactants…………………………..…………………………9 1.3.2.2 Particulate biosurfactants…………………………………………………...12 1.4 Application of biosurfactants………………………………………………….13 1.4.1 Petroleum Industry………………………………………………………….13 1.4.1.1 Microbial enhanced oil- recovery…………………………………………...13 1.4.1.2 Microbial de- emulsification of oil emulsions……………………….……...14 1.4.2 Food Industry……………………………………………………………….15 1.4.3 Cosmetic Industry…………………………………………………………..16 1.4.4 Textile Industry………………………………………………………….…..16 1.4.5 Therapeutic and biomedical applications……………………………………16 1.4.6 Pharmaceutical Industry…………………………………………………….17 1.5 Production of Microbial Biosurfactants…………………………………………18 1.5.1 Factors affecting biosurfactants production………………………………...19 1.5.1.1 Carbon source…..……………………………………………………...........19 1.5.1.2 Nitrogen source………………………………………………………..............20 1.5.1.3 Environmental factors…………………………………………………………20 1.6 Properties of Biosurfactants……………………………………………………….20 1.7 Biosurfactants interaction in an Aqueous solution…………………………...……23 1.7.1 Biosurfactants and oil bioremediation………………………….….…………..25 1.7.1.1 Increasing the surface area of hydrophobic water- insoluble substrate………..26 1.7.1.2 Increasing the bioavailability of hydrophobic water- insoluble substrate……..27

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1.7.2 Recovery of Biosurfactants…………………………………………………….27 1.8 Rationale for study……………………………………………………..…….30 1.9 Aims and objectives…..…………………………………………………..……….30

CHAPTER TWO: MATERIALS AND METHODS 2.1 Materials………………………………………………………………….………..31 2.2 Preparation of solutions……………………………………………………………31 2.3 Isolation of biosurfactant- producing bacteria……………………………………..32 2.4 Characterization of isolates…………………………………………………………33 2.4.1 Gram’s staining……………………………………………………………………33 2.4.2 Catalase test……………………………………………………………………….34 2.4.3 Motility test……………………………………………………………………….34 2.4.4 Indole test…………………………………………………………………………35 2.4.5 Hydrogen sulphide test………………………………............................................35 2.4.6 Sugar fermentation test……………………………………………………………36 2.4.7 Oxidase test………………………………………………………………………..36 2.5 Diculture of the isolates…………..…….………………………………………….36 2.5.1 Preparation of amendment for the diculture………………………………………37 2.6 Biosurfactant activity assay……………………………………………………….37 2.6.1 Haemolysis test……………………………………………………………………37 2.6.2 Drop collapse test…………………………………………………………………38 2.6.3 Emulsification test…………………………………………………………………38 2.7 Purification and extraction of biosurfactants………………………………………39 2.7.1 Acid precipitation…………………………………………………………………..39 2.7.2 Cold acetone precipitation………………………………………………………….39 2.8 Characterization of biosurfactants………………………………………………….39 2.8.1 Biochemical composition of biosurfactants………………………………………...39 2.8.1.1 Determination of the protein content……………………………………………..40 2.8.1.2 Determination of carbohydrate content……………..……………………………40 2.8.1.3 Determination of lipid content…………………………………………………….41 2.9 Estimation of biomass……………………………………………………………..41 CHAPTER THREE: RESULTS

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3.1 Characterization of bacterial isolates………………………………………………..42 3.2 Time course of growth of the broth culture…………………………………………44 3.2.1 Time course of growth of the pure strain of Pseudomonas on Pseudomonas medium..44 3.2.2 Time course of growth of Azotobacter vinelandii on Azotobacter medium…………45 3.2.3 Time course of growth of the amended Pseudomonas culture………………………46 3.2.4 Time course of growth of the diculture………………………………………………47 3.2.5 Time course of growth of the diculture in medium supplemented with mannitol and Crude oil……………………………………………………………………………..48 3.3 Statistical analysis……………………………………………………………………….49 3.4 Test for biological nitrogen fixation…………………………………………………….52 3.5 Test for biosurfactant activity……………………………………………………………52 3.6 Characterization of biosurfactants……………………………………………………….53 CHAPTER FOUR: DISCUSSION AND CONCLUSION 4.1 Discussion……………………………………………………………………………….55 4.2 Conclusion………………………………………………………………………………58 REFERENCES……………………………………………………………………………...60APPENDIX………………………………………………………………………………….68

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LIST OF TABLES Table 1.1 Classification and microbial origin of biosurfactant……………………….12 Table 1.2 Downstream processes for recovery of important biosurfactants and their advantages…………………………………………………………………..29 Table 3.1 Characterization of bacterial isolates………………………………………..43 Table 3.2 Test for biosurfactant activity……………………………………………….53 Table 3.3 Characterization of biosurfactants…………………………………………..54

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LIST OF FIGURES Fig 1.1 Structure of rhamnolipid……………………………………………………..5 Fig 1.2 Structure of trehalolipid……………………………………………………...6 Fig 1.3 Structure of sophorolipid……………………………………………………..7 Fig 1.4 Structure of phoshatidylethanolamine………………………………………..8 Fig 1.5 Structure of cyclic surfactin………………………………………………….9 Fig 1.6 Structure of emulsan…………………………………………………………11 Fig 1.7 Schematic diagram of the variation of surface tension, interfacial and contaminant Solubility with surfactant concentration………………………………………24 Fig 3.1 Time course of growth of the pure strain of Pseudomonas spp on Pseudomonas Medium………………………………………………………………………..44 Fig 3.2 Time course of growth of Azotobacter vinelandii in Azotobacter medium........45 Fig 3.3 Time course of growth of Pseudomonas spp in Pseudomonas medium with Inadequate nitrogen source…………………………………………………….46 Fig 3.4 Time course of growth of the diculture of Pseudomonas spp with inadequate Nitrogen source and Azotobacter vinellandii…………………………………..47 Fig 3.5 Time course of growth of the diculture in medium supplemented with mannitol and crude oil…………………………………………………………………….48 Fig 3.6 Correlation analysis between isolate C1P and NC11P…………………………49 Fig 3.7 Correlation analysis between NC11P* and NC11P………………………….....50 Fig 3.8 Correlation analysis between NC11P and diculture………………………….....50 Fig 3.9 Correlation analysis between diculture and diculture + mannitol………………51 Fig 3.10 Correlation analysis between diculture and diculture + crude oil………………51

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CHAPTER ONE

INTRODUCTION

Biosurfactants are surface active agents or amphiphilic compounds which can

reduce surface and interfacial tensions of liquids. Biosurfactants produced mostly by

microbial cells are excreted extracellularly and contain hydrophobic/lipophilic and

hydrophilic moieties that reduce surface tension. However, for degradation of crude oil-

contaminated site, special microbial cultures are needed which can survive in the

contaminated environment and degrade the contaminant efficiently and completely. This

can be enhanced by bioremediation techniques which include aeration of the

contaminated site and nutrient additions, supplementing the source of microorganisms

capable of degrading the contaminant (bioaugmentation) and the enhancement of the

desorption of pollutants from particulates using biosurfactants which increase

hydrocarbon degradation (Christofi and Ivshina, 2002). Surface tension is defined as the

free surface enthalpy per unit area (OECD, 1995) and is the force acting on the surface of

a liquid leading to minimization of the area of that surface (Christofi and Ivshina, 2002).

Surfactants do this, by accumulating at the interface of immiscible fluids thereby

increasing the solubility, mobility, bioavailability and subsequent biodegradation of

hydrophobic or insoluble organic compounds (Rahman et al., 2003; Singh et al., 2006).

Surfactants are amphiphilic compounds that reduce the free energy of the system

by replacing the bulk molecules of higher energy at an interface (Mulligan et al., 2001;

Mulligan, 2005). They contain a hydrophobic portion with little affinity for the bulk

medium and a hydrophilic group that is attracted to the bulk medium. They are used

industrially as adhesive, flocculating, wetting and foaming agents, de-emulsifiers and

penetrants (Mulligan, 2005). The effectiveness of a surfactant is determined by its ability

to lower surface tension, which is a measure of the surface free energy per unit area

required to bring a molecule from the bulk phase to the surface. These surfactants can be

synthetic or naturally produced by microorganisms, hence the name biosurfactants.

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Both synthetic and biological surfactants have been shown to enhance the

apparent aqueous solubility of nonpolar organic contaminants resulting in increased

bioavailability and biodegradation. However, there are reports which suggest that some

synthetic surfactants inhibit biodegradation. This inhibition is generally attributed to

toxicity or reduction in bioavailability due to partitioning of contaminant into surfactant

micelles. They are often toxic and recalcitrant and pose the threat of additional

contamination (Banat, 1994; Giedraityte et al., 2001; Batista et al., 2005).

1.1 Biosurfactants

Biosurfactants are surfactants that are produced extracellularly or as part of the

cell membrane by bacteria, yeasts and fungi (Karanth et al., 1999, Mulligan 2005,

Tabatabaee et al., 2005). They are a structurally diverse group of surface-active

molecules synthesized by microorganisms. These molecules reduce surface and

interfacial tension in both aqueous solutions and hydrocarbon mixtures, which makes

them potential candidates for enhancing oil recovery and deemulsification processes

(Desai and Banat, 1997; Youssef et al., 2004; Muthusamy et al., 2008). They are

amphipathic molecules enabling the formation of specialized structures vital to their

action. They function by residing at the oil-water interface (Christofi and Ivshina, 2002).

Biosurfactants lower the interfacial tension between immiscible fluids enabling

them to be miscible through the creation of additional surfaces. A single interface

consisting of an immiscible and miscible constituent is transformed into smaller interface

of the two constituents (Christofi and Ivshina, 2002). They improve the bioavailability of

hydrocarbons to the microbial cells by increasing the area at the aqueous-hydrocarbon

interface. This increases the rate of hydrocarbon dissolution and their utilization by

microorganism (Tuleva et al., 2001). The main physiological role of biosurfactants is to

permit microorganisms to grow on water immiscible substrates by reducing the surface

tension at the phase boundary, thus making the substrate more readily available for

uptake and metabolism (Thambivajah, 1998).Therefore, the use of biosurfactant should

be a promising means to emulsify polluted oils prior to biodegradation (Maneerat and

Pheetrong, 2007).

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1.1.1 Biosurfactant- producing bacteria

Petroleum bioremediation is carried out by microorganisms capable of utilizing

hydrocarbons as a source of energy and carbon. These microorganisms are ubiquitous in

nature and are capable of degrading the various types of hydrocarbons – short-chain, long

chain and numerous aromatic hydrocarbons. All these compounds have low solubility in

water. This fact, coupled to the fact that the first step in hydrocarbon degradation

involves a membrane-bound oxygenase, makes it essential for bacteria to come in direct

contact with the hydrocarbon substrates (Ron and Rosenberg, 2002). One biological

strategy that can enhance contact between bacteria and water-insoluble hydrocarbons is

emulsification of the hydrocarbon. Therefore, it is not surprising that bacteria growing on

petroleum usually produce potent emulsifiers. These surfactants help to disperse the oil,

increase the surface area for growth, and help detach the bacteria from the oil droplets

after the utilizable hydrocarbon has been depleted (Rosenberg, 1993; Ron and Rosenberg,

2002). Biosurfactants generally increase the adhesion of cells to the substrate (Calvo et

al., 2004). The various biosurfactant – producing bacteria are:

Torulopsis bombicola, Pseudomonas aeruginosa, Bacillus licheniformis, Bacillus subtilis

Arthrobacter paraffineus, Pseudomonas flurescens, Torulospis petrophilum, Candida

tropicalis, Corynebacterium lepus, Acinetobacter calcoaceticus, Candida lipolytica,

Candida petrophilum, Nocardia erythropolis, Rhodococcus eryithropolis,

Corynebacterium hydrocarboclatus, Micrococcus spp,, Saccharomyces.

(Das et al., 1997; Kosaric, 2001).

1.2 Types of biosurfactants

Biosurfactants are generally categorized mainly by their chemical composition

and microbial origin (Desai and Banat, 1997; Muthusamy et al., 2008). Their structure

includes a hydrophilic moiety consisting of amino acids or peptides, anions or cations;

mono-, di-, or polysaccharides or phosphate, and a hydrophobic moiety consisting of

unsaturated, saturated, or fatty acids. Desai and Banat, (1997) and Muthusamy et al.,

(2008) quoted that “biosurfactants can be divided into low- molecular-mass molecules,

which efficiently lower surface and interfacial tension and high molecular-mass

polymers, which are more effective as emulsion stabilizing agents”.

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1.2.1 Low Molecular-Mass Biosurfactants:

The low molecular-mass surfactants generally lower surface and interfacial

tension of liquids efficiently. These include the following:

(a) Glycolipids

(b) Phospholipids

(c) Lipopeptides and lipoproteins

1.3.1.1 Glycolipids

Glycolipids are the most common type of biosurfactant. These are compounds

made up of carbohydrates and lipid (Healy et al., 1996). They are carbohydrates in

combination with long-chain aliphatic acid or lipopeptides (Ron and Rosenberg, 2002),

and hydroxyl aliphatic acids (Desai and Banat, 1997). The linkage is by means of either

ether or an ester group (Healy et al., 1996; Desai and Banat, 1997). The glycolipids

contain the rhamnose and trehalose sugars (Christofi and lvshina, 2002).

The constituent mono-, di-, tri- and tetrasaccharides include glucose, mannose,

galactose, glucuronic acid, rhamnose and galactose sulphate. Among the glycolipids, the

best known are:

i) Rhamnolipids

ii) Trehalolipids

iii) Sophorolipids

Rhamnolipids

One or two molecules of rhamnose are linked to one or two molecules of β-

hydroxydecanoic acid as shown in Fig 1.1 (Desai and Banat, 1997: Ron and Rosenberg,

2002). While the OH group of one of the acids is involved in glycosidic linkage with the

reducing end of the rhamnose disaccharide, the OH group of the second acids is involved

in ester formation. (Karanth et al., 1999; Muthusamy et al., 2008).

The production of rhamnose containing glycolipids was first described in

Pseudomonas aeruginosa by Jarvis and Johnson in 1949 (Desai and Banat, 1997). L-

Rhamnosyl- L- rhamnosyl- β -hydroxydecanoyl-β-hydroxydecanoate and L-rhamnosyl- β

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- hydroxydecanoyl- β –hydroxydecanoate referred to as rhamnolipid 1 and 2, are the

principal glycolipids produced by P. aeruginosa (Banat and Desai, 1997).

Fig. 1.1: Structure of rhamnolipid

(Source: Urum and Pekdemir, 2004)

Trehalolipids

Trehalolipids are disaccharides that are acylated with long-chain fatty acids or

hydroxyl fatty acids as shown in Fig 1.2 (Ron and Rosenberg, 2002). Trehalolipids from

different organisms differ in the size and structure of mycolic acid, the number of carbon

atoms and the degree of unsaturation. They link at C-6 and C-61 to mycolic acids which

is associated with most species of Mycobacterium, Nocardia and Corynebacterium.

(Desai and Banat, 1997).

O – CH – CH2 –C – O – CH –CH – CH2– C – O – H

(CH2)6 CH3

OH

(CH2)6 CH3

O OH

CH3 OH

O

C

O

C

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CH2

(CH2)n

CH2O CO CH CHOH (CH2)m CH3

O

OH

OHOOH

O

OH

OHOH

H3C (H2C)m HOHC CH CO OCH2

(CH2)n

CH3

m+n=27 to 31.

Fig. 1.2: Structure of trehalolipids

(Source: Desai and Banat, 1997)

Sophorolipids

Sophorolipids consist of a dimeric carbohydrate sophorose linked to a long-chain

hydroxyl fatty acid by glycosidic linkage as shown in Fig 1.3 (Desai and Banat, 1997;

Muthusamy et al., 2008).

They are produced mainly by yeast such as Torulopsis bombicola. Sophorolipids

occur as a mixture of macrolactones and free acid form. These biosurfactants are a

mixture of at least six to nine different hydrophobic sophorolipids.

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ROH2C

OO

CH3

(CH2)15

COOH

CH

OO

ROH2C

OH

OHHO

R=CH2CO

Fig. 1.3: Structure of Sophorolipids

(Source: Desai and Banat, 1997)

1.3.1.2 Phospholipids

These are the esters formed between the alcohol groups on a lipid and a phosphate

as shown in Fig 1.4 (Healy et al., 1996). They are major components of cellular or

microbial membranes (Bengmark., 1998).

Several bacteria and yeasts produce large quantities of fatty acid and phospholipid

surfactants during growth on hydrocarbon (alkane substrates). The hydrophilic and

lipophilic balance (HLB) is directly related to the length of the hydrocarbon in their

structures (Desai and Banat, 1997; Muthusamy et al., 2008). Although there are very few

examples of extracellular production, the most notable one being the biosurfactants

produced by Corynebacterium lepus (Bognolo, 1999). In Acinetobacter spp, phosphatidyl

ethanolamine-rich vesicles are produced which form optically clear microemulsions of

alkanes in water (Desai and Banat, 1997).

OH

OH

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Fig. 1.4: Structure of phosphatidylethanolamine

1.3.1.3 Lipopeptides and Lipoproteins

These biosurfactants consist of a lipid attached to a polypeptide chain (Healy et

al., 1996). It is an amphiphilic biosurfactant containing lipid and peptide as hydrophobic

and hydrophilic moiety (Lee et al., 2004). A large number of cyclic lipopeptides,

including decapeptide antibiotics (gramicidins) and lipopeptide antibiotics (polymyxins)

are produced by microorganisms.

The best known lipopeptides and lipoproteins include.

i) Surfactin

ii) Lichenysin

Surfactin

It has an amphiphilic structure associated with high surfactant activity and

extensive biological properties (Christofi and Ivshina, 2002). The cyclic lipopeptide

surfactin produced by Bacillus subtilis is one of the most potent biosurfactants as shown

in Fig 1.5. It is composed of a seven amino-acid ring structure coupled to a fatty acid

chain via lactone linkage (Desai and Banat, 1997).

H 2 C - O - C - R 1

O

O

H C - O - C - R 2

O

HC-O-C-R2

H2C - O- P - O - CH2 - CH2 - NH3

O

O

+

+

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L - ASP - D-leu - L-Leu - O

D-leu - L-Leu - L- Leu - L- Glu - C = O

L - ValHC(CH2)9 - CH

CH2

CH3

CH3

Fig. 1.5: Structure of cyclic surfactin

(Source: Desai and Banat, 1997)

Lichenysin

Lichenysin is produced by Bacillus licheniformis and it exhibits excellent

temperature, pH and salt stability. It is similar in structural and physio-chemical

properties to surfactin. The detailed characterization of lichenysin showed that isoleucine

is the C-terminal amino acid instead of leucine, and an asparagine residue present instead

of aspartic acid as in the surfactin peptide (Desai and Banat, 1997).

1.2.2 High-Molecular Mass Biosurfactants

High molecular weight biosurfactants are amphiphilic (amphipathic), (lipo)

polysaccharides, (lipo) protein or combination of these (Ron and Rosenberg 2002;

Christofi and Ivshina, 2002). These biosurfactants are associated with the production of

stable emulsions but do not lower the surface tension of liquids. The production of stable

emulsions enables bacteria to adhere to hydrophobic surface very strongly (Christofi and

Ivshina, 2002). They are efficient at coating the oil droplet and preventing their

coalescence (Ron and Rosenberg, 2002). These include the following;

a) Polymeric

b) Particulate

1.3.2.1 Polymeric biosurfactants

These are products formed between saccharide units and fatty acid residues; but

they are polymeric in nature (Healy et al., 1996). These biosurfactants are polymeric

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heterosaccharide containing proteins. The best studied are emulsan, liposan, alasan,

lipomanan and other polysaccharide – protein complexes.

Acinetobacter calcoaceticus RAG-1 produces an extracellular potent polyanionic

amphipathic heteropolysaccharide bioemulsifier called emulsan. The

heteropolysaccharide backbone contains a repeating trisaccharide of N-acetyl-D-

galactosamine, N-acetylgalactosamine uronic acid and an unidentified N-acetyl amino

sugar. Fatty acids are covalently linked to the polysaccharides through O-ester linkages.

(Desai and Banat, 1997, Muthusamy et al., 2008). The structure of emulsan is shown in

Fig 1.6. Its surface activity results from the presence of fatty acids that are attached to the

polysaccharide backbone via O-ester and N-acyl linkages (Garti, 1999; Ron and

Rosenberg, 2002).

Alasan, produced by a strain of Acinetobacter radioresistens, is a complex of an

anionic polysaccharide and protein. The polysaccharide component of alasan is unusual

in that it contains covalently bound alanine (Desai and Banat, 1997; Ron and Rosenberg,

2002; Muthusamy et al., 2008). Liposan is an extracellular water-soluble emulsifier

synthesized by Candida lipolytica and is composed of 83% carbohydrate and 17%

protein. The carbohydrate portion is a heteropolysaccharide consisting of glucose,

galactose, galactosamine and galacturonic acid (Desai and Banat, 1997; Muthusamy et

al., 2008).

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Fig. 1.6: Structure of emulsan

(Source: Desai and Banat, 1997)

CH3

(CH2)9

CHOH

C = O

CH

CH3

(CH2)3

CHOH

C = O

CH

CH2

CH2

C = O

CH2

O

OH

O

NH

O

O

C

CH3

NH

C

(CH2)12

= O

CH3

NH

C

CH3

O - -- -

O O

C= O C= O

HO

HO C

O O

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1.3.2.2 Particulate biosurfactants

Extracellular membrane vesicles partition hydrocarbons to form a microemulsion

which plays an important role in alkane’s uptake by microbial cells (Garti, 1999). The

vesicles of Acinetobacter spp strain are composed of protein, phospholipid and

lipopolysaccharide (Desai and Banat, 1997; Muthusamy et al., 2008). The table below

shows the classes of biosurfactant and its microbial origin.

Table 1.1: Classification and microbial origin of biosurfactant (source: Garti, 1999; Mulligan et al, 2001; Mulligan, 2005) Surfactant class Microorganism Trehalose lipids Arthrobacter paraffineus, corynebacterium spp.

Mycobacterium spp. Rhodococus erythropolis

Rhamnolipids Pseudomonas aeruginosa Sophorolipids Candida apicola

Candida bombicola Candida lipolytica

Glucose-, fructose-, saccharose lipids

Arthrobacter spp. Corynebacterium spp. Rhodococus erythropolis

Cellobiose lipids Ustilago maydis Diglycosyl diglycerides Lactobacillus fermentii Lipopolysaccharides Acinetobacter calcoaceticus (RAGI)

Pseudomonas spp Candida lipolytica

Lipopeptides Arthrobacter spp. Bacillus pumilis Bacillus subtilis Bacillus licheniformis Pseudomonas fluorescens

Ornithine, lysine peptides Thiobaccilus thioxidans Streptomyces sioyaensis Gluconobacter cerinus

Phospholipids Thiobaccilus thiooxidans Corynebacterium alkanolyticum Capnocytophaga spp.

Fatty acids (Corynomycolic acids, spiculisporic acids, etc.)

Penicillium spiculisporum Corynebacterium lepus Arthrobacter parafineus Talaramyces trachyspermus

Polyol lipids Rhodotorula glutinous Rhodotorula graminus

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1.4 Application of biosurfactants

Rapid advances in biotechnology over the past decades have led to considerable

interest in the development and use of microbial surfactants. This is due to their diversity,

environmental friendly nature or characteristics, low toxicity, the possibility of their

production through fermentation, and biodegradation rate when compared to their

synthetic counterparts (Puntus et al., 2004; Rashedi et al., 2005; Muthusamy et al., 2008).

Moreover, they are ecologically safe and can be applied in such areas as environmental

protection, healthcare, the food processing industries, in bioremediation and waste water

treatment (Puntus et al., 2004; Rashedi et al., 2005).

The role of biosurfactants in various industries is outlined below:

1.4.1 Petroleum Industry

In the petroleum industry, biosurfactants are applied in enhanced oil recovery and

de-emulsification. These biosurfactants increase the pseudo solubility of petroleum

components in water (Singh et al., 2006). They are effective in reducing the interfacial

tensions of oil and water in situ and they can also reduce the viscosity of the oil and

remove water from the oil prior to processing.

1.4.1.1 Microbial Enhanced oil-Recovery (MEOR)

Poor oil recovery in oil producing wells may be due to either low permeability of

some reservoirs or high viscosity of the crude oil resulting in poor mobility (Banat, 1994;

Singh et al., 2006). The ability of indigenous or injected microorganisms to synthesize

useful fermentation products to improve oil recovery from the oil reservoirs is exploited

in MEOR processes. MEOR-participating microorganisms produce a variety of products

such as biosurfactants, polysaccharides, carbon dioxide, methane and hydrogen.

Enhanced oil recovery of the residual oil in reservoirs can also be achieved by plugging

of highly permeable watered out regions of oil reservoirs with bacterial cells and

biopolymers. Biosurfactants aid oil emulsification and detachment of oil films from

rocks. Microorganisms are capable of synthesizing biosurfactants from crude oil, pure

hydrocarbons and a variety of non- hydrocarbon substrates such as simple carbohydrates,

acids and alcohols. Biosurfactant applications in MEOR are:

24

(a) Biosurfactant production in batch or continuous culture and addition to the

reservoir using the conventional way of MEOR.

(b) Production of biosurfactants by injected microbes at the cell-oil interface

within the reservoir

(c) Injection of selected nutrients into the reservoir to stimulate growth of

indigenous biosurfactant producing bacteria

Biosurfactants play an important role in MEOR by improving oil drainage into

well bore, stimulating the release of oil entrapped by capillaries, wetting of solid surfaces,

reduction of oil viscosity and oil pour point, dissolving of oil and lowering of interfacial

tension (Singh et al., 2006).

1.4.1.2 Microbial De-Emulsification of Oil Emulsions

Oilfield emulsions, both oil-in-water and water-in-oil, are formed at various

stages of exploration, production, oil recovery and processing, and constitute a major

problem for the petroleum industry (Onwurah and Nwuke, 2004; Singh et al., 2006). A

process of de-emulsification is required to recover oil from these emulsions. Factors that

influence the stability of emulsions include viscosity, droplet size, phase volume ratio,

temperature, pH, age of emulsion, type of emulsifying agent present, density difference

and agitation.

Traditional de-emulsification methods include centrifugation, heat treatment,

electrical treatment and chemicals containing soap, fatty acids and long chain alcohols.

Since biological processes can be carried out at non-extreme conditions, an effective

microbial de-emulsification process could be used directly to treat emulsions at the

wellhead, thus saving on transport and high capital equipment costs. Microbes exploit the

dual hydrophobic/hydrophilic nature of biosurfactants or hydrophobic cell surfaces to

displace the emulsifiers that are present at the oil-water interface. Biosurfactants act by

the de-emulsification of emulsions, thereby reducing the viscosity of the oil phase. They

also solubilize the oil and serve as wetting agents (Singh et al., 2006).

25

1.4.2 Food Industry

Apart from the role of biosurfactants as agents that decrease surface and

interfacial tension, thus promoting the formation and stabilization of emulsions,

biosurfactants have several other functions in food. For example, to control the

agglomeration of fat globules, stabilize aerated systems, improve texture and shelf life of

starch-containing products, modify rheological properties of wheat dough and improve

consistency and texture of fat-based products (Muthusamy et al., 2008). Biosurfactants

serve as additives in the food industry where they act as emulsifiers, solubilizers,

demulsifier, suspension, wetting, foaming, defoaming, thickener and lubricating agents

(Desai and Banat, 1997; Singh et al., 2006). An Emulsifier is defined as “a single

chemical component, or mixture of components, having the capacity for promoting

emulsion formation and stabilization by interfacial tension”, (Garti, 1999). Lecithin

(phosphatidyl choline ) and its derivatives, fatty acid esters containing glycerol, sorbitan

or ethylene glycol and ethyoxylated derivatives of monoglycerides are used as

emulsifiers (Desai and Banat, 1997).

In bakery and ice-cream formulations biosurfactants act by controlling

consistency, retarding staling and solubilizing flavor oils (Kosaric 2001, Bednarski et al.,

2004, Muthusamy et al., 2008). They are also utilized as fat stabilizers and antispattering

agents during cooking of oil and fats (Kosaric, 2001; Muthusamy et al., 2008). In the

crystallization of sugar, biosurfactants act by improving washing and processing time,

they are used for cleaning in food processing plants (Kosaric, 2001). A novel

bioemulsifier from candida utilis has shown potential for use in salad dressing (Desai and

Banat, 1997).

Biosurfactants are also applied as functional ingredient in the food industry,

where they act by interacting with lipids, proteins and carbohydrates; they are also

protecting agents (Singh et al., 2006). They act as antiadhesive agents thus controlling the

adherence of microorganisms to food-contact surfaces. A surfactant released by

Streptococcus thermophilus has been used for fouling control of heat-exchanger plates in

pasteurizers, as it retards the colonization of other thermophilic strains of Streptococcus

responsible for fouling (Muthusamy et al., 2008).

26

1.4.3 Cosmetic Industry

Biosurfactants are applied in health and beauty products. They act as emulsifiers,

foaming agents, solubilizers, wetting agents, cleansers, antimicrobial agent and mediators

of enzyme action (Singh et al., 2006). A product containing 1mole of sophorolipid and 12

moles of propylene glycol has excellent skin compatibility and is used commercially as a

skin moisturizer (Desai and Banat, 1997; Bognolo, 1999). A large number of compounds

for cosmetic applications are prepared by enzymatic conversion of hydrophobic

molecules by various lipases and whole cells. The cosmetic industry demands

biosurfactants with a minimum shelf life of 3 years (Desai and Banat, 1997).

Sophorolipid is commercially used by Kao Co. Ltd. as a humectant for cosmetic

makeup brands such as sofina. This company has developed a fermentation process for

sophorolipid production and after a two-step esterification process; the product finds

application in lipstick and as moisturizer for skin and hair products (Desai and Banat,

1997). Formulations for anti-dandruff shampoos, hair gels, deodorant sticks, aftershave

lotions, hair and body shampoos and rinse aids are based on sophorolipids (Bognolo,

1999).

1.4.4 Textile Industry

Biosurfactants are used in textile industry for the preparation of fibers where they

act as detergents and emulsifier in raw wool scoring, dispersant in viscose rayon spin

bath, lubricant and anti stat in spinning of hydrophobic filaments (Kosaric, 2001). For

dyeing and printing of fibers, biosurfactants act as wetting, penetration, solubilization,

emulsification, dye leveling, detergency and dispersion agents (Kosaric, 2001). They are

also utilized in finishing of textile, where they act as wetting and emulsification in

finishing formulations, softening, lubricating and antistatic additives to finishes

(Bognolo, 1999; Kosaric, 2001).

1.4.5 Therapeutic and Biomedical Applications

The main commercial use of biosurfactants is in pollution remediation because of

their ability to stabilize emulsion. Several biosurfactants have strong antibacterial,

antifungal and antiviral activity (Singh and Cameotra, 2004; Muthusamy et al., 2008).

27

Other medical relevant uses of biosurfactants include their role as anti-adhesive agents to

pathogens, making them useful for treating many diseases and as therapeutic and

probiotic agents. The lipopetide iturin A from Bacillus subtilis has a potent antifungal

activity (Singh and Cameotra 2004, Muthusamy et al., 2008). In yeast cells, iturin A

disrupts the plasma membrane by the formation of small vesicles and the aggregation of

intramembranous particles. It also releases electrolytes and high molecular mass

products, and degrades phospholipids (Singh and Cameotra, 2004).

Surfactin, a cyclic lipopeptide produced by B. subtilis strains, is one of the other

biosurfactants with well known antimicrobial properties. Apart from antifungal and

moderate antibacterial properties, surfactin inhibits fibrin clot formation, induces

formation of ion channels in lipid bilayer membranes, inhibits platelet and spleen

cytosolic phospholipase A2 and exhibits antiviral and antitumor activities (Sullivan, 1998;

Singh and Cameotra, 2004).

Seven microbial extracellular glycolipids, including mannosylerythritol lipids-A

(MEL-A), mannosylerythritol lipids-B (MEL-B), polyol lipid, rhamnolipid, sophorose

lipid, succinoyl trehalose lipid (STL)-1 and succinoyl trehalose lipid-3 were found to

induce cell differentiation instead of cell proliferation in the human promyelocytic

leukemia cell line HL60. They also induce the human myelogenous leukemia cell line

K562 and the human basophilic leukemia cell line KU812 to differentiate into

monocytes, granulocytes and megakaryocytes (Cameotra and Makkar, 2004; Singh and

Cameotra, 2004; Muthusamy et al., 2008). Sophorolipids are promising modulators of the

immune response (Deleu and Paquot, 2004; Muthusamy et al., 2008).

1.4.6 Pharmaceutical Industry

In the pharmaceutical industry, lecithins are used in much diluted solution for the

formation of liposomes (Garti, 1999). Lecithin is hydrophilized by attaching to its tail

chains hydrophilic functional groups such as hydroxyl or epoxy, or by hydrolyzing (and

removing) one of its tails (fatty acid) to form lysolecithin which is by far more

hydrophilic than lecithin and can act as a good oil-in-water emulsifier (Bognolo, 1999).

Biosurfactants have the potential to form an important part of food supplements. Instead

of pure minerals and vitamins as food supplements, it is now advisable to use

28

homeostatic nutrient complexes, which are a mixture of vitamins and minerals liberated

and maintained in their natural form by probiotic microorganisms and their byproducts,

including enzymes and organic acids (Singh and Cameotra, 2004). This increases the

bioavailability, adsorption, usefulness and effectiveness of the minerals and vitamins, in

addition to providing many other nutritionally important compounds. Also created in the

complex are numerous essential biochemicals that are required by the body, including the

antioxidant, superoxide dismutase, as well as various immune supportive β glucans,

antimicrobial peptides, bacteriocins, biosurfactants, biotins, coenzymes, conjugated

linolenic acids, glutathione, chromium compounds, hydrogen peroxide, lactic acid and

lysozymes (Singh and Cameotra, 2004).

1.5 Production of Microbial biosurfactants

Microbial biosurfactants are produced through fermentation processes.

Fermentation is a type of energy-converting metabolism in which the substrate is

metabolized without the involvement of an exogenous (i.e. external) oxidizing agent.

(Singleton, 2004). Fermentation is any process involving the mass culture of

microorganisms, either anaerobic or aerobic. It involves the breaking down of complex

organic substances into simpler ones.

Microbial biosurfactants production is embedded in the cell metabolism as a

whole (Angelova and Schmauder, 1999). Microorganisms can use a variety of substrates

such as sugars, oils, alkanes and agro wastes to produce either sugars or hydrocarbons

(Mulligan, 2005; Singh et al., 2006). Where the starting material is glucose, glucose 6-

phosphate is produced, which in turn can be converted to other saccharides such as

trehalose, sophorose or rhamnose or can be split via glyceraldehyde 3-Phosphate and

pyruvate to acetyl-CoA. This in turn can add to oxaloacetate to produce malonyl CoA

and thence to higher fatty acids. This extension of acetyl CoA to longer fatty acids is a

fundamental reaction in prokaryotes and eukaryotes. At each stage in the cycle, an acetyl

group is added to the growing chain to produce a slightly longer fatty chain (Karsa,

1990). Alternatively hydrocarbon substrates can be utilized to produce fatty acids or

saccharides. Alkanes can be oxidized via the alcohol and aldehyde to the fatty acid.

Oxidation at the β position produces acetyl-CoA, from which various fatty acids can be

29

synthesized. Thus sugars, alkanes or mixtures of both can be utilized to synthesize

biosurfactants, either from the feedstock directly or via intermediate compound (Karsa,

1990).

The biosurfactants are usually produced as cultures reach the stationery stage of

growth; its production is concurrent with the increase in cell density and the onset of the

stationary phase of growth (Ron and Rosenberg, 2002). As the bacteria grow, the cells

use nutrients and they also produce waste products which accumulate in the medium.

Eventually, therefore, growth slows down and stops due either to a lack of nutrients or to

the accumulation of waste products (or both); the phase in which there is no overall

increase in the number of living cells is called stationary phase (Singleton, 2004).

Production can be growth associated; in this case, they can either use the emulsification

of the substrate (extracellular) or facilitate the passage of the substrate through the

membrane (cell membrane associated). Biosurfactants however, are produced from

carbohydrates, which are very soluble (Mulligan, 2005). For example, lipopeptides are

synthesized by many Bacillus as well as other species. Glycolipids are produced by

Pseudomonas and Candida species, while Thiobacillus thiooxidans produce phospholipid

biosurfactants. Microbes often synthesize these biosurfactants during growth on water-

immiscible substrates, to facilitate uptake of the substrates by the cell (Desai and Banat,

1997; Singh et al., 2006).

Rhamnolipid production is dependent on the central metabolic pathways for fatty

acid synthesis and for dTDP (Thiamine diphosphate) – activated sugar formation and on

enzymes which participate in the production of the exopolysaccharide alginate.

Biosynthesis is regulated by a complex genetic regulatory system which is also involved

in the control of virulence –associated characteristics (Singh et al., 2006).

1.5.1 Factors Affecting Biosurfactant Production

The factors controlling the production of biosurfactants include the quality and

quantity of carbon and nitrogen constituents in culture and the natural physical

environment of soil systems (Christofi and Ivshina, 2002).

30

1.5.1.1 Carbon Source

Carbon is the structural backbone of living matter; it is needed for all the organic

compounds that make up living cell (Tortora et al., 1990). Microorganisms utilize a

variety of organic compounds as source of carbon and energy for growth. When the

carbon source is an insoluble substrate like a hydrocarbon (CxHy), microorganisms

facilitate their diffusion into the cell by producing a variety of substances, the

biosurfactants (Karanth et al., 1999). Water-soluble carbon sources such as glycerol,

glucose, mannitol and ethanol are used for rhamnolipid production by Pseudomonas spp.

(Desai and Banat, 1997). The source of carbon determines the maximum production of

biosurfactants.

1.5.1.2 Nitrogen Source

The synthesis of deoxyribonucleic acid (DNA) and RNA requires nitrogen and

some phosphorus. Nitrogen is used primarily to form the amino group of the amino acids

of proteins (Tortora et al., 1990). The type of nitrogen present (whether ammonia (NH4+),

Nitrate (NO3-), urea or amino acid) influences the biosurfactants produced. An interesting

observation relates to the effect of nitrogen limitation that appears to stimulate

biosurfactant production and over production by some microorganisms (Desai and Banat,

1997; Christofi and Ivshina, 2002). This has implications for bioremediation as systems

are often supplemented with nitrates and phosphates to alleviate nitrogen and phosphorus

limitation and to enhance microbial activity (Onwurah, 1996; Christofi and Ivshina,

2002; Onwurah, 2004).

1.5.1.3 Environmental Factors

These factors are pH, temperature, agitation and oxygen availability which also

affect biosurfactant production through their effects on cellular growth or activity.

Pseudomonas spp can grow in the presence of oxygen but can still grow under anaerobic

conditions (i.e in the absence of oxygen) and are called facultative anaerobes. It has been

observed that biosurfactants are very effective at extreme temperatures and pH (Kosaric,

2001; Deleu et al., 2004; Singh and Cameotra 2004,).

31

1.6 Properties of Biosurfactants

Biosurfactants are of increasing interest for commercial use because of their

continual growing spectrum of available substances. There are many advantages of

biosurfactants compared to their chemically synthesized counterpart. The main distinctive

features of biosurfactants include:

(i) Biodegradability

The extent and rate of crude oil biodegradation depend on the oil low aqueous

solubility and strong adsorptive capacity to the soil (Giedraityte et al., 2001).

Biosurfactants increase the rate of biodegradation by increasing their bioavailability to

hydrophobic compounds, (Bai et al., 1998; Hua et al., 2003) and thus facilitates their

assimilation by microbial cells (Kuyukina et al., 2004).

In general, biosurfactants are easily degraded and particularly suited for

environmental applications such as bioremediation and dispersion of oil spills

(Muthusamy et al., 2008), thus, it is environmentally acceptable and ecologically safe.

(ii) Emulsion Forming and Emulsion Breaking

An emulsion is formed when one liquid phase is dispersed as microscopic

droplets in another liquid continuous phase (Desai and Banat, 1997). Biosurfactants

stabilize emulsion, thus they either emulsify or de-emulsify emulsions. High molecular-

mass biosurfactants are in general better emulsifiers than low molecular-mass

biosurfactants (Muthusamy et al., 2008). Its ability to emulsify hydrocarbon enhances

water solubility and increases the displacement of oily substances from soil particles

(Rahman et al., 2002; Hua et al., 2003). This property is especially useful for making

oil/water emulsions for cosmetics and food.

(iii) Surface and Interface Activity

A good surfactant can lower surface tension of water from 72 to 35mN/m and the

interfacial tension of water/ hexadecane (Muthusamy et al., 2008). This effect is known

as the changing surface-active phenomenon (kosaric, 2001).

The lowering of interfacial tension between immiscible fluids enable them to be

miscible through the creation of additional surfaces which is as a result of biosurfactants.

32

A single interface consisting of an immiscible and miscible constituent is transformed

into smaller interface of the two constituents (Christofi and Ivshina, 2002).

(iv) Temperature, pH and Ionic Strength Tolerance

Many microbial surfactants and their surface activities are not affected by

environmental conditions such as temperature and pH (Muthusamy et al., 2008).

Biosurfactants have been observed to be very effective at extreme temperatures, pH and

salinity (Kosaric, 2001; Deleu and Paquot, 2004; Singh and Cameotra, 2004).

(v) Availability of Raw Materials

Surfactants can be produced from cheap raw materials, which are available in

large quantities (Kosaric, 2001). The carbon source may come from hydrocarbons,

carbohydrates, glucose, sucrose, mannitol and or lipids-glycerol, which may be used

separately or in combination with each other. Surfactants can be derived from both

petrochemical feedstock and renewable resources (plant and animal oil, microorganisms)

(Deleu and Paquot, 2004).

(vi) Metal Sequestration

Biosurfactants enhance the mobility and removal of sorbed heavy metals from

soil by complexation (Pepper et al., 1996 ; Bai et al., 1998). In sorption, metal-ligand

complexation occurs. The complexation with soil constituents and cation exchange

processes are involved in affecting access of the metals to microorganisms.

Biosurfactants remove metals by forming a complex, thereby enhancing surface removal

by the Le chatelier’s principle. The use of anionic surfactant which brings contact with

metals can lead to desorption from the surface (Miller 1995; Christofi and Ivshina, 2002).

(vii) Low Toxicity

Biosurfactants or microbial surfactants are generally considered as low, less or

non-toxic products and therefore, appropriate for pharmaceutical, cosmetic and food uses

(Abraham et al., 2002; Muthusamy et al., 2008).

33

(viii) Hydrophylicity and hydrophobicity actions

This cell surface property of biosurfactants helps in bacterial cell adhesion to

surfaces (Youssef et al., 2004) and promotes attachment of the cells to hydrocarbon

droplets (Noordman et al., 2002).

(ix) Biocompatibility and Digestibility

This property allows the use of surfactants in cosmetics, pharmaceuticals and as

functional food additives (Muthusamy et al., 2008). Other properties exhibited by

biosurfactants are, its wetting and penetrating actions, enhancement of microbial growth,

and bioavailability of the substrate, specificity and acceptable production economics

(Desai and Banat, 1997; Vasileva-Tonkova and Gesheva, 2004; Muthusamy et al., 2008).

1.7 Biosurfactant interaction in an aqueous solution;

Surfactants are amphoteric molecules consisting of a non polar tail and polar/ionic

head. In an aqueous solution, surfactants reduce surface tension by accumulating at

interfaces and facilitating the formation of emulsions between liquids of different

polarities (Miller, 1995). Water tends to hydrate the hydrophilic portion of an amphiphile,

but it also tends to exclude the hydrophobic portion (Voet et al., 2006).

At low concentration, surfactants are present as individual molecules. However,

as the concentration of the surfactant is increased, a concentration is reached where no

further change in interfacial properties takes place. The amount of surfactant needed to

reach this concentration is called the critical micelle concentration (CMC). CMC is

defined as the minimum concentration necessary to initiate micelle formation (Mulligan

et al., 2001; Urum and Pekdemir, 2004; Mulligan, 2005). At the CMC, surfactant

molecules aggregate to form structures such as bilayers and micelles (Miller, 1995;

Christofi and Ivshina, 2002; Voet et al., 2006).

Micelles are globules of up to several thousand amphiphilic molecules arranged

so that the hydrophilic groups at the globule surface can interact with the aqueous solvent

while the hydrophobic groups associate at the centre, away from the solvent (Voet et al.,

2006). The molecular arrangement of micelles eliminates unfavourable contact between

water and the hydrophobic tails of the amphiphiles and yet permits the solvation of the

34

polar head groups (Voet et al., 2006). These micelles arise when the lipophilic part of the

surfactant molecule that is unable to form hydrogen bonding in an aqueous phase causes

an increase in the free energy of the system. One way to alleviate this free energy

increase is for the hydrocarbon tail to be isolated from water by adsorption onto surfaces,

absorption into an organic matrix or the formation of micelles and vesicles where the

hydrocarbon moiety of the surfactant become situated towards the centre with the

hydrophilic part in contact with water (Christofi and Ivshina, 2002).

Micelle formation allows the partitioning of hydrophobic structures into the

central hydrophobic pseudophase core enabling solubility. This can lead to increased

dispersion of a compound in solution above its water solubility limit. This solubilization

can also lead to mobilization of sorbed and absorbed hydrophobic soil contaminants by

the lowering of capillary forces (Bai et al., 1998; Christofi and Ivshina, 2002). Fig 1.7

below shows an illustration to boost the points mentioned above.

Surfactant concentration

Fig. 1.7: Schematic diagram of the variation of surface tension, interfacial and contaminant solubility with surfactant concentration

(Source: Mulligan et al., 2001)

CMC

Micelle

Solubility

surface tension

Phys

ical

pro

perti

es

Interfacial tension

35

1.7.1 Biosurfactants and Oil Bioremediation

Bioremediation involves the acceleration of natural biodegradative processes in

contaminated environments by improving the availability of materials (eg. Nutrients and

oxygen), conditions (eg. pH and moisture content), and prevailing microorganisms (Ron

and Rosenberg, 2002). Hua et al., (2003) described bioremediation as the conversion of

chemical compounds by viable organisms, especially microorganisms with novel

catabolic functions derived through selections or by the introduction of genes encoding

such functions into energy cell mass and harmless biological waste product.

Bioremediation typically involves the augmentation of soil or other media, contaminated

with pollutants, with nutrients and sometimes microorganisms to improve the processes

for biodegradation of the contaminant. Biodegradation rate of a contaminant in the soil

depends on its bioavailability to the metabolizing organisms which is influenced by

factors such as desorption, diffusion and dissolution (Singh et al., 2006). Therefore, the

extent and rate of crude oil biodegradation depend on the oil low aqueous solubility and

strong adsorptive capacity to a soil (Gredraityte et al., 2001). In general, biodegradation

of hydrocarbons at any given site will depend upon the indigenous soil microbial

population, hydrocarbon variety and concentration, soil structure, nutrient availability and

oxygen availability (Kosaric, 2001; Christofi and Ivshina, 2002).

Petroleum bioremediation is carried out by microorganisms capable of utilizing

hydrocarbons as a source of energy and carbon. These microorganisms are ubiquitous in

nature and are capable of degrading the various types of hydrocarbons-short-chains, long-

chains and numerous aromatic compounds including polycyclic aromatic hydrocarbon.

All these compounds have low solubility in water (Ron and Rosenberg, 2002). Poorly

soluble in water, hydrocarbons remain partitioned in a separate non aqueous phase liquid

(NAPL), which may be present as droplets or films on soil particles and thus hardly

available to micro-organisms. Many studies on biodegradation of such compounds in soil

have shown that this slow release from the soil matrix to the aqueous phase is often the

rate limiting step in the process (Hua et al., 2003; Vasileva-Tonkova and Gesheva, 2004).

The first step in hydrocarbon degradation involves a membrane-bound oxygenase which

makes it essential for bacteria to come in direct contact with the hydrocarbon substrates

(Ron and Rosenberg, 2002). One promising approach to increasing bioavailability of

36

hydrophobic organic compounds is the addition of biosurfactants (Vasileva-Tonkova and

Gesheva, 2004).

The mechanism behind biosurfactants enhanced removal of oil from soil or which

biosurfactants are involved in bioremediation have been proposed to occur in two ways.

(i) Mobilization (Increasing the surface area of hydrophobic water-

insoluble substrates

(ii) Solubilization (Increasing the bioavailability of hydrophobic

compounds). (Ron and Rosenberg, 2002; Mulligan and Eftekhari,

2003; Urum and Pekdemir, 2004).

1.7.1.1 Increasing the surface area of hydrophobic water-insoluble substrates

Mobilization mechanism occurs at concentrations below the surfactant CMC.

Phenomena associated with this mechanism include reduction of surface and interfacial

tension, reduction of capillary force, wettability and reduction of contact angle (Urum et

al., 2004). When the interfacial tension is lowered mobility of petroleum or bound

hydrophobic molecules is enhanced (Mulligan et al., 2001; Ron and Rosenberg, 2002).

For bacteria growing on hydrocarbons, the growth rate can be limited by the interfacial

surface area between water and oil. When the surface area becomes limiting, biomass

increases arithmetically rather than exponentially. Emulsification is a cell-density-

dependent phenomenon; that is, the greater the number of cells, the higher the

concentration of extracellular product. The concentration of cells in an open system, such

as an oil-polluted body of water, never reaches a high enough value to effectively

emulsify oil. Furthermore, any emulsified oil would disperse in the water and not be more

available to the emulsifier producing strain than the competing microorganisms. If

emulsion occurs at, or very close to the cell surface and no mixing occurs at the

microscopic level, then each cluster of cells creates its own microenvironment and no

overall cell-density dependence would be expected (Ron and Rosenberg, 2002).

37

1.7.1.2 Increasing the bioavailability of hydrophobic water-insoluble substrates

Biosurfactants are more effective than chemical surfactants in increasing the

bioavailability of hydrophobic compounds. In addition, they are selective,

environmentally friendly and generally less stable than most synthetic surfactants (Calvo

et al., 2004; Ron and Rosenberg, 2002). One of the major reasons for the prolonged

persistence of high molecular weight hydrophobic compounds is their low water

solubility, which increases their sorption to surfaces and limits their availability to

biodegrading microorganisms. When organic molecules are bound irreversibly to

surfaces, biodegradation is inhibited. Biosurfactants can enhance growth on bound

substrates by desorbing them from surfaces or by increasing their apparent water

solubility (Ron and Rosenberg, 2002). Low molecular weight biosurfactants that have

low critical micelle concentration (CMC) increase the apparent solubility of

hydrocarbons by incorporating them into the hydrophobic cavities of micelles.

The solubility of oil increases dramatically due to the aggregation of surfactant

micelles. The hydrophobic end of the surfactant molecules cluster together inside the

micelle structure with the hydrophilic end exposed to the aqueous phase on the exterior.

Consequently, the interior of a micelle constitutes a compatible environment for

hydrophobic organic molecules, the process of incorporation of these molecules into a

micelle is known as solubilization (Mulligan and Eftekhari, 2003; Urum et al., 2004).

Solubilization occurs above the CMC, when contaminants are partitioned from soil into

the hydrophobic core of surfactant micelle (Kuyukina et al; 2004). Micellar phase

bioavailability of hydrophobic organics means that contaminants partitioned into the

micellar phase are biodegradable without having to transfer to the dissolved phase first

(Kuyukina et al., 2004).

1.7.2 Recovery of Biosurfactants

The recovery of biosurfactants depends mainly on their ionic charge, water

solubility and location (Intracellular, extracellular or cell bound) (Muthusamy et al.,

2008). The recovery and concentration of biosurfactants from fermentation broth largely

determines the production cost. Often, low concentration and the amphiphilic nature of

38

microbial surfactants limit their recovery. Different methods used for biosurfactants

isolation include high speed centrifugation, ultrafiltration, acid and salt precipitation,

crystallization, solvent extraction, adsorption chromatography and ammonium sulphate

precipitation (Kuyukina et al., 2001; Muthusamy et al., 2008).

The method used for the isolation of a certain product depends on the nature of

the compound; there are no rules for the isolation of biosurfactants (Kosaric, 1993). A

wide variety of organic solvents for example, methanol, ethanol, diethyl ether, pentane,

acetone, chloroform, dichloromethane, hexane and butanol have been used, either singly

or in combination for biosurfactant extraction (Kosaric, 1993; Desai and Banat, 1997;

Kuyukina et al., 2001). Most effective are the mixtures of chloroform and methanol in

various ratios, which facilitate adjustment of polarity of extraction agent to the target

extractable material. However, the solvents that are generally used for biosurfactant

recovery, for example, methanol and chloroform (a highly toxic chloro-organic

compound), are regarded as harmful to the environment and human health (Kuyukina et

al., 2001; Muthusamy et al., 2008). Cheap and less toxic solvents such as methyl tertiary-

butyl ether have been successfully used to recover the biosurfactants produced by

Rhodococcus (Muthusamy et al., 2008).

The advantages of ultrafiltration, adsorption-desorption on polystyrene resins and

ion exchange chromatography and adsorption-desorption on wood-based activated carbon

as a biosurfactant recovery strategy is their ability to operate in a continuous mode for

recovering biosurfactants with high level of purity (Muthusamy et al., 2008). Table 1.2

below shows the various processes of biosurfactant recovery and their advantages.

39

Table 1.2: Downstream processes for recovery of important biosurfactants and their

advantages

Process Biosurfactant Advantages

1 Ammonium sulfate

precipitation

Emulsan Effective in isolation of certain

type of polymeric biosurfactant

2 Acetone precipitation Bioemulsifier Efficient in crude biosurfactant

recovery and partial

purification, reusable nature

3 Acid precipitation Surfactin Low cost, efficient in crude

biosurfactant recovery

4 Solvent extraction Trehalolipids

Sophorolipids

Liposan

Efficient in crude biosurfactants

recovery and partial

purification, reusable nature

5 Crystallization Cellobiolipids

Glycolipids

6 Continuous mode

centrifugation

Glycolipids Reusable, effective in crude

biosurfactant recovery.

7 Adsorption Rhamnolipids

Lipopeptide

Glycolipids

Fast, one step recovery, high

level of purity and reusability.

8 Foam separation and

precipitation

Surfactin Useful in continuous recovery

procedures

9 Diafiltration and

precipitation

Glycolipids

10 Ultrafiltration Glycolipids Fast, one step recovery high

level of purity.

40

1.8 Rationale for Study

The cost of biosurfactant production is relatively high due to the source of

nitrogen compound used in its production. To produce biosurfactants economically,

increased yields are necessary. This study was aimed at exploring the use of cheaper

substrates for the production of biosurfactants.

1.9 Objectives

The ultimate goal of this research was to:

i. selectively isolate and characterize Pseudomonas species and compatible

Azotobacter from crude oil - contaminated sites.

ii. make a diculture of the Pseudomonas species and Azotobacter vinelandii for

the sole purpose of biosurfactant production whereby the diazotroph supplies

the needed nitrogen compound in a continuous energy-saving process

(continuous culture).

iii. precipitate and characterize the biosurfactants produced by the diculture.

41

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Soil Identity: Sandy loam soil.

2.1.2 Soil Sample: A portion of the soil was mapped out (20×30cm) and 200g of crude

oil (Bonny light) was used to contaminate the soil and left for four weeks.

2.1.3 Sampling of Soil: Soil samples (16.0g) were randomly collected from the

contaminated soil.

2.1.4 Equipment: All the apparatus used were available at the Microbiology and

Biochemistry Departments and also Lion’s water Ltd. in the University of Nigeria,

Nsukka Campus.

2.1.5 Chemicals: The chemicals used were of analytical grade. The agars (agar No. 2

and bacteriological peptone) were purchased from Lab M England, mannitol and other

mineral salts from Lab Tech Chemicals England, glycerol and sodium molybdate (Na2

MoO4) from May and Baker England and were all supplied by Joechem Chemicals Ltd.

Nsukka. The distilled water used in this study was purchased from Lion’s water and the

National centre for equipment maintenance and development, University of Nigeria,

Nsukka.

2.2 Preparation of Solutions

2.2.1 Methyl Violet

Methyl violet (0.5g) was measured and dissolved in 100ml of distilled water and

then filtered, stored in a labeled reagent bottle (Baker et al., 1998).

2.2.2 Lugol’s Iodine

Potassium iodide (4.0g) was dissolved in 50ml of distilled water, iodine (2.0g)

was added to it and was dissolved by shaking, and the volume was brought to 200ml

(Baker et al., 1998).

42

2.2.3 Acetone- Alcohol

A given quantity, 10 ml, of acetone was measured and added to 5ml of ethanol,

the volume was brought to 60ml by adding distilled water (Baker et al., 1998).

2.2.4 10% Hydrogen Peroxide

A volume of 50ml of 40% hydrogen peroxide (H2O2) was brought to a volume of

200ml using distilled water.

2.2.5 Bromothymol Blue Indicator

Bromothymol blue (0.1g) was dissolved in 2.5 of 0.1mol/L NaOH, the volume

was brought to 50ml by adding 47.5ml of sterile distilled water and was mixed

thoroughly.

2.2.6 Safranin

Safranin (1.0g) was dissolved in 200ml of distilled water, filtered and stored in a

well labelled reagent bottle.

2.3 Isolation of Biosurfactant-producing bacteria

2.3.1 Preparation of Pseudomonas broth

A selective medium broth was prepared for the isolation of Pseudomonas species.

The broth medium contains peptone- 2.2% (w/v), glycerol-1.1% (v/v), MgSO4.7H2O-

0.17% (w/v), K2HPO4-1.5% (w/v) and pH was adjusted to 7.2±0.2

2.3.2 Preparation of Azotobacter medium

Azotobacter medium was prepared for the isolation of Azotobacter vinelandii. The

broth medium contains Sucrose-1%(w/v), MgSO4.7H2O-0.02%(w/v), K2HPO4-

0.005%(w/v), KH2PO4-0.15%(w/v), CaCl2-0.002%(w/v), FeCl2-10ˉ7%(w/v), Na2MoO4-

0.002%(w/v).

The Pseudomonas and Azotobacter media was autoclaved at 121oC at 15psi for

sterility. Contaminated soil samples (2.0g) were added to 125ml of the broth in four

43

different conical flasks which served as the test. This procedure was carried out on the

Pseudomonas and Azotobacter broth media. It was thoroughly mixed by shaking and left

to stand for 96 hours at room temperature. After the 96 hours of incubation, the samples

were inoculated into appropriate Pseudomonas and Azotobacter agar media in sterile

Petri-dishes. The plates were incubated at room temperature for 24 hours. The colonies

were isolated and transferred into a fresh Pseudomonas and Azotobacter agar media in

bijou bottles for stocking.

2.4 Characterization of Isolates

Isolates were characterized and identified using conventional microbiological

procedures such as culture morphology, Gram staining reactions and biochemical tests

such as catalase test, sugar fermentation test, indole test, hydrogen sulfide test, motility

test and oxidase test.

2.4.1 Gram’s staining

Principle

Gram staining of bacteria divides bacteria into two categories, depending on

whether they can be decolourized with acetone alcohol after staining with one of the

rosaniline dyes such as crystal violet, methyl violet and treating with iodine. Those that

resist decolourization remain blue or violet in colour, and are designated Gram positive,

while those that are decolourized are termed Gram negative.

Method

A drop of sterile normal saline was dropped on a clean slide and the isolate was

collected using an inoculating loop. The isolate was emulsified on the slide containing the

normal saline forming a thin smear. It was left to dry and then heat fixed using a Bunsen

burner flame.

a) The slides were placed on a staining rack.

b) Methyl violet was applied on it for 30 – 60 seconds, drained off and washed with

water.

c) Lugol’s iodine was applied and allowed to act for 1 minute.

44

d) The above was rinsed off with water and acetone alcohol applied until no colour

appears to flow from the preparation for 30 seconds, before washing with water.

e) Safranin was applied for 2 minutes.

f) The above was rinsed with water, blotted carefully and fixed with gentle heat.

Finally, a drop of immersion oil was dropped on the slide and observed under

×100 objective lens of the microscope (Baker et al., 1998).

2.4.2 Catalase Test

Principle

Catalase is an iron- containing enzyme which catalyzes the decomposition of

hydrogen peroxide (H2O2) to water and oxygen. It is formed by most aerobic bacteria,

and it de-toxifies hydrogen peroxide produced by aerobic metabolism. The catalase test is

used to detect the presence of catalase in a given strain of bacterium. The presence of

catalase is indicated by bubbles of gas (oxygen) (Singleton, 2004).

Method

A loopful of isolates was individually emulsified on a clean slide with a drop of

sterile distilled water. A drop of 10% hydrogen peroxide was added. The presence of

catalase was indicated by a bursting bubble which gives rise to an effervescence.

2.4.3 Motility Test

Motility was tested by stab-inoculating the test organisms into a semi-solid

medium (SIM Medium) using a straight wire. A single straight stab was made at the

centre of the test tubes containing the SIM medium about half the depth of the medium.

The medium was then incubated at room temperature for 24 hours. Motility was detected

by the migration of the organism from the stab line and diffusion into the medium

causing turbidity and rendering the medium opaque. Non motile organisms gave growth

confined to the path of inoculation.

45

2.4.4 Indole Test

Principle

This is used in determining the ability of bacteria to break down the amino acid

tryptophan and liberate indole using the enzyme tryptophanase (Singleton, 2004). Indole

is generated by reductive deamination from tryptophan via the intermediate molecule

indole pyruvic acid. Tryptophanase catalyzes the deamination reaction during which the

amino (NH2) group of the tryptophan molecule is removed. Final products of the reaction

are indole, pyruvic acid, ammonia and energy. Pyridoxal phosphate is required as a co-

enzyme.

Method

This was carried out using the sulfide - indole – motility medium (SIM) in which

the organism was stab inoculated for motility. After 24 hours of incubation, 0.5ml

Kovac’s indole reagent was added and gently shaked to observe for a colour change. The

appearance of a red ring at the surface of the medium indicates a positive test, while for a

negative test, the orange colour of the Kovac’s reagent is retained.

2.4.5 Hydrogen Sulfide Test

Principle

It determines the ability of the microorganism to produce hydrogen sulfide. For

example, by the reduction of sulphate or from the metabolism of sulphur – containing

amino acids (Singleton, 2004).

Method

Sulfide–indole– motility medium was prepared and poured into test- tubes before

sterilization at 121oC for 15 minutes. The test organisms were aseptically inoculated into

the medium after cooling by stabbing using a sterile straight wire. The stab – inoculated

media were incubated at 37oC for 24 to 48 hours and then examined for a black colour

which indicates a positive result.

46

2.4.6 Sugar Fermentation Test

The sugar fermentation tests were carried out using 1% (w/v) of the sugars in

normal peptone water containing bromothymol blue indicator. The indicator was used to

detect acidification due to metabolism of the sugar (Singleton, 2004). The solutions were

dispensed in screw – capped bijou bottles with inverted Durham tubes for the collection

of gas that may be produced during the metabolism of the sugar. The contents in the

screw – capped bijou bottles were sterilized at 121oC for 15 minutes and allowed to cool

before inoculation. The test organisms were inoculated into the bottles and incubated for

24 hours at room temperature. A positive result was noted by a change in colour from

green to yellow for acid production, while gas production was indicated by a

displacement of the medium in the Durham tubes. Before incubation of the media,

absence of gas bubbles in the Durham tubes were confirmed. An uninoculated sterile

medium served as a control. The procedure was carried out for 6 (six) sugars namely

glucose, fructose, lactose, sucrose, sorbitol and mannitol.

2.4.7 Oxidase Test

Principle

This test detects a particular type of respiratory chain: one containing a terminal

cytochrome c and its associated oxidase (Singleton, 2004).

Method

A piece of filter paper was placed in a sterile dish and was flooded with oxidase

reagent (Tetra- methyl - paraphenylene - diamine dihydrochloride) and the test organism

smeared across the impregnated paper. A positive result is indicated by a deep purple

after 30 seconds.

2.5 Diculture of the Isolates

2.5.1. Preparation of amendment for the diculture

A Pseudomonas medium with reduction in the amount of nitrogen source was

prepared as follows; Peptone-1.1% w/v, Glycerol-1.1% w/v, MgSO4.7H2O-0.17% w/v

47

and K2HPO4-1.5% w/v. This is to find out the role of Azotobacter vinelandii present in

the diculture.

The isolates were grown to 500ml each.

Experimental design:

a) 500ml of pure culture of Pseudomonas species in four different conical flask.

b) 500ml of pure culture of Azotobacter vinelandii in four different conical flask.

c) The diculture containing 150ml of Pseudomonas species and 300ml of

Azotobacter vinelandii.

The various conical flask mentioned above was placed in a rotary shaker and

allowed to shake at a minimal speed for 168 hours.

The growth of the diculture was measured at 24 hours interval and a serial

dilution of 1:10 was made, the optical densities were read at a wavelength of 600nm and

their plate counts were taken on nutrient agar, Azotobacter and Pseudomonas agar media.

The plates were inoculated with 100µl of the dilutions using the pour plate method

described by Prescott et al., 2008. The plates were incubated for 120 hours before the

colonies were counted. This procedure was repeated for the Azotobacter vinelandii pure

culture and Pseudomonas pure culture. A serial dilution of 1:10 was made for the

Pseudomonas culture and its plate was incubated for 24 hours. A calibration curve was

drawn i.e, a graph of optical density against cell number.

2.6 Biosurfactant Activity Assay

Cells in the flasks were harvested by centrifugation at 6000rpm for 15 minutes and the

supernatant used as the biosurfactant solution. The test for the potency of the

biosurfactant was based on the following:

2.6.1 Haemolysis Test

An association between haemolytic activity and surfactant production was stated by

Carrillo et al. (1996) and Banat (1994) and they recommended the use of blood agar lysis

as a primary method to screen for biosurfactant activity (Youssef et al., 2004). The

supernatants were screened by plating cells on blood agar plates containing 5% (v/v)

human blood and incubated at room temperature for 24 hours. Haemolytic activity was

48

detected by the occurrence of a defined clear zone around a colony which is indicative of

biosurfactants (Maneerat and Phetrong, 2007).

2.6.2 Drop Collapse Test

Principle

It involves examining the capacity of a drop of culture broth from a putative

surfactant producer to collapse an aqueous droplet formed on a hydrophobic surface. The

surfactant in this case increases the contact angle and droplet collapse can be estimated as

a function of surfactant. The method is easy, rapid and yields relatively low numbers of

false positives (Dua et al., 2002). Therefore the drop collapse technique depends on the

principle that a drop of a liquid containing biosurfactant will collapse and spread

completely over the surface of oil (Youssef et al., 2004). A drop of water applied to a

hydrophobic surface in the absence of surfactants will form a bead. The bead is formed

because the polar water molecules are repelled from the hydrophobic surface. In contrast,

if the water droplet contains surfactant, the force or interfacial tension between the water

drop and the hydrophobic surface is reduced, which results in the spreading of the water

drop over the hydrophobic surface.

Method

Mineral oil (2µl) was added to each well of a 96 microwell plate lid. The lid was

equilibrated for 1 hour at room temperature, and then 5µl of the culture supernatant was

added to the surface of oil. The shape of the drop on the oil surface was inspected after 1

minute. Biosurfactant – containing cultures gave flat drops, thus a positive result.

2.6.3 Emulsification Test

It is the ability of a molecule to form a stable emulsion. The emulsification

activity is defined as the height of the emulsion layer divided by the total height and

expressed in percentage (Onwurah and Nwuke, 2004; Tabatabaee et al., 2005).

49

10024 xheightTotal

layeremulsiontheofHeightE

Method

Sterile biosurfactant solution (2ml) was added into each test-tube (in a set of

three) containing the substrate (crude oil and kerosene) 2ml. The content of the tubes

were vigorously shaked for uniformity for 2 minutes and left undisturbed for 24 hours.

The volume of oil that separated after 24 hours of standing was measured.

2.7 Purification and Extraction of Biosurfactants

2.7.1 By Acid Precipitation

The culture was centrifuged at 7000g for 15 minutes to remove cells. The

supernatant was then precipitated by acidification with hydrochloric acid to pH 2.0. It

was centrifuged at 7000g for 30 minutes, the precipitate was extracted with chloroform-

ethanol solvent (2:1). The precipitate was collected by extraction and was weighed

(Banat, 1994; Chen et al., 2004).

2.7.2 By Cold Acetone Precipitation:

The culture broth was refrigerated at 4oC and then centrifuged at 4000g for 30

minutes to remove the cells and filtered with sterile whatman No 1. filter paper. The clear

sterile supernatant served as the source of crude biosurfactant. The biosurfactant was

recovered from the cell free culture supernatant by cold acetone precipitation,2ml of

chilled acetone was added and allowed to stand for 10 hours at 4oC. The precipitate was

collected by centrifugation and evaporated to dryness to remove residual acetone after

which it was re-dissolved in sterile water (Okafor and Ejiofor, 1985; Healy et al., 1996;

Ilori et al., 2005).

2.8 Characterization of Biosurfactants

2.8.1 Biochemical composition of biosurfactant

The supernatant and precipitate was analyzed for protein, carbohydrate and lipid

content.

50

2.8.1.1 Determination of the protein content of the biosurfactant

Determination of the protein content of the biosurfactant was by the Biuret

commercial kit method as described by Lowry et al., 1951; Ohnishi and Barr, 1978).

Principle

This method is based on the principle that cupric ions in an alkaline medium

interact with peptide bonds of proteins resulting in the formation of a coloured complex.

Procedure

Distilled water (0.02ml) was pipetted into reagent blank (B) test-tubes only.

Standard solution (0.02ml) was added to another set of test tubes labeled STD (standard)

only. After which 0.02ml of the biosurfactant solution from the diculture and pure

cultures of the Pseudomonas and Azotobacter vinelandii were added to different test

tubes labeled SA (sample) only. Biuret reagent (1.0ml) was added to all the three sets of

test tubes. The content was mixed thoroughly and incubated for 30 minutes at 25oC.

Absorbance of the sample (Asample ) and the Standard (Astandard) against the reagent

blank was read at a wavelength of 530nm.

The Total Protein concentration was calculated as follows:

dardSofionConcentratAbsAbs

dlgoteinTotalofionConcentratdards

sample tan)/(Prtan

2.8.1.2 Determination of Carbohydrate Content by the Anthrone method (Snell and

Snell, 1962; Ilori et al., 2005)

Procedure

To 2ml of biosurfactant solution was added 3ml of distilled water and 10ml of

0.2% solution of Anthrone reagent (contains 0.2% of Anthrone in 95% H2SO4). Distilled

water served as the blank and the absorbance was read at 520nm. Glucose (2g/dl) was

used as standard.

dardSofionConcentratdardSofAbs

TestofAbsdlgsampletheinCHOofionConcentrat tantan

)/(

51

2.8.1.3 Determination of Lipid content by following the procedure of Chabrole and

Charnnat, 1937.

Principle

It is based on the colorimetric determination of total lipids with sulfo- phosphovanilic

mixture.

Procedure

To 100µl of biosurfactant solution was added 2.9ml of Conc. H2SO4 and boiled

for 10 minutes. It was left to cool and 2.5ml of phosphovanillin reagent (contains 20.0ml

of 0.6% vanillin in 80ml of phosphoric acid)was added to 0.1ml of the cooled solution

and was incubated in the dark for 45 minutes at room temperature. The absorbance was

read at 532nm against the reagent blank.

The result was compared with the Standard curve.

2.9 Estimation of Biomass

Discarded cells from the purification of the biosurfactant process were centrifuged

at 4000rpm for 25 minutes and extracted with a mixture of acetone/hexane (3:1) to

remove adhering hydrocarbon. This was followed with drying overnight to obtain the dry

biomass which was weighed.

52

CHAPTER THREE

RESULTS

3.1 Characterization of bacterial isolates

A total of nine (9) isolates were isolated but for the purpose of this study three (3)

isolates were used. Namely;

C1A – Azotobacter vinelandii strain

C1P – Pseudomonas spp strain 1

NC11P – Pseudomonas spp strain 11

The biosurfactant – producing bacteria isolates C1P and NC11P from the oil –

contaminated soil had a creamy raised colony and smooth edged, Gram negative rods,

oxidase and catalase positive, an acid – gas organism that is able to utilize the following

sugars namely; mannitol, sorbitol, sucrose, lactose, fructose and glucose. The bacteria

Pseudomonas spp was identified by the Bergey’s manual.

The microscopic and biochemical examinations of Azotobacter vinelandii isolates

showed a short rod that is motile, catalase and indole positive, utilizes mannitol and

sorbitol and the extract readily fixed atmospheric nitrogen as ammonia.

The physiological and biochemical characteristics of Pseudomonas spp and Azotobacter

vinelandii isolated are shown in Table 3.1.

53

Table 3.1: Characterization of bacterial isolates.

Isolates

Dia

met

er

Colony & Morphology Shape

Gra

m st

ain

Mot

ility

Cat

alas

e

Oxi

dase

Indo

le

Hyd

roge

n Su

lphi

de Sugar Fermentation Test

Man

nito

l

Sorb

itol

Sucr

ose

Lact

ose

Fruc

tose

Glu

cose

C1A 1-2mm

Colourless flat colony, Smooth

edge Short rod -ve +ve +ve +ve +ve Acid Gas Acid Gas Acid Acid Gas Acid

NC11A 1-2mm

Colourless raised colony, Smooth

edge Cocco-bacilli -ve +ve +ve -ve +ve

+ve (Black) -ve Gas -ve -ve -ve Gas

C1P 1-4mm

Creamy raised colony, Smooth

edge Long rod -ve +ve +ve +ve +ve +ve

(Black) Gas Acid Gas

Acid Gas Acid -ve

Acid Gas

NC11P 1-3mm

Creamy raised colony, Smooth

edge Long rod -ve +ve +ve +ve +ve +ve

(Black) Acid Gas Acid Gas Acid

Acid Gas Acid

Acid Gas

NCIVA 1-2mm

Colourless flat colony, Smooth

edge short rod -ve +ve +ve +ve -ve -ve Gas -ve -ve Acid

C1VA 1-2mm

Colourless flat colony, Smooth

edge Cocci +ve +ve +ve +ve +ve -ve Acid Gas Acid -ve Acid

C111P 1-3mm

Creamy raised colony, Smooth

edge Cocci +ve +ve +ve -ve +ve -ve Acid Acid Gas

Acid Gas

Acid Gas

Acid Gas

NC1P 1-3mm

Creamy raised colony, Smooth

edge Long rod -ve +ve +ve -ve +ve +ve

(Black) Acid Gas Acid Gas

Acid Gas Acid

Acid Gas

Acid Gas

NC111P 1-4mm

Creamy raised colony, Smooth

edge Long rod -ve +ve +ve +ve +ve +ve

(Black) Gas Gas Acid Gas Gas

Acid Gas Acid

54

3.2 Time course of growth of the broth cultures

3.2.1 Time course of growth of the pure strains of Pseudomonas spp on

Pseudomonas medium:

Figure 3.1 shows the growth of isolate C1P and NC11P identified as Pseudomonas

spp in a selective medium for Pseudomonas. The growth was rapid and turbidity was

observed within 24 hours of inoculation.

Fig 3.1: Time course of growth of the pure strains of Pseudomonas spp on Pseudomonas medium.

55

3.2.2. Time course of growth of Azotobacter vinelandii in Azotobacter medium:

Figure 3.2 shows the growth of the Azotobacter vinelandii strain – C1A. The growth

was quite slow which reached a peak on the 8th day after initial inoculation.

Fig 3.2: Time course of growth of Azotobacter vinelandii in Azotobacter medium.

56

3.2.3. Time course of growth of Pseudomonas spp on Pseudomonas medium with

inadequate nitrogen source

Figure 3.3 shows the growth of the isolate NC11P in Pseudomonas medium with

inadequate nitrogen source. Turbidity was observed after 24 hours of initial inoculation

but the growth was not as rapid like that of Fig 3.1. The peak growth was reached on the

6th day but the maximum peak was reached on the 10th day. It was observed that nitrogen

as a nutritional requirement for the growth of organisms is needed for the proper growth

and reproduction of microorganisms

Fig 3.3: Time course of growth of Pseudomonas spp in Pseudomonas medium with inadequate nitrogen source.

57

3.2.4. Time course of growth of the diculture of Pseudomonas spp with inadequate

nitrogen source and Azotobacter vinelandii:

Figure 3.4 shows the growth of the diculture (i.e. a mixed culture containing the

Pseudomonas spp with inadequate nitrogen and Azotobacter vinelandii). The Azotobacter

vinelandii co – existed in this medium by fixing the nitrogen needed for the growth of the

culture.

It was observed that the diculture exhibited a better growth in the production of

biosurfactants. The peak growth was reached at an optical density of 1.770 when the cells

got to their early stationary phase before it showed a decline. The decline could be as a

result of the accumulation of waste in the culture medium that was produced by the

organism; this does not favour the growth of microorganism.

Fig 3.4: Time course of growth of the diculture of Pseudomonas spp with inadequate nitrogen source and Azotobacter vinelandii.

58

3.2.5. Time course of growth of the diculture in medium supplemented with

mannitol and crude oil

Figure 3.5 shows the growth of the diculture in the supplemented medium. The

growth with the diculture + mannitol gave similar trend with Fig 3.4 but the diculture +

crude oil produced the highest growth yield in the production of biosurfactants. Crude oil

was a better source of carbon because the culture has adapted to it. The sharp decline

observed could be as a result of the depletion of nutrients due to the rate of growth,

thereby leading to the accumulation of toxic metabolic products which inhibited the free

flow of oxygen into the medium, thus leading to a decline in the growth of the organism.

Fig 3.5: Time course of growth of the diculture in medium supplemented with mannitol and crude oil.

59

3.3. Statistical Analysis

The time course of growth of the bacteria was statistically analyzed using

correlation analysis.

3.3.1. Statistical analysis of the pure strains of Pseudomonas spp with adequate

nitrogen:

The analysis of the growth of isolates NC11P and C1P showed that they are positively

and strongly correlated. This is because the two isolates have identical external factors

which increased simultaneously and proportionally.

The result of Figure 3.6 could be said to highly positively correlated.

Fig 3.6: Correlation analysis between isolate C1P and NC11P with adequate nitrogen 3.3.2. Statistical analysis between the amended Pseudomonas medium with

inadequate nitrogen and isolate NC11P:

It was observed that the Pseudomonas spp in Fig 3.1 gave a rapid growth when compared

to Fig 3.3. due to sufficient nitrogen source as bacteriological peptone which has 14%

nitrogen constituent.

From the correlation graph of Figure 3.7, it shows that they are weakly but positively

correlated.

60

Fig 3.7: Correlation analysis between NC11P* and NC11P

3.3.3. Statistical analysis of the growth of the pure Pseudomonas strain isolate

NC11P and the diculture:

The correlation analysis between the time course of growth of the Pseudomonas

strain isolate NC11P and diculture as shown in Figure 3.8 gave a weak but positive

correlation.

Fig 3.8: Correlation analysis between NC11P and diculture

61

3.3.4. Statistical analysis of the diculture and diculture + mannitol:

The correlation analysis between the diculture and the diculture medium

supplemented with mannitol as shown in Figure 3.9 gave a positive but fairly weak

correlation.

Fig 3.9: Correlation analysis between diculture and diculture + mannitol 3.3.5. Statistical analysis of diculture and the diculture supplemented with crude oil:

The correlation analysis gave a weak or low but positive correlation but when

compared to Fig 3.1, it gave a strong and positive correlation.

Fig 3.10: Correlation analysis between diculture and diculture + crude oil

62

In summary, the statistical analysis showed that for the growth of Pseudomonas spp

adequate nitrogen source is needed.

3.4 Test for biological nitrogen fixation

The ability of Azotobacter vinelandii to fix nitrogen was investigated. The pure

culture of the Azotobacter vinelandii showed minimal nitrogen fixation but nitrogen

fixation occurred in the diculture medium (containing the amended Pseudomonas spp

medium and Azotobacter vinelandii medium), as identified by moistened red litmus

paper. The medium turned the red litmus paper to blue indicating the presence of

ammonia.

3.5 Test for Biosurfactant Activity

3.5.1. Drop Collapse Test

A drop of water applied to a hydrophobic surface in the absence of surfactants will

form a bead. The bead is formed because the polar water molecules are repelled from the

hydrophobic surface. In contrast, if the water droplet contains surfactant, the force or

interfacial tension between the water drop and the hydrophobic surface is reduced, which

results in the spreading of the water drop over the hydrophobic surface.

The bacterial isolates C1P, C1A and NC11P, including the diculture tested positive to the

drop collapse test as shown in Table 3.2. The diculture was highly positive which could

be as a result of the mixed culture. It is also important to know that the amount of

biosurfactant required to cause drop collapse is dependent on the ability of the surfactant

to reduce surface and interfacial tension.

3.5.2. Emulsification Test (E24)

The emulsification values were taken after 24 hours. The emulsification of the

hydrocarbons were in the order of crude oil< kerosene < water as shown in Table 3.2.

NC11P gave the highest emulsification value of 84% whereas the diculture gave the

highest emulsification value for kerosene and water of 74.4% and 94.87% respectively.

63

3.5.3. Haemolysis Test:

All the isolated strains were tested for haemolytic activity, which is regarded by

some authors as indicative of biosurfactant production and used as a rapid method for

bacterial screening (Tabatabaee et al., 2005; Sepahi et al., 2008). The test isolates were

positive to the haemolysis test which gave clear zones on the solid agar medium.

Table 3.2: Test for Biosurfactant Activity

3.6 Characterization of Biosurfactants

Biochemical composition of the biosurfactant revealed that it is a mixture of

carbohydrate, lipid and protein in a combination of 61%: 30%: 9% respectively as shown

in Table 3.3.

Test For Biosurfactant Activity

Isolates Drop Collapse

Emulsification Test

Oil Spreading Haemolysis Crude oil Kerosene Water

C1P ++ 72.20% 64.10% 92.11% 0.7 ++

C1A ++ 64.30% 51.72% 94.74% 0.5 ++

DICULTURE +++ 83.30% 74.40% 94.87% 1.2 ++

NC11P ++ 84.20% 71.10% 94.74% 0.8 ++

64

Table 3.3: Characterization of Biosurfactant.

Characterization Of Biosurfactants Biochemical Composition

Isolates Protein Carbohydrate Total Lipid C1A 0.17g/dl 2.79g/dl 0.60g/dl

NC11P 0.51g/dl 1.05g/dl 1.20g/dl DICULTURE 0.34g/dl 2.06g/dl 0.80g/dl

CHARACTERIZATION AFTER PURIFICATION AND EXTRACTION

Isolate Protein Carbohydrate Total Lipid DICULTURE 0.34g/dl 2.43g/dl 1.20g/dl

Weight of precipitate= 1.180g Weight of precipitate (crude) = 3.830g

Weight of the biomass of the cells= 0.031g Weight of the biomass of the cells (crude) = 0.066g

65

CHAPTER FOUR

DISCUSSION

Pseudomonas spp are hydrocarbonoclastic microorganisms and are the best

known bacteria capable of utilizing hydrocarbons as carbon and energy sources and

producing biosurfactants to enhance the uptake of such immiscible hydrophobic

compounds (Yataghene et al, 2008). Crude oil served as a good source of carbon for

biosurfactant production by the Pseudomonas spp. Nitrogen is an important nutrient that

often limits microbial activity because it is an essential part of many key microbial

metabolites and building blocks including proteins and amino acids (Pepper et al., 1996).

The Pseudomonas medium with inadequate nitrogen source showed a slow growth rate,

while maximum growth was reached at the 6th day before the cells were transferred into a

fresh medium of identical composition while they were still growing exponentially,

thereby causing maintenance of cells (Brooks et al., 2007) This was done to keep the

cells active for the mixed culture.

The associative growth of Azotobacter vinelandii with Pseudomonas spp gave

greater growth and nitrogen fixation than the pure cultures as was previously reported

(Onwurah, 1999). This goes to suggest that Azotobacter vinelandii provided fixed

nitrogen that was utilized by Pseudomonas spp during growth and subsequent production

of biosurfactants. The pure culture of Azotobacter vinelandii showed minimal nitrogen

fixation and this could be as a result of nutrient depletion relative to the diculture. In

contrast, nitrogen- fixing potential increased in the diculture with the Azotobacter

vinelandii medium to support the growth of Pseudomonas spp and the increased

production of biosurfactants (Eze and Onwurah, 2004). It was explained that Azotobacter

vinelandii has a greater propensity to survive in natural crude oil polluted environment

containing hydrocarbonoclastic bacteria. Allison and Burris (1957) reported that actively

growing cells of the Azotobacter excrete a small and constant proportion of the nitrogen

they fix, thus enabling events in the extracellular metabolic pool to be traced. Nitrogen is

reduced directly to ammonia in the fixation process. This ammonia is the primary source

of the organically bound nitrogen in Azotobacter vinelandii cultures which are actively

fixing molecular nitrogen as the sole source of nitrogen. This is seen in the reaction

catalyzed by nitrogenase,

66

N2 + 6H+ + 6e- + 12ATP — 2NH3 + 12ADP + 12Pi

The nitrogenase complex which carries out nitrogen fixation is derived from

Azotobacter vinelandii. The nitrogenase is a complex of two enzymes; one enzyme

contains iron and the other contains iron and molybdenum. Increased nitrogen fixation is

well correlated with cell growth and proliferation, hence increased nitrogen fixation in

the diculture medium reflected a corresponding increase in the Azotobacter vinelandii

population.

The Pseudomonas spp as previously suggested utilized the nitrogen produced by

Azotobacter vinelandii in the medium. It should be recalled that since nitrogen is usually

not a part of the precursor metabolites or intermediate building blocks in most bacterial

biosynthetic pathways, it must be provided for externally, either through inorganic or

organic sources. It is because of this that nitrogen is considered a limiting nutrient in most

bacterial growth experiments (Neidhardt et al., 1990). Nitrogen and phosphorus are also

said to be the most limiting inorganic nutrients for crude oil degrading bacteria

(Onwurah, 1999) and are needed for growth of microorganisms. The interaction between

Pseudomonas spp and Azotobacter vinelandii in the diculture is as a result of co-

metabolism which is the simultaneous metabolism of two compounds, in which the

degradation of the second compound (the secondary substrate) depends on the presence

of the first compound (the primary substrate) or the metabolic transformation of a

substance while a second substance serves as primary energy or carbon source (Ryoo et

al., 2000). When the strains were tested for biosurfactant activity, it was observed that

Azotobacter vinelandii also produced biosurfactants. Hence, the diculture gave a greater

yield of biosurfactants since Azotobacter vinelandii produces biosurfactant containing

fatty acid and exopolysaccharide compound (Helmy et al., 2008) and it is possible that

the increase was due to the presence of the nitrogen from Azotobacter which provided a

better environment for the growth of the Pseudomonas. The significant growth of the

diculture was due to the availability of carbon and nitrogen sources. Christofi and Ivshina

(2002) stated that the factors controlling the production of biosurfactants include the

quality and quantity of carbon and nitrogen constituents in cultures and natural physical

67

environment of soil systems (such as temperature, pH) and that the type of nitrogen

present (whether ammonia, nitrate, urea or amino acid) influences the biosurfactant

produced. The diculture showed a mutual relationship. In this case, Pseudomonas spp

made carbon source available for the utilization and growth of Azotobacter vinelandii,

while Azotobacter vinelandii fixed nitrogen. There was continuous supply of fixed

nitrogen in the diculture medium to support the growth of Pseudomonas spp and the

increased production of biosurfactants. Excreted nitrogen compounds which are found as

high molecular weight compounds have been shown to be available to bacteria such as

Pseudomonas, growing in association with Azotobacters and they include aspartic acid

and traces of hydroxylamine (Onwurah, 2004). It has been shown that biosurfactants are

produced during the early stationary phase of cell growth (Thavasi et al., 2007). At this

phase, new cell materials are usually synthesized at a constant rate and the mass increases

in an exponential manner, and this continues until one of two things happens; either one

or more nutrients in the medium become exhausted or toxic metabolic products

accumulate and inhibit growth. For aerobic organisms like Pseudomonas and Azotobacter

vinelandii, the nutrient that becomes limiting is usually oxygen.

The exhaustion of these nutrients or the accumulation of toxic products causes

growth to cease completely. This is noticed by the decline in the growth that is observed

in Figures 3.1 to 3.5 showing the time course of growth of the organism. This decline

maybe as a result of depletion of metabolites and nutrients as a result of unfavorable

conditions that do not support growth.

Crude oil served as a good substrate for emulsification by the biosurfactant.

Microbial molecules which exhibit high surface activity and emulsifying activity are

classified as biosurfactants. These molecules reduce surface and interfacial tensions in

both aqueous solutions and hydrocarbon mixtures making them potential agents for

bioremediation. In the estimation of the emulsification index (E24), after 24 hours, an

84% increase was observed for isolate NC11P while the diculture had 83%. Most

microbial surfactants are substrate specific, solubilizing or emulsifying different

hydrocarbons at different rates. An emulsion is formed when one liquid phase is

dispersed as microscopic droplets in another liquid continuous phase. The high

emulsification rate seen in the diculture and isolate NC11P could be as a result of the

68

exopolysaccharide compound contained in the biosurfactant from both Azotobacter

vinelandii and Pseudomonas spp. Exopolysaccharide increases the dispersion and

solubility of oil (Helmy et al., 2008). Relatively poor emulsification of kerosene might be

due to the fact that the strains are adapted to crude oil from their cultivation and the

inability of the biosurfactant to stabilize the microscopic droplets.

The solubility of biosurfactant to water was observed. Water is a polar solvent,

therefore, the aqueous solubility depends strongly on the degree of polarity of their

molecules. The biosurfactant was miscible with water. A miscible organic liquid is one

that can be mixed with water such that a single liquid phase results. Azotobacter

vinelandii was the best source of nitrogen for biosurfactant production by the

Pseudomonas spp since it is an organic source of nitrogen. Mannitol, a water soluble

carbon source affected biosurfactant production by the organism. This clearly indicates

that biosurfactants may be produced using non-hydrocarbon substrates. Biosurfactants

produced from water-soluble substrates have been reported to be inferior to that obtained

with water immiscible substrates (Ilori et al., 2005). Such biosurfactants may however be

cheaper to produce and useful in food and pharmaceutical industries as it will not require

extensive purification. The best growth was observed in Fig 3.5 showing that diculture

medium supplemented with crude oil could be used to produce greater quantity of

biosurfactants, this is because the organism has been adapted to crude oil.

The biosurfactant produced by the diculture was classified as a carbohydrate –

protein – lipid complex. Some Pseudomonas spp produce this type of biosurfactant

(Desai and Banat, 1997; Garti, 1999; Mulligan et al., 2001). Other microorganisms that

produce this type of biosurfactant include Ustilago maydis, and Bacillus subtilis. One

interesting aspect of this method of biosurfactant production is that once the right source

of cheap hydrocarbon is in place, the diculture continues to produce biosurfactant in an

energy-saving, continuous process.

In conclusion, biosurfactant – producing bacteria appear to be found in soil which

has been exposed to hydrocarbon contamination and crude oil seems to be the best

substrate for the production of biosurfactants. Results obtained in the biosurfactant

production with a diculture of Pseudomonas species and Azotobacter vinelandii

suggested the possibility of greater production of biosurfactant using Azotobacter

69

vinelandii as the sole nitrogen source in a continuous process. From the study, isolates

NC11P, C1P, and C1A which are Pseudomonas spp and Azotobacter vinelandii have the

potential to produce biosurfactants for use in bioremediation of crude oil – polluted

environment, and other important industrial processes.

70

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APPENDIX

Appendix I: The calibration curve for the serial dilutions of the culture medium

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Appendix II: Calibration curve for total lipid