i

Digitally Signed by: Content manager’s Name

DN : CN = Weabmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

Nwamarah Uche

Faculty of Biological Science,

Department of Biochemistry

PRODUCTION AND CHARACTERIZATION OF

BIOSURFACTANT BY PSEUDOMONAS AERUGINOSA

USING RED CASHEW POMACE AS SUBSTRATE

OKUDA, FRANK

(PG/MSc/12/63547)

ii

TITLE PAGE

PRODUCTION AND CHARACTERIZATION OF BIOSURFACTANT BY PSEUDOMONAS

AERUGINOSA USING RED CASHEW POMACE AS SUBSTRATE

A RESEARCH PROJECT SUBMITTED IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE (M.Sc) IN

INDUSTRIAL BIOCHEMISTRY AND BIOTECHNOLOGY

BY

OKUDA, FRANK

(PG/MSc/12/63547)

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

iii

CERTIFICATION

This is to certify that this research work titled “Production and Characterization of Biosurfactant

by Pseudomonas aeruginosa using Red cashew pomace as Substrate” was carried by Okuda

Frank, under my supervision in the Department of Biochemistry, Faculty of Biological Science,

University of Nigeria, Nsukka.

---------------------------------------- ----------------------------------------

Prof. O.U. Njoku Dr. V.N. Ogugua (Supervisor) (Supervisor)

---------------------------------------- ------------------------------------- Prof. OFC Nwodo External Examiner (Head of Department)

iv

DEDICATION

This project is dedicated to the glory of God Almighty whose light has shone constantly and led

me to the path of success. I also dedicate it to my beloved parents, Rev and Mrs. Okuda, O.

Sunday, who supported me morally, financially and in prayers throughout the programme.

v

ACKNOWLEDGEMENT

I owe my gratitude to numerous individuals whose support, assistance and

encouragement made this research work a reality. First of all, my sincere gratitude goes to my

supervisors, Prof. O.U. Njoku and Dr. V.N. Ogugua who despite other academic activities and

commitments guided me throughout this research work

I am indeed very grateful to my parents, Rev and Mrs Sunday Okuda for their

unconditional love, support and provision throughout the period of this programme. My success

won’t be complete if I fail to appreciate my siblings, Mrs. Abigail Isoje, Oghenekaro Ferdinand,

Eucharia Oreva and Christiana Aghoghomena for their love and encouragement

I am deeply grateful to the Head, Department of Biochemistry, Prof. OFC Nwodo and to

all the staff for their love, contributions and solutions to the challenges encountered during this

research and for the knowledge they imparted on me.

I want to say a big thank you to Okechukwu Iroha, Iruoghene Onosakponome, Okezi

Obara, Jamila Ekpete, Solomon Odiba, Chimere Ukegbu, Jennifer Kanu, Sandra Ekpechi and to

all my friends who have in one way or the other contributed to the success of this research work.

My heartfelt appreciation goes to my best friend Enifome Okumor, for her encouragement and

support throughout the duration of this work. Above all, I thank Almighty God for His blessings

and protection.

vi

ABSTRACT

Biosurfactants are amphipathic compounds produced extracellularly by microorganisms on cell surfaces, or excreted extracellularly. They contain hydrophilic and hydrophobic moieties that reduce surface and interfacial tension between molecules at the surface and interface respectively. The present study was focused on development of economical methods for biosurfactant production by the use of unconventional substrates. The research investigated the potential of utilizing agroindustrial (red cashew pomace) wastes to replace synthetic media for cultivation of Pseudomonas aeruginosa and biosurfactant production. The organism was able to grow and produce surfactant. The pseudomonas strains were screened for biosurfactant activity using haemolysis and oil spreading test. The surfactant was able to form emulsions with various vegetable oils and hydrocarbons being more effective against palm oil (70.3 ±0.57), olive oil (65.3 ±0.57) and kerosene (60.0 ±0.57). The surface-active compound retained its properties during exposure to elevated temperatures (up to 100°C), relatively high salinity (8% NaCl) and a wide range of pH values (2-12). The biosurfactant was extracted after 10 days using chlorofoam: methanol and the dry weight was calculated as 1.0g/L. Preliminary characterization by the use of basic biochemical tests revealed that the compound is a glycolipid. The biosurfactant produced was used in this study to explore the possible potential for cleaning up pesticides (chlorpyrifos) residue in tomatoes. Different concentration of biosurfactant solution (5ppm, 10ppm, 20ppm and 40ppm) were able to reduce 100ppm pesticide (chlorpyrifos) contaminated tomatoes to below maximum residue limit of 0.5ppm. The results of this study suggest the possible use of red cashew pomace in biosurfactant production and its useful properties in environmental and industrial application.

vii

TABLE OF CONTENT

Title Page i

Certification ii

Dedication iii

Acknowledgement iv

Abstract v

Table of Content vi

List of Figures x

List of Tables xi

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW

1.0 Introduction 1

1.1 Biosurfactant and Classification 2

1.1.1 Glycolipids 2

1.1.2 Phospholipids, Lipopeptides and Polymeric Biosurfactants 4

1.2 Biosurfactant Producing Micro-Organisms 4

1.3 Properties of Biosurfactants 6

1.3.1 Surface and Interface Activity 6

1.3.2 Temperature, pH and Ionic Strength Tolerance 7

1.3.3 Biodegradability 7

1.3.4 Low Toxicity 7

1.4 Factors Affecting Biosurfactant Production 8

1.4.1 Nature of Carbon Source 8

1.4.2 Nitrogen Source 9

1.4.3 Effect of pH 9

1.4.4 Effect of Temperature 9

1.4.5 Effect of Agitation and Aeration of Biosurfactants 10

1.4.6 Metal ion Concentration 10

viii

1.5 Applications of Biosurfactants 10

1.5.1 Bioremediation Applications 10

1.5.1.1 Application in Biodegradation Process 10

1.5.1.2 Application in Microbial Enhanced Oil Recovery 12

1.5.1.3 Application in Agriculture 12

1.5.2 Therapeutic and Biomedical Applications 13

1.5.3 Miscellaneous Applications of Biosurfactants 13

1.5.3.1 Application in Cosmetics Industries 13

1.5.3.2 Application in Food-processing Industries 13

1.5.3.3 Application in Commercial Laundry Detergent 14

1.5.3.4 Application as Biopesticides 14

1.6 Economic Factors of Biosurfactant Production 14

1.7 Cashew (Anacardium occidentale) 16

1.7.1 Origin, Taxonomy and Morphology 16

1.7.2 Chemical Composition and Uses of Cashew Fruits 16

1.8 Fermentation Process for Biosurfactant Production 16

1.9 Aim and Objectives 17

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials 18

2.1.1 Plant Materials 18

2.1.2 Microorganism 18

2.1.3 Instruments/Equipment 18

2.1.4 Chemicals 19

2.2 Methods 20

2.2.1 Preparation and Processing of Plant Material 20

2.2.2 Proximate Analysis 20

2.2.2.1 Moisture Content 20

2.2.2.2 Fibre Content 20

ix

2.2.2.3 Ash Content 20

2.2.2.4 Protein Content 21

2.2.2.5 Lipid Content 21

2.2.2.6 Carbohydrate Content 21

2.2.3 Reactivation of Pseudomonas aeruginosa by Subculture 21

2.2.4 Preparation of Pseudomonas aeruginosa Seed Culture and its Growth Rate

Determination 22

2.2.5 Media Preparation 22

2.2.5.1 Preparation of Basal Mineral Medium (B.M.M) and Carbon source 22

2.2.5.2 Preparation of the Different Culture Media 23

2.2.6 Screening the Culture Broth for Growth of Pseudomonas aeruginosa 23

2.2.7 Screening of the Supernatant for Biosurfactant Activity 23

2.2.7.1 Haemolysis Test 24

2.2.7.2 Oil Spreading Test 24

2.2.7.3 Emulsification Index Test 24

2.2.7.4 Stability Test 24

2.2.8 Extraction of Biosurfactants 25

2.2.9 Characterization of the Isolated Biosurfactant 25

2.2.9.1 Carbohydrate Content 25

2.2.9.2 Protein Content 25

2.2.9.3 Lipid Test 25

2.2.10 Study of Biosurfactant as a Cleaning Agent 26

2.2.10.1 Preparation of Chlorpyrifos Stock Solution and Standard Curve 26

2.2.10.2 Preparation of Chlorpyrifos Solution 26

2.2.10.3 Preparation of Washing Solutions 26

2.2.10.4 Extraction of Pesticide Residue 26

2.2.10.4 Determination of Pesticide Residue 27

2.2.11 Statistical Analysis 27

CHAPTER THREE: RESULTS

3.1 Proximate Composition 28

3.2 Determination of Growth in the Seed Culture 28

3.3 Growth Rate Determination of Pseudomonas aeruginosa in the Different

Culture Media 28

x

3.4 Screening for Biosurfactant Activity in Culture Broth 28

3.4.1 Haemolysis Test 28

3.4.2 Oil Spreading Test 28

3.4.3 Emulsification Index Test (E24) 34

3.4.4 Stability Studies 34

3.4.4.1 Effect of Temperature on Biosurfactant and SDS Activity 34

3.4.4.2 Effect of pH on Biosurfactant Activity 34

3.4.4.3 Effect of Salinity on Biosurfactant Activity 34

3.5 Quantification of Biosurfactant 39

3.6 Biochemical Composition of Isolated Biosurfactant 39

3.7 Effect of Washing Solutions on Pesticide Residue 39

CHAPTER FOUR: DISCUSSION

4.0 Discussion 41

4.1 Conclusion 45

REFERENCES 46

APPENDICES 53

xi

LIST OF FIGURES

Figure 1.1: The Four Major Rhamnolipids 3

Figure 1.2: Mechanisms of Hydrocarbon removal by Biosurfactants 11

Figure 1.3: Mechanism of oil recovery by Biosurfactants 12

Figure 3.1: Growth of Pseudomonas aeruginosa in the seed culture at OD600nm 30

Figure 3.2: Growth curve of Pseudomonas aeruginosa in each of the culture broths 31

Figure 3.3: Emulsification indices (%) of culture broth supernatant and SDS 35

Figure 3.4: Determination of temperature stability of culture broth supernatants and SDS 36

Figure 3.5: Effect of pH on Biosurfactant activity 37

Figure 3.6: Effect of salt on Biosurfactant activity 38

Figure 3.7: Effect of different washing solutions 40

xii

LIST OF TABLES

Table 1.1: List of Biosurfactant Producing Organisms 5

Table 1.2: Substrate for Microbial Surface Active Agents and Their End Products 15

Table 3.1: Proximate Composition of Red Cashew Fruit Pomace 29

Table 3.2: Red Blood Cell Lysis by the Various Culture Broth Supernatants 32

Table 3.3: Determination of Oil Spreading Capacity of the Various Culture Broth Supernatant 33

1

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.0 Introduction

Biosurfactants are naturally surface-active compounds derived from microorganisms

(Anandaraj and Thivakaran, 2010). They are amphiphilic compounds produced mostly on

microbial cell surfaces or excreted extracellularly and contain hydrophobic and hydrophilic

moieties that reduce surface and interfacial tensions between two immiscible fluids like oil and

water (Anyanwu et al., 2011; Govindammal, 2013). Biosurfactants are classified based on their

chemical structure, molecular weight, physico-chemical properties and mode of action and

microbial origin (Calvo et al., 2009). Their chemical composition is very unique in that they

contain a hydrophilic moiety, comprising an acid, peptide cations or anions, mono-, di- or

polysaccharides and they also contain a hydrophobic moiety comprising of unsaturated or

saturated hydrocarbon chains or fatty acids (Saharan et al., 2011). The upsurge on replacement

of synthetic surfactant with their biological counterparts (Biosurfactants) is due to the latter’s

better characteristics such as low toxicity, higher biodegradability and mild process conditions,

higher foaming capacity, temperature, pH and salinity stability and synthesis under user-friendly

conditions (Parveen et al., 2011; Chandran and Das, 2010). On the other hand, different

microorganisms are known to synthesize different types of biosurfactants when grown on

several carbon sources, therefore the type, quality and quantity of biosurfactant produced are

also influenced by the nature of the carbon substrate and the culture conditions such as pH,

temperature, agitation and dilution rate in continuous culture (Lakshmipathy et al., 2010).

Considerable attention has been given in the past to the production of surface-active molecules

of biological origin because of their potential utilization in food processing, pharmacology,

cosmetic, biomedical and petroleum industries (Emine and Aysun, 2009).

In spite of their numerous advantages over synthetic surfactants, biosurfactant has not yet

been employed in industries due to their relatively high production and recovering cost involved

(Makkar et al., 2011), hence the need for inexpensive and renewable carbon sources and highly

efficient microorganisms for biosurfactants production (Plaza et al., 2011). Certain substances

are used as sources of energy for microbial fermentation with the aim of producing

biosurfactants. In the bid to diversify these substances recent advances have focused on the use

of agricultural products, byproducts and wastes. Red cashew (Anacardium occidentale) fruits are

widely distributed and are rich in carbohydrate, vitamins, proteins and mineral salts (Akinhanmi

and Atasie, 2008) which make them an interesting and inexpensive renewable carbon source for

2

microbial fermentation. A large percentage of the red cashew (Anacardium occidentale) fruits

are wasted in Nigeria annually as people are only interested in the nuts, hence the need to

harness these raw materials for biosurfactant production. Pseudomonas aeruginosa is one of the

widely studied microorganisms used in the production of biosurfactants. It is a bacterium that is

able to thrive in various environments and conditions. It can also use a wide range of organic

materials as source of energy and carbon. Pseudomonas species has been identified to degrade

hydrocarbons and produce biosurfactants predominately glycolipids (Beal and Betts, 2000). In

the current study, biosurfactants produced by Pseudomonas aeruginosa in submerged

fermentation system using red cashew fruit pomace as substrates will be characterized and

applied in cleaning of insecticide residue in vegetables.

LITERATURE REVIEW

1.1 Biosurfactant and Classification

Biosurfactants are suface active compounds produced on microbial cell surfaces or

excreted extracellularly by a wide variety of microorganisms (Priya and Usharani, 2009; Jamal

et al., 2012). The classification of biosurfactants is dependent on their chemical structure and

molecular weight. Based on their chemical structure, biosurfactants are determined by the

different molecules forming the hydrophobic and hydrophilic moieties. The hydrophobic

moieties may contain saturated or unsaturated fatty acids while the hydrophilic moieties may

contain peptide anions or cations, mono-, di-, or polysaccharides, or amino acids (Makkar and

Cameotra, 2002). Based on molecular weight, they are divided into low-molecular-mass

biosurfactants which include glycolipids, phospholipids and lipopeptides and into high-

molecular-mass biosurfactants containing amphipathic polysaccharides, proteins,

lipopolysaccharides, lipoproteins or complex mixtures of these biopolymers. Low-molecular-

mass biosurfactants are efficient in lowering surface and interfacial tensions, whereas high-

molecular-mass biosurfactants are more effective at stabilizing oil-in-water emulsions (Calvo et

al., 2009).

1.1.1 Glycolipids

Glycolipids are the most common types of biosurfactants. They consist of carbohydrates

in combination with long chain aliphatic and hydroxyaliphatic acids and are further divided into

rhamnolipids, trehalose-lipids and sophorolipids, of which rhamnolipids are of utmost

importance. Rhamnolipids are biosurfactants produced by Pseudomonas aeruginosa and some

3

other Pseudomonas strains. Rhamnolipids have rhamnose sugars as hydrophilic moiety and fatty

acids as hydrophobic moiety. New technologies have been used to discover up to 28

homologues of rhamnolipids (Benincasa et al., 2004) with four of these being more important

than others. These four homologues are usually designated as R1, R2, R3 and R4 (where R

represents rhamnolipids) (see Fig. 1). These four rhamnolipids are distinct from each other by

the amount of rhamnose sugar and fatty acid chain each one of them contains. They usually

contain two or more important rhamnose and fatty acid chain (Lang and Wullbrandt, 1999).

Rhamnolipids are said to enhance the degradation and dispersion of different classes of

hydrocarbons by lowering surface tension. They emulsify hydrocarbons and vegetable oils and

induce the growth of Pseudomonas on n-hexadecane (Whang et al., 2008). Trehalose lipids are

produced from different species of Mycobacterium tuberculosis, Arthrobacter and Nocardia.

They enhance the bioavailability of hydrocarbons (Franzetti et al., 2010). Sophorolipids are

produced by different strains of the yeast, Torulopsis. The sugar unit is the disaccharide

sophorose which consists of two β-1, 2-linked glucose units(Perfumo et al., 2010).

FIG 1.1: The Four Major Rhamnolipids.

Source: Lang and Wullbrandt, (1999)

4

1.1.2 Phospholipids, Lipopeptides and Polymeric Biosurfactants

Phospholipids are major components of microbial membranes. They contain a phosphate

group and fatty acid chain and are further divided into corynomycolic acid, spiculisporic acid

and phosphotidylethanolamine. The level of phospholipids increases greatly (40-80% w/w)

when some micro-organisms like bacteria, yeast, Acinetobacter species, Arthrobacter species,

Aspergillus species are grown in hydrophobic substrates (Pooja and Cameotra 2004).

Phospholipids promote the enhancement of bitumen recovery, removal of metal ions from

aqueous solution and dispersion of hydrophilic pigments. They are utilized in the preparation of

new emulsion-type organogels, super fine microcapsules (liposomes or vesicles) and heavy

sequestrants. Phospholipids increase the tolerance of bacteria to heavy metals (Ishigami et al.,

2000).

Lipopeptides are biosurfactants which are produced by organisms like Pseudomonas,

Bacillus and Streptomyces species. They are comprised of fatty acids attached to an amino acid

chain (Kiran et al., 2010). They are classified into surfactin and lichenysin. Lipopeptides

enhance oil recovery, biodegradation of hydrocarbons and chlorinated pesticides, removal of

heavy metals from a contaminated soil, sediment and water; thus, increasing the effectiveness of

phytoextraction (Chakraborty et al., 2011).

Polymeric biosurfactants are very complex molecules which usually contain a backbone

of three to four repeating sugars having fatty acid chains attached to them. They consist of

lipopolysaccharides, lipoproteins, proteins and polysaccharides. Polymeric biosurfactants are

classified into emulsan, alasan, biodispersan, liposan and mannoprotein. Polymeric

biosurfactants are implicated with functions like stabilization of the hydrocarbon-in-water

emulsions and dispersion of limestone in water (Toren et al., 2001).

1.2 Biosurfactant Producing Microorganisms

Biosurfactants produced by a variety of microorganisms mainly bacteria, fungi and

yeasts are diverse in chemical composition and their nature and the amount depend on the type

of microorganism producing a particular biosurfactant. Many microorganisms for industrial

utilization for waste products have been isolated from contaminated soils, effluents and waste

water sources. Thus, these have the ability to grow on substrates considered potentially noxious

for other non-producing microorganisms (Saharan et al., 2011).

5

Table 1.1: List of biosurfactants producing organisms.

S.NO

Biosurfactant Microorganism(s) Current economic

importance

1 Cellobiose lipids

Ustilago maydis Antifungal Compounds

2 Rhamnolipids

Pseudomonas aeruginosa, Pseudomonas

chlororaphis, Serratia rubidea.

Bioremediation,

Antimicrobial and

biocontrol properties

3 Trehalose lipids

Rhodococcus erythropolis, Arthrobacter

sp., Nocardia erythropolis,

Corynebacterium sp., Mycobacterium sp

Dissolution of

hydrocarbons

4 Sophorolipids

Candida bombicola, C. antartica,

Torulopsis petrophilum C. botistae, C.

apicola, C. riodocensis, C. stellata, C.

bogoriensis

Antimicrobial,

Antiviral, Spermicidal

5 Phospholipids Acinetobacter sp. Bioremediation

Acinetobacter Bioremediation

6 Emulsan A. calcoaceticus

Microbially enhanced

oil recovery (MEOR )

7 Alasan A. radioresistens Biodegradation

of polyaromatic

compounds

A. radioresistens

Biodegradation of

polyaromatic

compounds

8 Peptide lipids

B. licheniformis

Antimicrobial

properties

9 Carbohydrate lipids P.fluorescens, Debaryomyces polmorphus

Bio-emulsifiers

10 Fatty acids /neutral

lipids

Clavibacter Bio-emulsifiers

Source: Saharan et al., (2011)

6

1.3 Properties of Biosurfactants

Biosurfactants are of increasing interest for commercial use because of the continually

increasing spectrum of available substances. There are various advantages of biosurfactants

compared to their chemically produced counterpart. The major distinctive features of

biosurfactants and a brief description of each property are given below:

1.3.1 Surface and Interface Activity

Biosurfactants are substances with very strong surface active characteristics which

accumulate at the interface between two immiscible fluids or between a fluid and a solid. They

have the ability to lower surface and interfacial tension in water, gases, liquids and solids.

Biosurfactant activities depend on the concentration of the surface-active compounds until the

critical micelle concentration (CMC) is obtained. The concentration at which the rate of surface

tension reduction results in the formation of micelles and vesicles is known as the critical

micelle concentration (CMC). This concentration determines the efficiency in the rate of surface

tension reduction ability of biosurfactants. Biosurfactants have CMC values ranging from 1 to

200mg/L (Puntus et al., 2004) and are said to have 10-40 fold lower CMC value than synthetic

surfactants, which means that less biosurfactant is required to decrease the surface tension. The

most active biosurfactants can lower the surface tension of water from 72 to 30 mN·m−1

and the

interfacial tension between water and n-hexadecane from 40 to 1 mN·m−1

(Signh et al., 2006).

At concentrations above the CMC, biosurfactant molecules associate to form micelles, bilayers

and vesicles. Micelle formation enables biosurfactants to reduce the surface and interfacial

tension and increase the solubility and bioavailability of hydrophobic organic compounds

(Whang et al., 2008). Micelle formation has a significant role in microemulsion formation

(Nguyen et al.,2008). Microemulsions are clear and stable liquid mixtures of water and oil

domains separated by monolayer or aggregates of biosurfactants. Microemulsions are formed

when one liquid phase is dispersed as droplets in another liquid phase, for example oil dispersed

in water (direct microemulsion) or water dispersed in oil (reversed microemulsion).

Biosurfactants are also identified as biologically active substances, having biocidal

activity against some microbes like yeast, bacteria, viruses and fungi. This is expressed in the

zone of inhibition or minimal inhibitory concentration (MIC) (Muthusamy et al., 2008).

Biosurfactants achieve this effect by influencing the bacterial cell surface hydrophobicity (CSH).

This ability has been reported by Al-Tahhan et al. (2000), who studied chemical and structural

modifications in the cell surface hydrophobicity (CSH) of Pseudomonas aeruginosa by a

rhamnolipid in the presence of hexadecane. Results of their study demonstrated that

7

rhamnolipid, at very low concentration, caused release of lipopolysaccharide (LPS) from the

outer membrane resulting in an increase of cell surface hydrophobicity. In contrast, Sotirova et

al. (2009) reported that rhamnolipid at the concentrations below CMC did not affect the LPS

component of the bacterial outer membrane but instead changed the composition of outer

membrane proteins (OMP). However, all of the changes in the structure of the bacterial cell

surface cause increase of accessibility of hydrocarbons to microbial cells.

1.3.2 Temperature, pH and Ionic Strength Tolerance

Many biosurfactants and their surface activities are not affected by environmental

conditions such as temperature and pH. McInerney et al., (1990) suggested that lichenysin

produced by B. licheniformis was not affected by temperature (up to 50°C), pH (4.5–9.0) and by

NaCl and Ca concentrations up to 50 and 25 g/l respectively. A lipopeptide produced by B.

subtilis was stable after autoclaving (121°C/20 min) and after 6 months at –18°C; the surface

activity did not change from pH 5 to 11 and NaCl concentrations up to 20% (Charkraborty et.al.,

2011).

1.3.3 Biodegradability

Unlike synthetic surfactants, microbial-produced compounds surfactants are easily

degraded (Mohan et al., 2006) and chiefly suited for the environmental applications such as

bioremediation (Mulligan, 2005) and dispersion of oil spills.

1.3.4 Low Toxicity

Very little data are available in the literature regarding the toxicity of biosurfactants.

They are in general considered as low or non-toxic products and therefore are appropriate for

pharmaceutical, food and cosmetic uses. A biosurfactant from P. aeruginosa was compared to a

synthetic surfactant that is widely used in the industry, regarding toxicity and mutagenic

properties. Both assays indicated a higher level of toxicity and mutagenic effect of the

chemically derived surfactant, whereas the biosurfactant was considered to be slightly non-toxic

and non mutagenic (Cooper and Cavalero, 2003). Experiment conducted by Anyanwu et.al.,

2011, lipopeptide biosurfactant was non-toxic to mice at the 5.0g/kg body weight dose tested,

which was the highest dose recommended by the Food and Agricultural Organization/World

Health Organization for food additives. This is indicative of its non-toxic nature even when used

as food additive or accidentally consumed. The low toxicity of biosurfactants has been

recommended as a veritable advantage over synthetic surfactants.

8

1.4 Factors Affecting Biosurfactant Production

Biosurfactants are produced by a number of microorganisms, predominantly during their

growth on water-immiscible substrates. However, some yeast may produce biosurfactants in the

presence of different types of substrates, such as carbohydrates. The use of different carbon

sources alters the structure of the biosurfactant produced and its properties and can be exploited

to get products with desired properties for particular applications. There are a number of studies

in biosurfactant production involving the optimization of their physicochemical properties

(Sarubbo et al., 2006). The composition and characteristics of biosurfactants are influenced by

the nature of the nitrogen source as well as the presence of iron, magnesium, manganese,

phosphorus and sulphur in the media (Sarubbo et al., 2001). Environmental factors are also

extremely important in the yield and characteristics of the biosurfactant produced. In order to

obtain large quantities of biosurfactant it is necessary to optimize the process conditions because

the production of a biosurfactant is affected by variables such as pH, temperature, aeration and

agitation speed.

1.4.1 Nature of Carbon Source

Till date, biosurfactants are unable to compete inexpensively with chemically

synthesized compounds due to their high production costs and recovery system. These costs may

be significantly reduced by the use of alternative sources of nutrients. Zinjarde and Pant (2002)

demonstrated the biosynthesis of surfactant by Y. lipolytica NCIM 3589 using soluble carbon

source such as glucose, glycerol and sodium acetate. Sarubbo et al. (2001) identified for the first

time a biosurfactant produced by Y. lipolytica IA 1055 using glucose as carbon source and

concluded that the induction of biosurfactant production is not dependent on the presence of

hydrocarbons. Biosurfactant production by B. subtilis MTCC 2423 was monitored by measuring

the reduction in surface tension of the cell-free broth. Surface tension reduction was better when

glucose, sucrose, tri sodium citrate, sodium pyruvate, yeast extract, and beef extract were used

as carbon sources. The maximum bioemulsifiers production was observed when the strain C.

glabrata isolated from mangrove sediments was cultivated on cotton seed oil (7.5%) and

glucose (5.0%), reaching values of 10 g L-1 after 144 hr. The soy molasses, a byproduct from the

production of soybean oil, plus oleic acid were tested as carbon sources for the production of

sophorolipids by the yeast C. bombicola (Solaiman et al., 2004). The purified SLs were obtained

at 21 g l−1 and were 97% in lactone form. The surface properties of the SLs obtained from the

soy molasses/oleic acid fermentation had minimum surface-tension values of 37 mN m−1

(pH 6)

and 38 mN m−1

(pH 9), and critical micelle concentration values of 6 mg l−1

(pH 6) and 13 mg

9

l−1 (pH 9). The carbon sources such as glucose, glycerol, acetates and other organic acids, as

well as pure n-alkanes are quite expensive and cannot reduce the cost of biosurfactant

production. An approach to lessen the cost is partial or complete replacement of pure reagents

with industrial/agricultural mixtures. The substrate does merely determine the amount of

biosurfactants produced but also determines the kind of biosurfactant produced.

1.4.2 Nitrogen Source

Nitrogen is important in the biosurfactant production medium because it is an essential

component of the proteins that are essential for the growth of microbes and for production of

enzymes for the fermentation process. Several sources of nitrogen have been used for the

production of biosurfactants, such as urea, peptone, ammonium sulphate, ammonium nitrate,

sodium nitrate, meat extract and malt extract (Mata-Sandoval et al., 2001). Yeast extract is the

most widely used nitrogen source for biosurfactant production, but its required concentration

depends on the nature of microorganism and the culture medium to be used. The production of

biosurfactants often occurs when the nitrogen source is depleted in the culture medium, during

the stationary phase of cell growth (Thanomsub et al, 2004).

1.4.3 Effect of pH

Production of biosurfactants occurs best at a pH of 8.0, which is the natural pH of sea

water. The reported pH for rhamnolipid production by Pseudomonas aeruginosa was all in the

neutral range. Lower production with lower cell growth rates could occur as a result of the pH

being lower than 6.5 or higher than 7.5. It is important to have a proper control of the pH

throughout the production process to avoid retardation in the process (Chen et al. 2007).

1.4.4 Effect of Temperature

Most of the biosurfactant productions reported so far have been performed in a

temperature range of 25 to 30˚C. Casas and Garcia-Ocho (1999) reported that the amount of

sophorolipids obtained in the culture medium of C. bombicola at temperature of 25˚C or 30˚C is

similar. Nevertheless, the fermentation at 25˚C presents a lower biomass growth and a higher

glucose consumption rate in comparison to the fermentation at 30˚C. In the culture of C.

antarctica, temperature causes variations in the biosurfactant production. The highest

mannosylerythritol lipid production was observed at 25˚C for the production with both growing

and resting cells (Kitamoto et al., 2001).

10

1.4.5 Effect of Agitation and Aeration on the Production of Biosurfactants

Aeration and agitation are important factors that influence the production of

biosurfactants as both facilitate the oxygen transfer from the gas phase to the aqueous phase. It

may also be linked to the physiological function of microbial emulsifier, it has been suggested

that the production of bioemulsifiers can enhance the solubilization of water insoluble substrates

and consequently facilitate nutrient transport to microorganisms. In Agitation rates between

50rpm and 250rpm, it was observed that the best production was achieved at 250rpm (Wei et al.,

2007).

1.4.6 Metal Ion Concentration

Metal ion concentrations play a very important role in the production of some biosurfactants

as they form important cofactors of many enzymes. The overproduction of surfactin

biosurfactant occurs in the presence of Fe2+ in mineral salt medium. The properties of surfactin

are modified in the presence of inorganic cations such as overproduction (Wei et al., 2007).

1.5 Applications of Biosurfactants

Biosurfactants are implicated in a wide range of applications. Most biosurfactants produced

by micro organisms are utilized in the remediation of crude oil and pesticide-contaminated soils,

hydrocarbons and heavy metals, oil recovery and as emulsifiers in food industries and in skin

conditioning (Suwansukho, 2008). They are also utilized in medicine, agriculture and petroleum

industries.

1.5.1 Bioremediation Applications

In recent times, biosurfactants have been utilized in bioremediation. Bioremediation is

the use of micro organisms’ metabolism to remove pollutants. This process is achieved due to

certain properties which the biosurfactants possess. Such properties may include their low

toxicity, ability to disperse a wide range of hydrophobic pollutants like crude oil, pesticides and

other chemicals and biocompatibility (Makkar et al., 2011).

1.5.1.1 Application in Biodegradation Process

A promising method that can improve bioremediation effectiveness of hydrocarbon

contaminated environments is the use of biosurfactants. They can enhance hydrocarbon

bioremediation by two mechanisms. The first includes the increase of substrate bioavailability

for microorganisms, while the other involves interaction with the cell surface which increases

the hydrophobicity of the surface allowing hydrophobic substrates to associate more easily with

11

bacterial cells (Mulligan and Gibbs, 2004). By reducing surface and interfacial tensions,

biosurfactants increase the surface areas of insoluble compounds leading to increased mobility

and bioavailability of hydrocarbons. In consequence, biosurfactants enhance biodegradation and

removal of hydrocarbons. Addition of biosurfactants can be expected to enhance hydrocarbon

biodegradation by mobilization, solubilization or emulsification (see Fig. 2) (Nievas et al.,

2008). The mobilization mechanism occurs at concentrations below the biosurfactant CMC. At

such concentrations, biosurfactants reduce the surface and interfacial tension between air/water

and soil/water systems. Due to the reduction of the interfacial force, contact of biosurfactants

with soil/oil system increases the contact angle and reduces the capillary force holding oil and

soil together. In turn, above the biosurfactant CMC the solubilization process takes place. At

these concentrations biosurfactant molecules associate to form micelles, which dramatically

increase the solubility of oil. The hydrophobic ends of biosurfactant molecules connect together

inside the micelle while the hydrophilic ends are exposed to the aqueous phase on the exterior.

Consequently, the interior of a micelle creates an environment compatible for hydrophobic

organic molecules. The process of incorporation of these molecules into a micelle is known as

solubilization (Urum and Pekdemir, 2004).

FIG 1.2: Mechanisms of hydrocarbon removal by biosurfactants

Source: Urum and Pekdemir (2004).

Emulsification is a process that forms a liquid, known as an emulsion, containing very

small droplets of fat or oil suspended in a fluid, usually water. The high molecular weight

biosurfactants are efficient emulsifying agents. They are often applied as an additive to stimulate

bioremediation and removal of oil substances from environments (Urum and Pekdemir, 2004).

12

1.5.1.2 Application in Microbial Enhanced Oil Recovery

Biosurfactants can be utilized in oil recovery in a process called Microbial Enhanced Oil

Recovery (MEOR). Here, the microorganisms in the reservoir are stimulated thereby causing

them to yield biosurfactants and polymers which lower interfacial tension at the oil-rock

interface and thus, increasing the production of oil from subtly-producing reservoirs. The

mechanism responsible for the release of oil is the acidification of the solid phase. Micro

organisms like Pseudomonas aeruginosa, Bacillus subtilis and Torulopsisbombicola utilize

crude oil and hydrocarbons as carbon sources and can be utilized in cleaning oil spillages while

micro organisms produced in situ are provided with low-cost substrates like molasses and

inorganic nutrients in order to improve their growth and biosurfactant production (Das and

Mukherjee, 2007).

FIG 1.3: Mechanism of oil recovery by biosurfactants.

Source: Das and Mukherjee (2007)

1.5.1.3 Application in Agriculture

Biosurfactants when applied as mobilizing agents increases the apparent solubility of

hydrophobic organic contaminants (HOC) in the soil by enhancing solubility of lethal chemical

compounds like polycyclic aromatic hydrocarbons (PAH). Biosurfactants also aid in adsorbing

microorganisms to soil particles occupied by pollutants and thereby reducing the diffusion path

length between the site of biouptake and the site of absorption by the microbes (Makkar and

Rockne, 2003).

Surfactants are utilized for hydrophilization of heavy soils to obtain good wet ability and

to achieve even distribution of fertilizer in the soil. They also prevent the caking of certain

fertilizer during storage and promote spreading and penetration of the toxicants in pesticides

(Makkar and Rockne, 2003).The rhamnolipid biosurfactant, mostly produced by the genus

Pseudomonas is known to possess potent antimicrobial activity. Further, no adverse effects on

13

humans or the environments are anticipated from aggregate exposure to rhamnolipid

biosurfactants. Biosurfactants can also be applied as cleaning agent for pesticide residue in

vegetables. Churdchai and Nguyen, 2010, explore the possible potential of biosurfactant for

cleaning up cypermethrin residue in lettuce.

1.5.2 Therapeutic and Biomedical Applications

Biosurfactants present good opportunity to be developed as new antibiotics, although the

first biosurfactants to be produced are now being produced as commercial antibiotics. Their

antimicrobial activity has been reported against bacteria, fungi, algae and viruses. Biosurfactants

have other applications as anti-cancer and anti-adhesive agents, agents for stimulating stem

fibroblast metabolism, gene delivery and immunomodulatory action agents, immunological

adjuvant (Gomaa, 2012).

1.5.3 Miscellaneous Applications Of Biosurfactants

Biosurfactants are also known to be applied in other areas other than bioremediation and

biomedicine. They are equally implicated in having roles as anti-foaming, foaming, wetting,

emulsifying, dispersing and cleaning agents in many products and applications such as

cosmetics (toothpastes, hair shampoo and conditioner), biopesticides, quantum dot coatings,

paints, detergents, emulsions, adhesives, laxatives, fabric softeners, inks, agro chemical

formulations (some herbicides and insecticides), anti-fogs, leak detectors in pipelines,

ferrofluids, ski and snowboard waxes etc. They are also utilized in pipelines as liquid drag

reducing agent, in mobilizing oil in oil wells and in firefighting.

1.5.3.1 Application in Cosmetics Industries

Due to the emulsifying character of biosurfactants such as foaming, water binding

capacity, spreading and wetting properties effect on viscosity and on product consistency,

biosurfactant have been proposed to replace chemically synthesized surfactants in cosmetics

industries. These surfactants are used as emulsifiers, foaming agents, solubilizers, wetting

agents, cleansers, antimicrobial agents, mediators of enzyme action, in insect repellants,

antacids, bath products, acne pads, anti dandruff products, contact lens solutions, baby products,

mascara, lipsticks, toothpaste, dentine cleansers (Gharaei-Fathabad, 2011).

1.5.3.2 Application in Food Processing Industries

Biosurfactants have been used for various food processing applications but they usually

play a role as food formulation ingredient and anti-adhesive agents, as food formulation

14

ingredients they promote the formation and stabilization of emulsion due to their ability to

decrease the surface and interfacial tension. They are also used 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 (Krishnaswamy et al., 2008).

1.5.3.3 Application in Commercial Laundry Detergent

Almost all surfactants, an important component used in modern day commercial laundry

detergents, are chemically synthesized and exert toxicity to fresh water living organisms.

Growing public awareness about the environmental hazards and risks associated with chemical

surfactants has stimulated the search for ecofriendly, natural substitutes of chemical surfactants

in laundry detergents. Biosurfactants such as Cyclic Lipopeptide (CLP) are stable over a wide

PH range (7.0-12.0) and heating them at high temperature does not result in any loss of their

surface-active property. They showed good emulsion formation capability with vegetable oils

and demonstrated excellent compatibility and stability with commercial laundry detergents

favouring their inclusion in laundry detergents formulation (Das and Mukherjee, 2007).

1.5.3.4 Application as Biopesticides

Conventional arthropod control strategy involves applications of broad-spectrum chemicals

and pesticides, which often produce undesirable effects. Further, emergence of pesticide

resistant insect populations as well as rising prices of new chemical pesticides have stimulated

the search for new eco-friendly vector control tools. Lipopeptide biosurfactants produced by

several bacteria exhibit insecticidal activity against fruit fly Drosophila melanogaster and hence

are promising to be used as biopesticide (Mulligan, 2005).

1.6 Economic Factors of Biosurfactant Production

To overcome the expensive cost constraints associated with biosurfactant production, two

basic strategies are generally adopted worldwide to make it cost-effective: (i) the use of

inexpensive and waste substrates for the formulation of fermentation media which lower the

initial raw material costs involved in the process; (ii) development of efficient and successfully

optimized bioprocesses, including optimization of the culture conditions and cost-effective

recovery processes for maximum biosurfactant production and recovery. As millions of tons of

hazardous and non-hazardous wastes are generated each year throughout the world, a great need

exists for their proper management and utilization. The residues from tropical agronomic crops

15

such as cassava (peels), soybean (hull), sugar beet (Onbasli, 2009), sweet potato (peel and

stalks), potato (peel and stalks), sweet sorghum, rice and wheat (Krieger et al, 2010); hull soy,

corn and rice; bagasse of sugarcane and cassava; residues from the coffee processing industry

such as coffee pulp, coffee husks, spent coffee grounds; residues of the fruit processing

industries such as pomace and grape, waste from pineapple and carrot processing, banana waste;

waste from oil processing mills such as coconut cake, soybean cake, peanut cake, canola meal

and palm oil mill waste; saw dust, corn cobs, carob pods, tea waste, chicory roots etc. have been

reported as substrates for biosurfactant production. Additional substrates used for biosurfactant

production include water-miscible wastes, molasses, whey milk or distillery wastes. The various

substrates previously reported for biosurfactants production are listed (Table 2) with their

advantages.

Table 1.2: Substrate for microbial surface active agents and their end products

Source

Substrate part End product(s)

Cassava Flour Biosurfactant

Soybean Seed Rhamnolipids

Sugar beet Peels Biosurfactant

Cashew apple juice Pomace Biosurfactant

Diary whey Whey Bioemulsifier

Sweet potato Peels Biosurfactant

Sugar bagasse Stem husk Biosurfactant

Source: Saharan et al., 2011.

Despite possessing many industrially attractive properties and advantages compared with

synthetic ones, the production of biosurfactants on industrial scale has not been undertaken due

to high investment costs. This necessitates their profitable production and recovery on a large

scale.

Various aspects of biosurfactants, such as their biomedical and therapeutic properties

(Cameotra and Makkar, 2004) their natural roles, their production on inexpensive alternative

substrates and their industrial potential, have been reviewed. However their cost of production

continues to remain very high. Using low-cost raw materials is a possible solution for this

obstacle. Another approach is to use renewable low cost starting materials from various sources

including industrial wastes from frying oils, oil refinery wastes, molasses, starch rich wastes,

cassava waste water and distilled grape marc (Rivera et al., 2007).

16

1.7 Cashew (Anacardium occidentale)

1.7.1 Origin, Taxonomy and Morphology

Anacardium occidentale L. (cashew) is an evergreen shrub or tree up to 15 m in height

that originated from Brazil. Nowadays, cashew is distributed across tropical America, the West

Indies, India and Africa. The cashew tree bears two food products, the cashew nut and the

cashew apple. The cashew nut is in demand on international markets due to its sweet flavor

(Gordon et al., 2012).

Cashew fruits belongs to the family Anacardiaceae and is a pseudo-fruit formed by an

enlarged peduncle, and the true fruit, a kidney-shaped (reniform) achene is about 3cm long with

a hard grey-green pericarp (Sivaguru et al., 2010).

Botanically, the cashew nut is the embryo of the kidney-shaped drupe, which has a length of 3–5

cm. The cashew fruit is attached as an enlarged peduncle to the drupe. This false fruit has a

yellow to red skin and a juicy flesh. It is 6–8 cm long and approximately 4.5 cm in diameter

(Gordon et al., 2012).

1.7.2 Chemical Composition and Uses of Cashew Fruits

Cashew apple (juice and pomace) is a rich source of sugars, proteins, mineral salts,

vitamin C and other bioactive constituents such as polyphenols, flavonols, tannins and

carotenoids. For this reason, cashew fruits are highly nutritive and can be used in various dietary

applications such as juice and syrup production, feed formulation and as alternative raw material

for the production of industrially important products (Sivaguru et al., 2010; Adebowale et al.,

2011; Gordon et al., 2012). The utilization of cashew fruits for industrial purposes is meant to

increase its economic value as large quantities are wasted annually due to its perishable nature

and lack of proper storage facilities.

1.8 Fermentation Process for Biosurfactant Production

Microorganisms are capable of growing on a wide range of substrates under precise

cultural conditions and can produce a remarkable spectrum of products.

As with other microbial fermentations the goal in the production of biosurfactants is to

maximize the productivity, that is, grams/litre/hour yield of biosurfactants from the carbon

source thereby achieving high final concentrations. In addition it is important to reduce the

accumulation of other metabolic products that may interfere with the properties or recovery of

the product of interest (Georgiou et al., 1992). Microorganisms may be grown different types of

fermentation systems and certain conditions such as fermentation media components and

operating conditions must be met to ensure optimal yield of biosurfactants.

17

Fermentations may be carried out as batch, continuous and fed-batch processes. The mode of

operation is, to a large extent, dictated by the type of product being produced (Stanbury et al.,

2003).

Batch fermentation is a closed culture system which contains an initial, limited amount

of nutrient. The inoculated culture passes through the lag, log, deceleration and stationary

growth phases as a result of its physiological state and nutrient availability in the medium

(Stanbury et al., 2003). In a continuous fermentation system, substrate is constantly fed to the

reaction vessel, and a corresponding flow of fermented product broth is discharged to keep the

reactor volume constant. Furthermore, the balance between feed and discharge is maintained for

long enough times to achieve steady state operation with no changes in the conditions within the

reactor (Brethauer and Wyman, 2010).

In fed-batch process, nothing is removed from the reactor during the process, but one

substrate component is added in order to control the reaction rate by its concentration. When

high substrate level inhibits the growth and/or product formation or when undesired components

are produced in parallel with a desired product, the fed-batch operation is shown to be superior

to both batch and continuous operations (Radwan et al., 2011).

1.9 Aim and Objectives

The current study is based on the production of biosurfactant from pseudomonas

aeruginosa in a submerged fermentation using red cashew fruit pomace as carbon source and its

application as a cleaning agent for pesticide residue in vegetables.

The objectives are

1. To determine the proximate composition of the carbon source, red cashew pomace

(Anacardium occidentale)

2. To grow a pure strain of Pseudomonas aeruginosa in culture medium with appropriate

nutritients using red cashew (Anacardium occidentale) pomace as the carbon source.

3. To test the culture broth supernatants for biosurfactants activity using haemolysis test, oil

spreading technique, emulsification index test, stability studies (effect of temperature, pH and

salinity)

4. To extract the biosurfactant produced by the microorganism in the culture medium.

5. To characterize the biosurfactants produced for lipids, carbohydrate and protein contents

using basic biochemical procedures

6. To explore the potential of biosurfactant produced as a cleaning agent for pesticide

contaminated vegetables.

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CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Plant Materials

The plant material, red cashew pomace fruits (Anacardium occidentale) fruits were

collected from Ubogidi Cashew Plantation Nsukka, Enugu State. The fruits were washed with

normal saline and manually crushed to remove juice. The pomace was sun-dried, milled into

powder form then stored in an air-tight polythene bag.

2.1.2 Microorganism

The microorganism, Pseudomonas aeruginosa, was obtained from the Culture Selection

Unit of the Department of Microbiology, University of Nigeria, Nsukka. It was further

characterized and sub cultured to yield pure strains of the organism.

2.1.3 Instruments/Equipment

The equipment used for this study were those in the Department of Biochemistry,

Microbiology and Crop Sciences, University of Nigeria, Nsukka and they include:

Equipment/Apparatus Manufacturer

Autoclave Changzhou Boiler, Company, China

Bench top Centrifuge PAC Pacific

Cuvette Pyrex, England

Filter paper Whatman, England

Gas Cylinder Fabiano, Co., India

Hand gloves Neogloves, United Kingdom

Hot Air Oven (0-200◦C) Gallenkamp, England

Measuring Cylinders Pyrex, England

Micropipette (0-100µL) Hanna Instruments

Petri dishes ZDG med com, China

Refrigerator Thermocool

Spectrophotometer Spectronic 20D

Syringes Lifescan

Test tubes Pyrex, England

Thermometer Zeal, England

Weighing balance Melter HAS

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2.1.4 Chemicals

The chemicals used in this study were of analytical grade. The chemicals and their

manufacturers include:

Chemical Manufacturer

Ethanol BDH, England

Ammonium Molybdate ((NH4)6Mo7O24.4H2O) BDH, England

Ammonium Sulphate ((NH4)2SO4) Burgoyne, India

Anthrone Fisher Chemical LTD, USA

Dipotassium Hydrogen Phosphate (K2HPO4) JHD, China

Cobalt Sulphate (CoSO4.7H2O) Cartivalue, Holland

Copper Sulphate (CuSO4.5H2O) Cartivalue, Holland

Distilled water Lion water, UNN

Glucose BDH, England

Boric acid (H3BO3) BDH, England

Kerosene NNPC, Enugu, Nigeria

Magnesium Sulphate (MgSO4.7H2O) Merck, Germany

Manganese Sulphate (MnSO4.4H2O) BDH, England

Methanol ScharlauChemie, Spain

Nutrient Agar Zayo-Sigma, Germany

Olive oil Goya, Nigeria

Palm oil Nsukka, Nigeria

Phosphoric acid Sigma, Aldrich

Potassium Dihydogen Phosphate (KH2PO4) Riedel-de HaenAg, Germany

Sodium Hydroxide BDH, England

Vanillin Burgoyne, India

Zinc Sulphate (ZnSO4.7H2O) Riedel-deHaen Ag, Germany

Chlorpyrifos West Africa cotton Co Ltd.

Salt NPA Quavs, Nigeria

Sulfuric acid BDH, England

Sodium dithionite Aldrich chemicals

Sodium acetate BDH, England

Sodium dodecyl sulphate BDH, England

20

2.2 Methods

2.2.1 Preparation and Processing of Plant Material

The red cashew fruits were washed with normal saline and manually crushed to remove

the juice. The pomace obtained thereafter was sun-dried for 5 days and then milled into powder.

The powdered pomace was packaged in an air-tight polythene bag.

2.2.2 Proximate Analysis

The proximate composition of the Red cashew pomace was determined using the method

described by AOAC (1990).

2.2.2.1 Moisture Content

A glass Petri dish was placed on a tarred analytical balance and 10.0g of dried red

cashew pomace was placed in it. The container and its contents were placed in an air-circulating

oven set at 105˚C for 4 hours. At the end of the heating, the container and its contents were

placed in a dessicator to cool down to room temperature. Ensuring minimum exposure to air, the

container and its contents were weighed again. The overall procedure was repeated until two

consecutive weighing gave a constant value. The moisture content was calculated as:

% Moisture = (initial weight – dry weight) / initial weight × 100

2.2.2.4 Fibre Content

One gram (1.0g) (W1) of the sample was weighed into a tall form 300mL beaker. One

hundred and fifty milliliters (150mL) preheated 0.128M H2SO4 was added and heated to boil for

30min and then filtered. The residue was washed three times with hot water and returned to the

beaker. A volume, 150mL preheated 0.223M KOH was added and heated to boil slowly for

30min and filtered. The mixture was washed with hot water and three times with acetone. It was

dried at 130˚C for 1 hr and weighed (W2). The mixture was ashed at 500˚C for 3 hrs, cooled and

weighed (W3). Crude fibre content was calculated as:

% crude fibre = (W2 – W3) /W1 × 100

2.2.2.5 Ash Content

Empty heat resistant crucibles were dried at 500˚C for 1 hr and then cooled in a

dessicator and weighed then 2g of the ground sample were transferred into dish and the contents

were ignited, first gently and then at 500˚C for 3 hrs. After this time, the crucible is removed and

21

the lid replaced. It was allowed to cool before reweighing. The weight of the residue was

obtained and expressed as a percentage of the original sample weight

% Ash = weight of ash / original weight × 100

2.2.2.4 Protein Content

A known weight, 0.5g of oven-dried sample was weighed out into 30ml Kjeldahl flask

and 15.0mL of concentrated H2SO4 was added with 1g of the catalyst mixture. The mixture was

heated cautiously in digestion rack under a fume cupboard until a greenish solution appeared.

After the digest was cleared it was heated further for 30 minutes and allowed to cool. 10mL of

distilled water was added to avoid caking. The mixture was transferred to the Kjeldahl

distillation apparatus. A 50.0mL receiver flask containing 5.0mL boric acid was placed under

the condenser of the distillation apparatus. Ten milliliters (10.0mL) of 40% NaOH solution was

added to the digested sample. Distillation was stopped when the distillate reached the 35ml mark

on the receiver flask. The distillate was titrated to first pink colour with 0.1 M HCl. The crude

protein content was calculated as:

% Crude protein = (titre × 14.01× 0.1× 100 × 6.25 × dilution factor) / (1000 × sample weight)

2.2.2.7 Lipid Content

Soxhlet flask was dried in an oven at 100˚C, allowed to cool and weighed (W1) and 5g

(W2) of the sample was transferred to a thimble and its contents into the Soxhlet extractor.

Hexane was used for the extraction. After 3 hrs, the thimble was removed and the solvent

distilled off from the flask. The flask was disconnected and placed in an oven set at 60˚C for

2hr, cooled and weighed (W3). Lipid content was calculated as:

% Lipid = (W3 - W1) / W2 × 100

2.2.2.8 Carbohydrate Content

Total carbohydrate was determined by the difference method by summing the values for

other constituents and subtracting the sum from 100 as reported by Onyeike and Omubo-Dede

(2002).

2.2.3 Reactivation of Pseudomonas aeruginosa by Subculture

The pure strain of Pseudomonas aeruginosa in a Bijou bottle was reactivated by streak-

plating on nutrient agar contained in a petri dish. The serving nutrient agar was prepared by

dissolving 2.8g of powdered nutrient agar in distilled water and making up to 100ml mark. The

solution was autoclaved at 121˚C, 15psi for 15 minutes. Under an aseptic environment, the

22

autoclaved nutrient agar solution was allowed to cool for some time and then poured into a

sterile petri dish and allowed to stand for some minutes to gel. After gelling, the pure strain of

the Pseudomonas aeruginosa was inoculated by streaking of the gelled nutrient agar in the petri

dish with inoculum from the bijou bottle containing the stock culture of the organism. The

streaking process was achieved by the use of a heated/sterilized wireloop. The inoculated petri

dish was allowed to stand for 24hr at room temperature to enhance the formation of colonies

required for the preparation of the inoculum.

2.2.4 Preparation of Pseudomonas aeruginosa Seed Culture and Its Growth Rate

Determination

A known weight, 1.3g of nutrient broth was dissolved in 100ml distilled water. It was

boiled for 10min to dissolve properly, then autoclaved for 15minutes at 121oC. A loopful of

isolated colony previously maintained on nutrient agar was transferred to the nutrient broth and

incubated for 24hr at 37oC. This primary inoculum was grown until the absorbance at 600nm

wavelength reached 1.459 and was used to inoculate the production media (Rashedi et al.,

2005). Growth was indicative of the increase observed in absorbance.

2.2.5 Media Preparation

Three different culture media were prepared according to the method described by Atlas

(2010). In this experiment, three culture media with different compositions were prepared. Each

culture medium was inoculated with Pseudomonas aeruginosa.

• Medium I: Composed of red cashew pomace as substrate.

• Medium II: Composed of glucose as substrate.

• Medium III: Composed of nutrient broth as substrate.

Proper working environment was maintained by aseptic means.

2.2.5.1 Preparation of Basal Mineral Medium (B.M.M) and Carbon Source

The basal mineral medium was prepared as described by Atlas (2010). The trace element

solution was prepared first by adding components (0.232g H3BO3, 0.174g ZnSO4.7H2O, 0.116g

FeSO4(NH4)2SO4.6H2O, 0.096g CoSO4.7H2O, 0.022g (NH4)6Mo7O24.4H2O, 8.0mg

CuSO4.5H2O, 8.0mg MnSO4.4H2O) to distilled water and bringing its volume to 1.0L. The

solution was then mixed thoroughly. The basal mineral medium (B.M.M) was prepared by

adding components (12.5g K2HPO4, 3.8g KH2PO4, 1.0g (NH4)2SO4, 0.1g MgSO4.7H2O plus

5.0mL of the trace elements solution) to distilled water and bringing the volume to 900.0mL

23

mark. The solution was mixed thoroughly, gently heated and brought to boiling. It was then

autoclaved at 121oC, 15psi for 15min and cooled to 45-50˚C. Subsequently, the carbon source

was prepared by adding 14g of the red cashew pomace to distilled water, bringing its volume to

the 100.0mL mark and mixing thoroughly. Glucose was prepared by dissolving 14g in distilled

water and bringing the volume to 100mL.

2.2.5.2 Preparation of the Different Culture Media

This was carried out as described by Atlas (2010), In the first media, 100ml of the carbon

source (red cashew pomace) was added to 900ml the sterilized basal mineral medium, this was

shaken very well and distributed into three 250mL conical flasks, 1mL of seed culture

containing Pseudomonas aeruginosa was inoculated in each flask and labeled medium I. In the

second media, 100mL of glucose was added to 900mL basal mineral medium and shaken

thoroughly. This was distributed into three 250mL conical flasks, 1mL of seed culture

containing Pseudomonas aeruginosa was inoculated in each flask and labeled medium II.

The last media which served as the control was prepared by dissolving 13g of powdered nutrient

broth was dissolved in 1000L distilled water and sterilized by at 121oC, 15psi for 15min. This

was allowed to cool to room temperature; 1mL of seed culture containing Pseudomonas

aeruginosa was inoculated. The preparation was made in triplicates and the setup was labeled

medium III.

2.2.6 Screening the Culture Broth for Growth of Pseudomonas aeruginosa

Viable cell numbers were determined by submitting pseudomonas aeruginosa culture

broth to serial dilution of 1:7 and viable counts were performed by spread plate technique. A

known weight, 2.8g of nutrient agar was dissolve in 100mL distilled water and autoclaved at

121oC for 15 minutes. This was allowed to cool to room temperature and distributed into petri

dishes. Twenty microlitres (20µl) of the serial diluted culture broth was inoculated and spread

using glass rod. This was incubated for 24 hours (Marcia and Glaucia, 2006). This was carried

out for 10days.

2.2.7 Screening of the Supernatant for Biosurfactant Activity

The various culture supernatants were screened to observe biosurfactant activity using,

haemolysis test, oil spreading technique, emulsification index test and stability test (effect of

temperature,pH and salinity).

24

2.2.7.1 Haemolysis Test

The haemolysis test was carried out using blood agar plates as described by Carrillo et

al. (1996). They recommended the use of blood agar lysis as a primary method to screen for

biosurfactant activity. The supernatants were screened by plating cells on blood agar plates

containing 5% (v/v) human blood and incubated at room temperature for 24 hrs. A clear zone

(zone of haemolysis) around the colonies after 24hr was indicative of biosurfactants.

2.2.7.2 Oil Spreading Test

Oil displacement test is a method used to measure the diameter of the clear zone, which

occurs after dropping biosurfactant-containing solution on an oil-water interphase. The binomial

diameter allows an evaluation of the surface tension reduction efficiency of a given

biosurfactant. A fifty milliliters (50ml) of distilled water was added to a large petri dish (15cm

diameter) followed by addition of 20µl of oil to the surface of water and then 10µl of supernants

of culture broth. The diameter and the clear halo visualized under visible light were measured

after 30s (Rodrigues et al., 2006).

2.2.7.3 Emulsification Index Test

The emulsification test was carried out as described by Balogun and Fagade (2010). Two

milliliters (2ml) of oil was added to the amount of culture supernant and vortex for 2 minutes

and left to stand for 24 hours. The volume of oil that separated after 24 hrs of standing was

measured; this indicated the ability of a molecule to form a stable emulsion. The emulsification

index (E24) was determined and this is given as percentage of height of emulsified layer (in cm)

divided by total height of the liquid column (cm).

E24 = Height of emulsified layer / Total height x 100%

2.2.7.4 Stability Test

Stability studies were carried out by the procedure described by Preethy and Nilanjana

(2010). The cell-free broth was obtained by centrifuging the cultures at 4000rpm for 15 minutes.

The pH of the biosurfactant (4.0mL) was adjusted between pH 2.0-10 using HCl after which

E24 was determined. To test the heat stability of the biosurfactant, the broth was heated at

temperature of 10–100°C for 15 minutes, cooled at room temperature and emulsification index

(E24) was determined. The salinity stability was tested by the addition of different concentration

of salt to the biosurfactant and the E24 was determine.

25

2.2.8 Extraction of Biosurfactants

The culture broth was centrifuged at 4000rpm for 15min to remove the cells as well as

debris and the supernant was used for the extraction. The supernant was then precipated by

acidification with hydrochloric acid to pH 2.0. Equal volume of chlorofoam: methanol (2:1) was

added. This mixture was shaken well for mixing and left overnight for evaporation. White

coloured sediment was obtained as a result i.e. the crude biosurfactant. This was dried and

weighed (Anandaraj and Thivakarn, 2010).

2.2.9 Biochemical Composition of the Isolated Biosurfactant

The biosurfactants isolated were analyzed for carbohydrate, protein and lipid content

using some biochemical methods

2.2.9.1 Carbohydrate Content

The carbohydrate content of the produced biosurfactant was determined by the Anthrone

method as described by Ilori et al. (2005) and Umeji et al. (2010). Two millilitres (2.0mL) each

of the biosurfactant solution (0.1g biosurfactant in 1000ml distilled water) was added into

appropriately labeled sterile test tubes, after which 3.0ml distilled water and 10.0mL of 0.2%

solution of anthrone reagent (containing 0.2% anthrone in 95% H2SO4) was added to each of the

test tubes. The absorbance of each of the preparation was read at 630nm against a blank

composed of water and anthrone reagent. Each sample experiment was done in triplicate to

minimize experimental error. Glucose was used as standard and standard curve of its absorbance

at 630 nm against various glucose concentrations was prepared from whence the carbohydrate

concentration of each of the biosurfactant was extrapolated.

2.2.9.2 Protein Content

The protein content of the biosurfactants was determined by Biuret method as described

by Umeji et al. (2010). A quantity, 0.2 ml of each of the biosurfactants isolated from the culture

broth supernatants was collected into twelve appropriately labeled test tubes and 1% w/v CuSO4

(5drops) and 40% w/v NaOH (2ml) were added to each of the test tubes and shaken. For a

qualitative assay, purple colouration was indicative of protein.

2.2.9.3 Lipid Test

The lipid content of the biosurfactant was carried out as described by Umeji et al. (2010).

A volume, 100.0µL each of the biosurfactant was added to appropriately label sterile test tubes.

Concentrated H2SO4, 2.9mL was added and the mixture was boiled for 10min. The mixture was

left to cool and 2.5mL of phosphovanillin reagent (containing 20.0mL of 0.6% vanillin in

80.0mL of phosphoric acid) was added to 0.1mL of the cooled solution. The mixture was

26

incubated in the dark for 45min at room temperature. The absorbance was read at 532nm against

a blank. Cholesterol was used as the standard and a standard curve obtained by plotting various

absorbances at 532nm against corresponding cholesterol concentrations was prepared from

where the lipid concentration of each of the biosurfactant was extrapolated.

2.2.10 Study of Biosurfactant as a Cleaning Agent

Biosurfactant was studied as a potential cleaning agent for chlorpyrifos contaminated

toamatoes.

2.2.10.1 Preparation of Chlorpyrifos Stock Solution and Standard Curve

One gramme (1g) of chlorpyrifos was weighed into a 1000ml conical flask; distilled

water was added until it got to mark. Ten milliliters (10ml) of the stock solution was added into

100ml volumetic flask, distilled water was added to mark to get 100ppm. A standard calibration

graph was obtained by running different dilutions of the standard chlorpyrifos (0.05-8.0ppm).

(Venugopal et al., 2012).

2.2.10.2 Preparation of Chlorpyrifos Solution

100ppm chlorpyrifos solution was prepared by adding 10ml stock solution into 100ml

volumetric flask and distilled water was added to mark. Thirty five grammes (35g) tomatoes

were contaminated by soaking in chlorpyrifos solution for 10min and allowed to air dry

(Churdchai and Nguyen, 2010)

2.2.10.3 Preparation of Washing Solutions

Five (5) different washing solutions were prepared. Different concentrations of

biosurfactant solution were prepared by adding 0.5ml, 1ml, 2ml and 4ml biosurfactant solution

into 100ml volumetric flask and made up to 100ml mark with distilled water to get 5ppm,

10ppm, 20ppm and 40ppm respectively. The last washing solution was just water. Chylorprifos

contaminated tomatoes were washed in each washing solution for 5min (Churdchai and Nguyen,

2010).

2.2.10.4 Extraction of Pesticide Residue

Five (5) grammes (35g) of washed tomatoes was blended and centrifuged at 4000rpm for

30minutes. The supernant was used for the analysis. A known weight 2.5g of the supernant was

weighed into 25ml calibrated test tubes. A volume, 2.5ml 6.0M sulfuric acid was added and

vortexed for 2min. The mixture was then centrifuged at 4000rpm for 10minutes. Supernant was

carefully removed and kept at 4oC until used (Akinloye et al., 2011).

27

2.2.10.5 Determination of Pesticide Residue

Twenty microlitres (20µl) of the supernant was added into test tubes and 5ml of 0.01M

sodium acetate buffer pH 5.0 was added. Two milliliters (2.0ml) of 1% (w/v) aqueous sodium

dithionite was added and then 1ml of 0.1M sodium hydroxide was added. The mixture was

allowed to stand for 5min and absorbance was taken at 450nm using a spectrophotometer.

(Akinloye et al., 2011).

2.2.11 Statistical Analysis

Mean ± standard deviations of triplicate determinations were calculated or used to

analyze data.

28

CHAPTER THREE

RESULTS

3.1 Proximate Composition

The results of the proximate composition of red cashew pomace are presented in Table

3.1. The constituents ranged from 2.45% in ash to 51.8% in total carbohydrate.

3.2 Determination of Growth in the Seed Culture

The growth of the Pseudomonas aeruginosa in the culture medium was monitored at

600nm using spectrophotometer. The increase in absorbance from 0.418 to 1.459 with time

indicated growth of Pseudomonas aeruginosa and its active nature (Fig 3.1).

3.3 Growth Rate Determination of Pseudomonas aeruginosa in The Different Culture

Media

The growth of Pseudomonas aeruginosa in each of the culture broths as shown in Fig 3.2

over a period of 10 days. This was monitored using the spread plate technique by the

determination of the number of colony forming units. There was an increase in cell numbers in

all the culture media showing the log phase of growth from the second day to the fourth day

with medium III (nutrient broth) having the lowest growth rate.

3.4 Screening for Biosurfactant Activity in Culture Broth

3.4.1 Haemolysis Test

Haemolysis test was used to check for the activity of biosurfactant where formation of

clear zones indicated presence of biosurfactant. The culture broths were positive for haemolytic

activity as clear zones of haemolysis were observed on blood agar. The result in table 3.2

showed that more biosurfactants were produced in medium I than in medium II and medium III

had the lowest.

3.4.2 Oil Spreading Test

Oil spreading or displacement ability was observed in all culture supernatants as an

indication of biosurfactant activity. The oil spreading test is indicative of the surface and wetting

activities of a surfactant sample, thus a larger diameter represents a higher surface activity.

Table 3.3 shows that medium I exhibited high spreading capacity and medium III had the lowest

spreading capacity.

29

Table 3.1: Proximate composition of red cashew fruit pomace.

Constituents Proximate composition (%)

Moisture 8.95± 0.21

Fibre 2.85± 0.05

Ash 2.45± 0.05

Crude Protein 9.66± 0.54

Lipid 24.28± 0.50

Total Carbohydrate 51.81± 0.60

30

Fig 3.1: Growth of Pseudomonas aeruginosa in the seed culture at OD600 nm.

31

Fig 3.2: Growth curve of Pseudomonas aeruginosa in each of the culture broths

Key:

Medium I: Red cashew pomace

Medium II: Glucose

Medium III: Nutrient broth

32

Table 3.2: Red blood cell lysis by the various culture broth supernatants.

CULTURE SUPERNATANT HAEMOLYTIC REACTION

Medium I (Red cashew pomace) +++

Medium II (Glucose) ++

Medium III (Nutrient Broth) +

KEY: +++ High activity, ++ Moderate activity, + poor activity

33

Table 3.3: Determination of oil spreading capacity of the various culture broth supernatant

CULTURE SUPERNATANT DIAMETER (cm)

Medium I (Red Cashew pomace) 5.4 ± 0.05

Medium I (Glucose)

Medium III (Nutrient broth)

2.5 ± 0.10

2.0 ± 0.10

34

3.4.3 Emulsification Index Test (E24)

One major advantage of biosurfactant is its ability to form stable emulsion with

hydrocarbons and vegetable oils. The emulsification ability of the biosurfactant and synthetic

surfactant, sodium dodecyl sulphate (SDS) was analyzed on some hydrocarbons and vegetable

oil. The result of the emulsification index test in Fig 3.3 reveals that emulsion was formed when

tested with palm oil, olive oil, kerosene and engine oil. Biosurfactant was able to form more

stable emulsion with vegetable oil than hydrocarbons; Meanwhile, SDS was unable to form

stable emulsion with vegetable oil but with hydrocarbons. The difference in emulsification

indices is due to the concentration of biosurfactant in each of the medium.

3.4.4 Stability Studies

3.4.4.1 Effect of Temperature on Biosurfactant and SDS Activity

The stability of the biosurfactants was assessed after incubation across a range of

temperature (30– 1000C) using the emulsification index. The emulsification index was

thermostable compare to SDS that decreases in activity as the temperature increase as seen in

Fig 3.4.

3.4.4.2 Effect of pH on Biosurfactant Activity

Fig 3.5 shows the result of the stability studies of the biosurfactant in the culture broth

supernatant and synthetic surfactant, SDS assessed over a range of pH (2 – 12) using the

emulsification index. The biosurfactant activity reduced between pH 2-6 but still retained more

than 50% of its activity. It was more active between pH 7-12 with highest activity at pH 8.

3.4.4.3 Effect of Salinity on Biosurfactant Activity

Fig 3.6 shows the effect of salt concentration on biosurfactant activity. The biosurfactant

was able to retain its activity up to 8% concentration of sodium chloride unlike the synthetic

surfactant, SDS that lost its activity at 3-4% concentration.

35

Fig 3.3: Emulsification indices (%) of culture broth supernatants and SDS.

36

Fig 3.4: Determination of temperature stability of culture broth supernatants and SDS.

37

Fig 3.5: Effect of pH on biosurfactant and SDS activities

38

Fig 3.6: Effect of salt on biosurfactant and SDS activities.

39

3.5 Quantification of Biosurfactant

The concentration of the biosurfactants for medium I (red cashew), medium II (glucose)

and medium III (nutrient broth) were 1.00, 0.39 and 0.15g/l respectively.

3.6 Biochemical Composition of Isolated Biosurfactant

The biochemical composition of the isolated biosurfactants showed that media I, II and

III isolates, the concentration of carbohydrate was 6.95, 5.04 and 4.57g/l respectively while for

lipids, the values were 0.34, 0.29 and 0.28g/l respectively. Protein was not detected.

3.7 Effect of washing solutions on pesticide residue

Fig 3.7 shows the different concentration of biosurfactant solution used in washing off

chylorpyrifos residue in tomatoes. From the result, the higher the concentration of biosurfactants

the more effective it becomes in reducing chylorpyrifos residue to the maximum residue limit.

The maximum chylorprifos residue limit for tomatoes is 0.5ppm.

40

Fig 3.7: Effect of different washing solutions with initial chylopyrifos concentration of 100ppm

Key:

BS- Biosurfactant solution

41

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.0 Discussion

In this study, the biosurfactants produced by Pseudomonas aeruginosa in a submerged

fermentation system using Red cashew (Anacardium occidentale) pomace as substrate was

characterized and applied as a cleaning agent for pesticide residue in tomatoes. The main factor

limiting commercialization of biosurfactants is associated with non-economical large scale

production. To overcome the obstacle and to compete with synthetic surfactants, inexpensive

substrate and effective microorganism has to be intensively developed for biosurfactant

production.

Proximate analysis of red cashew (Anacardium occidentale) pomace presented in table

3.1 showed similar values obtained by Adebowale et al, (2011). The high content of

carbohydrate (51.81 ± 0.60) and lipids (24.28 ± 0.50) make it a good carbon source that supports

the optimum growth of Pseudomonas aeruginosa in a fermentation medium.

Pseudomonas aeruginosa used in this study was maintained in nutrient broth (seed culture) at

37°C in order to monitor its viability and ease of inoculation into fermentation media. Growth

determination of the seed culture by measurement of absorbance at 600nm showed there was an

increase in absorbance from 0.418-1.459.

The growth of the microorganism (P. aeruginosa) in the various culture media was

monitored by determination of number of colony forming units as shown in Fig 3.2. There was

an increase in cell numbers in all the culture media showing the log phase of growth from the

second day to the fourth day with medium III (nutrient broth) having lowest growth rate.

Biosurfactant production was found to be maximum at the early stationary phase in all culture

media. Higher concentration of biosurfactant at the early phase may be due to the release of cell-

bound biosurfactant into the culture broth which lead to the rise in extracellular biosurfactant

concentration (Preethy and Nilanjana, 2010).

The positive haemolytic test agreed with the works of Thavasi et al., (2011) who showed

that the bacteria strain (Pseudomonasa aeruginosa) grown on crude oil exhibited clear zone

(3.05cm) on blood agar plates. In this study, medium I exhibited larger clear zone of haemolysis

(3.2cm diameter) on blood agar showing that more quantity of biosurfactants were produced

using red cashew pomace as substrate.

In table 3.3, oil spreading or displacement ability was observed in all the culture

supernatants as indication of biosurfactant activity. The oil displacement test is indicative of the

42

surface and wetting activities of a surfactant sample, thus a larger diameter represents a higher

surface activity (Chandran and Das, 2