ii

ISOLATION AND APPLICATION OF VIOLET PIGMENT EXTRACTED FROM Chromobacterium violaceum

NUR ZULAIKHA BINTI YUSOF

UNIVERSITI TEKNOLOGI MALAYSIA

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I hereby declare that I have read this thesis and in my opinion this thesis is sufficient in terms of scope and quality for the award of the degree of Master of Science (Chemistry)

Signature

: _______________________

Name of Supervisor : Prof. Dr. Wan Azlina Ahmad Date :

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bahagian a PENGESAHAN KERJASAMA*

Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui kerjasama antara _______________________ dengan _______________________

Disahkan oleh: Tandatangan Nama Jawatan (Cop rasmi) : : : Tarikh :

* Jika penyediaan tesis/projek melibatkan kerjasama.

bahagian b UNTUK KEGUNAAN PEJABAT SEKOLAH PENGAJIAN SISWAZAH

Tesis ini telah diperiksa dan diakui oleh: Nama dan Alamat Pemeriksa Luar :

Nama dan Alamat Pemeriksa Dalam

Nama Penyelia lain (jika ada)

Disahkan oleh Timbalan Pendaftar di Sekolah Pengajian Siswazah: Tandatangan : Nama : Tarikh :

ISOLATION AND APPLICATION OF VIOLET PIGMENT EXTRACTED FROM Chromobacterium violaceum

NUR ZULAIKHA BINTI YUSOF

A thesis submitted in fulfilment of the requirements for the award of the degree of Master of Science (Chemistry)

Faculty of Science Universiti Teknologi Malaysia

NOVEMBER 2010

ii

I declare that this thesis entitled Isolation and Application of Violet Pigment Extracted from Chromobacterium violaceum is the result of my own research except as cited in the references. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.

Signature Name Date

: _______________________ : Nur Zulaikha Yusof :

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In the name of Allah, the Most Gracious, the Most Merciful, This page is especially dedicated to my beloved mum, Che Zaharah binti Yamin, dad, Yusof bin Mohammad, sisters and brother for their inspiration, support and happiness.

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ACKNOWLEDGEMENTS

In the name of Allah s.w.t, the Most Gracious, the Most Merciful, All praise to Allah s.w.t, for His Mercy has given me patience and strength to complete this work. All the praise to Allah s.w.t again. First and foremost, I would like to express my deepest thanks to my supervisor, Prof. Dr. Wan Azlina Ahmad, for the valuable guidance and advice during the course of this research. Without her time and patience, this work could not have been accomplished. I have gained a lot of knowledge and experience during doing this research. I would also like to express my gratitude to PM. Dr. Farediah Ahmad, Ms. Nor Akmalazura Jani and Mrs. Mek Zum for their guidance in FTIR and NMR analysis, as well as Mr. Mohd Fazlin Rezali, SIRIM Berhad for Mass Spectrometry analysis. Special thanks also goes to Malaysian Craft Development Corporation, Kelantan (especially Mr. Khamis bin Yassim) and Prof. Dr. Wan Yunus Wan Ahmad from Program of Textile Technology, Faculty of Applied Sciences, Universiti Teknologi Mara (UiTM), Shah Alam for their co-operation and assistance in completing some part of this research. A million thanks also go to Dr. Zainul Akmar Zakaria, K. Diana, K. Lini, K. Laily, Asop, K. Quek, K. Fad, Nisa, K.rozi, Mr. Ali, Jay, Mimie and Sue for their knowledge, encouragement and guidance throughout the research. Last but not least, I wish to express my sincere appreciation to my beloved family for their continuous support, advices and motivation for me to complete my research .Thank you so much.

ABSTRACT

The potential of natural pigments derived from bacteria to replace the toxic synthetic colourants was studied. The scope of study includes isolation and screening of bacterial pigment producers, characterization of isolated pigment through instrumental analysis and application of the pigment in textile dyeing. A total of seventeen coloured bacterial isolates were obtained from two sampling points which are fish breeding centre, Gelang Patah and oil refinery wastewater treatment plant, Negeri Sembilan. Of these, two bacteria which produced violet and orange pigment were identified as Chromobacterium violaceum and Chryseobacterium sp. using 16S rRNA analysis. The yellow-orange pigment was suspected as flexirubin via testing with 20 % (w/v) KOH whilst the violet pigment was confirmed as violacein through ultraviolet/ visible (UV/Vis), Fourier-transform infrared (FTIR), nuclear magnetic resonance (NMR) and mass spectrometry (MS). Highest violacein production was observed in sugarcane bagasse (SCB) supplemented with L-tryptophan either in batch study (0.82 g L-1) or in immobilized cell system (0.15 g L-1). In batch study, the optimum condition for violacein production was obtained using 3 g SCB supplemented with 10 % (v/v) L-tryptophan in distilled water at 30oC for 24 hours. From the textile dyeing study, violacein was capable to dye both natural and synthetic fibers. Use of different mordants yielded different colours on dyed fabrics and also increase the lightfastness rating. On cotton, Fe2(SO4)3 and CuSO4 managed to increase the lightfastness rating from 1 (control) to 2 and 2/3 respectively while on silk satin, alum and slaked lime increased the lightfastness rating from 1 (control) to 2. Higher mordant concentration also darkened the dyed fabric colour whilst pretreatment of fabric manage to increase the pigment performance on the fabric. The reactive dye showed intense colouration on both fabrics compared to violacein. Overall, violacein shows comparable performance with reactive dyes on fabric dyeing and can be used as an alternative to replace synthetic dye.

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ABSTRAK

Potensi pigmen semula jadi yang berasal daripada bakteria untuk menggantikan pewarna tiruan yang bertoksik telah dikaji. Skop kajian ini melibatkan pemencilan dan penyaringan bakteria berwarna, pencirian pigmen menggunakan analisis berinstrumen dan penggunaan pigmen dalam pewarnaan tekstil. Sejumlah tujuh belas bakteria pengeluar pigmen telah dipencilkan dari dua tempat persampelan iaitu pusat penternakan ikan, Gelang Patah dan loji rawatan air sisa pusat penapisan minyak, Negeri Sembilan. Dua bakteria yang menghasilkan pigmen berwarna ungu dan jingga masing-masing telah dikenal pasti sebagai Chromobacterium violaceum dan Chryseobacterium sp. melalui analisis 16S rRNA. Pigmen jingga telah diramalkan sebagai flexirubin melalui ujian menggunakan 20 % (w/v) KOH manakala pigmen ungu telah disahkan sebagai violacein melalui analisis ultralembayung/ nampak (UV/Vis), analisis spektroskopi inframerah (FTIR), resonans magnet nukleus (NMR) dan spektrometri jisim (MS). Penghasilan violacein tertinggi telah diperoleh dalam hampas tebu (SCB) ditambah dengan L-triptofan sama ada dalam kajian kelompok (0.82 g L-1) atau dalam sistem sel pegun (0.15 g L1 ). Dalam kajian kelompok, keadaan optimum untuk penghasilan violacein adalah dengan menggunakan 3 g SCB yang ditambah dengan 10 % (v/v) L-triptofan dalam air suling pada suhu 30oC selama 24 jam. Daripada kajian pewarnaan tekstil, violacein mampu mewarnakan dengan baik kedua-dua serat semula jadi dan sintetik. Penggunaan mordan yang berbeza memberikan warna yang berbeza pada fabrik dan juga meningkatkan tahap ketahanan terhadap cahaya. Pada kain kapas, Fe2(SO4)3 dan CuSO4 masing-masing berjaya meningkatkan tahap ketahanan terhadap cahaya daripada 1 (kawalan) kepada 2 dan 2/3 manakala pada satin sutera, tawas dan kapur mati pula berjaya meningkatkan tahap ketahanan terhadap cahaya daripada 1 (kawalan) kepada 2. Kepekatan mordan yang tinggi juga telah memberikan warna yang gelap terhadap fabrik dan pra-rawatan terhadap fabrik berjaya meningkatkan prestasi pigmen pada fabrik. Pencelup reaktif memberikan warna yang terang pada kedua-dua jenis fabrik berbanding dengan violacein. Keseluruhannya, violacein menunjukkan prestasi yang setanding dengan pewarna reaktif dalam pewarnaan fabrik dan ia boleh digunakan sebagai satu alternatif untuk menggantikan pewarna sintetik.

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TABLE OF CONTENTS

CHAPTER

TITLE

PAGE

DECLARATION DEDICATION ACKNOWLEDGEMENTS ABSTRACT ABSTRAK TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

ii iii iv v vi vii xiii xv xvii

GENERAL INTRODUCTION 1.1 1.2 Definition of Pigments Classification of Pigments and Its Applications 1.2.1 Natural Pigments 1.2.2 Synthetic Pigments 1.3 World Scenario on the Use of Natural Pigments 1.3.1 History of Colourants 1.3.2 Ill Effects of Using Synthetic Dyes 1.3.2.1 Effects on Health 1.3.2.2 Effects on Environment 1.4 Natural Pigments 1.4.1 Importance of Using Natural Pigments 1.4.2 Microbial Sources of Natural Pigments 1.4.2.1 Bacteria 1 2 2 4 5 5 6 6 7 8 8 10 10

viii 1.4.2.2 Fungi 1.4.2.3 Yeasts 1.4.2.4 Algae 1.4.3 Functions of Microbial Pigments 1.4.4 Advantages of using Bacterial Pigments 1.4.5 Applications of Bacterial Pigments 1.4.5.1 Food Colourants 1.4.5.2 Pharmaceutical 1.4.5.2 Textile Dyeing 1.5 1.6 Objectives of the Study The Scope of Study 12 13 13 14 14 16 16 17 18 20 20

ISOLATION AND CHARACTERIZATION OF PIGMENT FROM COLOURED BACTERIA 2.1 Chemistry of Pigments 2.1.1 Instrumental Analysis 2.2 Materials and Methods 2.2.1 Growth Medium 2.2.1.1 Nutrient Broth 2.2.1.2 Nutrient Agar 2.2.2 Collection of Samples 2.2.3 Cultivation and Isolation of Cultures 2.2.3.1 Liquid Samples 2.2.3.2 Soil Samples 2.2.4 Characterization of Microorganism 2.2.4.1 Gram Staining 2.2.4.2 Identification of Bacterial Isolates 2.2.5 Maintenance of Stock Culture 2.2.5.1 Short term Maintenance 2.2.5.2 Long term Maintenance 2.2.6 Preparation of Inoculum 2.2.7 Extraction of Pigments 2.2.7.1 Yellow Orange Pigment 21 22 23 23 23 24 24 25 25 25 25 25 26 27 27 27 27 28 28

ix 2.2.7.2 Violet Pigment 2.2.8 Pigment in Powdered Form 2.2.9 Characterization of Crude Violet Pigment 2.2.9.1 Antimicrobial Effect 2.2.9.2 UV/Vis Spectroscopy 2.2.9.3 Fourier-Transform Infrared Spectroscopy (FT-IR) 2.2.9.4 Thin Layer Chromatography (TLC) 2.2.9.5 Nuclear Magnetic Resonance (NMR) Spectroscopic 2.2.9.6 Mass Spectrometry (MS) Analysis 2.2.9.7 Spectroscopic Data for Violet Pigment 2.2.10 Primary Identification of Yellow Orange Pigment 2.2.10.1 Simple Test for Flexirubin Type Pigment 2.3 Results and Discussion 2.3.1 Characteristics of the Soil and Water Sample 2.3.2 Isolation and Characterization of Coloured Bacteria 2.3.3 2.3.4 2.3.5 Identification of Bacteria Maintenance of Stock Culture Characterizations of Crude Violet Pigments 2.3.5.1 Antimicrobial Effect 2.3.5.2 UV/Vis Spectroscopy 2.3.5.3 Infrared Spectroscopy 2.3.5.4 Thin Layer Chromatography (TLC) 2.3.5.5 Nuclear Magnetic Resonance (NMR) 2.3.5.6 Mass Spectrometry (MS) 2.3.6 Primary Identification of Yellow - Orange 44 48 33 36 37 37 37 39 41 44 32 32 32 32 31 31 31 30 30 28 28 29 29 30

x Pigment 2.3.6.1 Simple Test for Flexirubin Type Pigment 2.3.7 Conclusion 50 51 50

OPTIMIZATION OF CULTURE CONDITIONS FOR PIGMENT PRODUCTION BY C. violaceum 3.1 Utilization of a Cheap Growth Medium for Pigment Production 3.1.1 Immobilized Cell Systems 3.2 Materials and Methods 3.2.1 Preparation of Inoculum 3.2.2 Preparation of L-tryptophan Stock Solution 3.2.3 Growth Profile of C. violaceum 3.2.4 Effect of Temperature on Pigment Production 3.2.5 Production Profile of Violacein by C. violaceum in Complex Media 3.2.6 Evaluation of Various Agricultural Waste Substrates for Pigment Production 3.2.7 Laboratory Scale Column System 3.2.8 Immobilization of C. violaceum onto SCB 3.2.9 Determination of Pigment Concentration 3.3 Results and Discussion 3.3.1 Growth Profile of Bacteria 3.3.2 Effect of Temperature on Bacterial Growth and Pigment Production 3.3.3 Violacein Production on Various Complex Media 3.3.4 Utilization of Agriculture Wastes as an Alternative Medium for Violacein Production 3.3.5 Preliminary Study on the Immobilization of 63 61 60 56 57 57 58 58 58 55 54 52 53 54 54 54 54

xi C. violaceum on SCB 3.4 Conclusion 64 65

APPLICATION OF PIGMENT ISOLATED FROM C. violaceum IN TEXTILE DYEING 4.1 Natural Colourants in Textile Dyeing 4.1.1 Reason for Natural Colouration 4.2 Materials and Methods 4.2.1 Fabrics 4.2.2 Soap Solution 4.2.3 Perspiration Solution 4.2.4 Mordants 4.2.5 Dye Material 4.2.6 Mordanting Procedure 4.2.7 Method of Dyeing 4.2.8 Dyeability of Pigment on Different Fibers 4.2.9 Dyeing of Silk Satin and Cotton Fibres 4.2.10 Colourfastness Standard Tests 4.2.11 Preparation of Fabric for Testing 4.2.11.1 4.2.11.2 4.2.11.3 Colourfastness to Washing Colourfastness to Light Colourfastness to Rubbing/Crocking 4.2.11.4 Colourfastness to Perspiration 4.2.11.5 4.3 Colourfastness to Water 74 74 75 73 67 68 69 69 69 69 70 70 70 71 71 71 72 73 73 73

Results and Discussion 4.3.1 Dyeability of Violacein on Different Fabrics 4.3.2 Effect of Types of Mordant on Cotton and Silk Satin 4.3.3 Effect of Mordant Concentrations on Cotton and Silk Satin

75

77

80

xii 4.3.4 Effect of Pre-Treatment on Dyeing Performance 4.3.5 Comparison of Fastness Properties of Violacein with Reactive Dyes 4.4 Conclusion 83 85 81

CONCLUSION 5.1 5.2 Conclusion Suggestions for Future Work 87 87 89 108

REFERENCES APPENDIX

xiii

LIST OF TABLES

TABLE NO. 1.1

TITLE Naturally occuring pigments in biology (Hendry and Houghton, 1997).

PAGE

1.2

Microbial production of pigments (already in use as natural food colourants or with high potential in this field) (Liu and Nizet, 2009). 9

2.1

Characteristics of water and soil samples collected from Brackishwater Aquaculcure Research Centre, Gelang Patah, Johor. 33

2.2

Characteristics of soil samples collected from treatment pond of oil refinery at Port Dickson, Seremban. 33

2.3

Characteristics of bacterial colonies obtained from soil and water isolates grown on NA plate. 34

2.4

Morphological features of the coloured bacterial colonies on NA plate. 35

2.5

Antimicrobial testing of violet pigments on P. aeruginosa, S. aureus, B. cereus and E. coli. 38 44 48 OH (free) cm-1 and C-O cm-1 in alcohols and phenols
1

2.6 2.7 4.1

H and C data of violet pigment.

13

Shades and fastness properties of different fabrics dyed with natural pigment. 77

4.2

Shades and fastness properties of dyed cotton and silk satin in the presence of various mordants. 79

4.3

Colour of dyed fabrics mordanted using different concentration of mordant. 80

4.4

Shade and fastness properties of dyed cotton and silk satin

xiv mordanted using difference concentration of mordants. 4.5 Shades and fastness properties of fabrics after pretreatment process. 4.6 Comparison of fastness properties of natural dyes with reactive dyes. 85 83 81

xv

LIST OF FIGURES

FIGURE NO. 2.1

TITLE Morphology of isolate S8b on NA plate (a) and NB medium (b).

PAGE

34

2.2

Morphology of isolate S1a on NA plate (a) and NB medium (b). 35

2.3

UV/Vis spectrum for methanolic extract of violet pigment. 40

2.4

The charge delocalization by the contributing structures in the absence (a) and presence (b) of electron donatingNHR group. 41

2.5

FTIR spectrum for crude methanolic extract of violet pigment. 42

2.6

Structure of secondary amides: ciscoid-ciscoid associations (a) and transoid-transoid polymeric association (b). 42

2.7

Resonance structure of carbonyl containing group in double bond structure. 43 45 45 47 49

1

2.8 2.9 2.10 2.11 2.12

H NMR spectrum of violet pigment.

The structure of violacein.

13

C NMR spectrum of violet pigment.

The ESI - MS spectrum of violet pigment. The ESI MS / MS spectrum of violet pigment (m/z = 342.34).

49 59 59 60

3.1 3.2 3.3

Growth profile of C. violaceum in NB. Effect of time on pigment production on C. violaceum. Growth profile of C.violaceum in NB at 25, 30 and 37oC.

xvi 3.4 The absorption spectrum of the violet pigment obtained at different incubation temperatures. 3.5 Time course of cell growth and pH profile of C. violaceum cultivated in NB, TSB, PGB and LB. Symbols: ( OD600, ( ( 3.6 3.7 ) NB OD600, ( ) PGB pH, ( ) NB pH, ( ) PGB ) LB pH, 62 62 61

) LB OD600, ( ) TSB pH.

) TSB OD600 and (

Violacein production by C. violaceum in complex media. Colour intensity of pigments in a) liquid substrate and b) solid substrate.

63

3.8

Pigment production by C. violaceum grown in SCB, SPW, M and BS supplemented with 10% (v/v) Ltryptophan, in distilled water; SCB sugar cane bagasse, SPW - solid pineapple waste, brown sugar - BS; M molasses. 64 65

3.9 4.1

Violacein production in sugarcane bagasse. Dyeability of violacein on different fabrics using alum as mordant; a) pure cotton (PC), b) pure silk (PS), c) pure rayon (PR), d) rayon jacquard (RJ), e) silk satin (SS), f) cotton (C) and g) polyester (P).

75

4.2

Colour of dyed cotton and silk satin control (no mordant) (a) and mordanted using (b) alum, (c) Fe2(SO4)3, (d) CuSO4 and (e) Ca(OH)2. 78

4.3

Colour of dyed cotton and silk satin fabric after pretreatment process; (a) unmordanted and mordanted using (b) alum, (c) Fe2(SO4)3, (d) CuSO4 and (e) Ca(OH)2. 82

4.4

Colour performance of fabrics dyed with reactive dye and violacein, a) silk satin and b) cotton. 84

xvii

LIST OF ABBREVIATIONS

AATCC Abs AMES EBV

13

American Association of Textile Chemists and Colorists Absorbance Salmonella typhimurium reverse mutation assay EpsteinBarr virus Carbon-13 Electronspray Ionization Mass Spectrometry Electrospray Ionization Tandem Mass Spectrometry Proton Hertz Food and Drug Administration Fourier-Transform Infrared Spectroscopy Megahertz Molecular Mass Spectrometry Nuclear Magnetic Resonance Provoke allergic or pseudo-allergic reactions Retention factor Reactive oxygen species Ribosomal ribonucleic acid Standard and Industrial Research Institute of Malaysia Sugarcane bagasse Solid pineapple waste Tumor promoter 12-O-tetradecanoylphorbol-13- actate Thin layer chromatography United States dollar Ultraviolet/visible Pi

ESI-MS ESI-MS/MS
1

Hz FDA FT-IR MHz MS NMR PARs Rf ROS rRNA SIRIM SCB SPW TPA TLC USD UV/Vis

xviii J d s t dd m m/z max max kPa Coupling constant Gamma Chemical shift Doublet Singlet Triplet Doublet of doublet Micrometer Mass-to-charge ion Wavelength at an absorption maximum Maximum absorption frequency Kilo pascal

CHAPTER 1

GENERAL INTRODUCTION

1.1

Definition of Pigments

The word pigment has a Latin origin and initially denoted a colour (in the sense of coloured matter), but it was later extended to indicate the coloured objects such as makeup. In the beginning of the middle ages, the word was also used to describe the diverse plant and vegetable extracts, especially those used for the food colouring. The word pigment is still used in this sense in the biological terminology such as the coloured matter present in the animals or the plants, occurring in the granules inside the cells or cell membranes as the deposits on the tissues, or suspended in the body fluids (Ullmann, 1985). Its also include organic compounds isolated from cells and their modified structure which is to alter stability, solubility or their colour intensity (Hendry and Houghton, 1997). The examples of biological pigments include chlorophyll and haemoglobin.

The modern meaning associated to the word pigment has its origin in the twentieth century, meaning a substance constituted of small particles which is practically insoluble in the applied medium, and is used due to its colourant, protective or magnetic properties (Ullmann, 1985). Pigment also changes the colour of light it reflects as a result of selective colour absorption. This definition applies well to the pigments of the mineral origin, such as titanium dioxide or carbon black, but for the soluble dyestuffs, usually the organic compounds, the expressions dye, colourant or simply colour (as in the food colours) is more adequate. The terms

2 pigment and colour are usually applied for the food colouring matters, sometimes indistinctly (Timberlake and Henry 1986).

1.2

Classification of Pigments and Its Applications

Pigments are classified as either organic or inorganic and natural or synthetic (Turner, 1993).

1.2.1

Natural Pigments

Natural organic pigments or biological pigments are used since the middle ages by cave artists to decorate the walls of caves. It is commonly extracted from naturally occurring fruits, vegetables, animals and others, by means of a simple solvent extraction process (Frank, 2001). Biological pigments can be classified into two methods of classifications, which are based on structural affinities and based on the natural occurrence of pigment in biology. Examples of natural occurring pigments are anthocyanins (blue-red), carotene (yellow-red), chlorophylls (green) and tannins (brown-red) (Babitha et al., 2004). Basically, most of the natural pigments have several features to distinguish them from the larger number of colourless compounds found in biological material. Almost all biological molecules are composed of no more than seventeen elements within the periodic table. Only four out of seventeen elements predominate notably H, C, N and O; most pigmented compounds, particularly those other than yellow, contain either N or O, often both and most of them are relatively large molecules. Among the more common pigmented compounds, their molecular weights range from about 200

(anthraquinones), 300 (anthocyanidins), 400 (betalaines), 500 (carotenoids) to 800 (chlorophylls). Much greater molecular weight is found in pigment polymers such as melanins (Hendry and Houghton, 1997). Almost all biological pigments can be reduced to no more than six major structural classes: tetrapyrroles, tetra-terpenoids,

3 quinines, O-heterocyclic, N-heterocyclic and metallo-proteins. The naturally occurring pigments in biology is summarized in Table 1.1.

Table 1.1: Naturally occuring pigments in biology (Hendry and Houghton, 1997). Group Tetrapyrroles Alternative or familiar name Porphyrins and porphyrin derivatives Major example Chlorophylls Heams (hemes) Bilins (Bile pigments) Carotenes Xanthophylls Anthocyanins Flavonols Naphtaquinones Anthraquinones Predominant colour Green Red Blue-greenyellow-red Yellow-red Yellow Blue-red Yellow-white Red-bluegreen Re-purple Yellow-red Black-brown White-yellow Opaque white Blue-green Red Brown-grey Various but commonly yellow

Tetraterpenoids

Carotenoids

O-heterocyclic compounds Quinines

Flavonoids

Phenolics compounds

N-heterocyclic compounds

Indigoids and indole derivatives Substituted pyrimidines

Betalaines Eumelanins Pterins Purines Blue-green Red Lipofuscins Fungal pigments

Metalloproteins

Cu-proteins Haemerythrin

miscellaneous

Natural inorganic pigments, derived mainly from mineral sources, have been used as colourants since prehistoric times and a few, notably iron oxides, remain of some significance today. The colour of inorganic pigments arises from electronic transitions which are quite diverse in nature and different from those responsible for the colour of organic colourants. Inorganic pigments generally exhibit high inherent opacity, a property which may be attributed to the high refractive index which results from the compact atomic arrangement in their crystal structure. Various methods are

4 employed in the manufacture of inorganic pigments. Frequently, the chemistry is carried out in aqueous solution from which the pigments can precipitate directly in a suitable physical form. In some cases, high temperature solid state reactions are used, while gas-phase processes because of their suitability for continuous large-scale manufacture are of importance for the manufacture of the two largest tonnage pigments, which are titanium dioxide and carbon black. Other examples of natural inorganic pigments were cadmium sulfides, lead chromates and ultramarines (Christie, 2001).

1.2.2

Synthetic Pigments

The synthetic organic pigments were prepared from water-soluble dyes rendered insoluble by precipitation onto colourless inorganic substrates such as alumina and barium sulphate where these products were referred to as lakes. A critical event in the development of the organic pigment industry was the discovery of copper phthalocyanine in 1928. Organic pigments generally provide higher intensity and brightness of colour than organic pigments. These pigments are unable to provide the degree of opacity which is typical of inorganic pigments, because of the lower refractive index associated with organic crystals. The range of commercial organic pigments exhibit variable fastness properties which are dependant both on the molecular structure and on the nature of the intermolecular association in the solid state. Since organic molecules will commonly exhibit some tendency to dissolve in organic solvents, organic pigment molecules incorporate structural features with design to enhance solvent resistant. The examples of synthetic organic pigments include azo pigments, copper phthalocyanines and high-performance organic pigments (Christie, 2001).

5 1.3 World Scenario on the Use of Natural Pigments

1.3.1

History of Colourants

Before the turn of the century natural dyes were the only source of colour available and, therefore, they were widely used and traded, providing a major source of wealth creation around the globe. It has been used for many purposes such as the colouring of natural fibers wool, cotton and silk as well as fur and leather. The dyes were also used to colour cosmetic products and to produce inks, watercolours and artists paints (Cristea and Vilarem, 2006). Since the introduction of synthetic dyes by Perkin in 1856, many convenient and cheap synthetic pigments have appeared, and the use of natural dyes has decreased due to their poor price competitiveness (Zollinger, 1991).

Synthetic dyes are extensively used in many fields of upto- date technology, for example in various branches of the textile industry, of the leather tanning industry in paper production, in food technology, in agricultural research, in light-harvesting arrays, in photoelectrochemical cells, and in hair colourings. Moreover, synthetic dyes have been employed for the control of the efficacy of sewage and wastewater treatment, and for the determination of specific surface area of activated sludge for ground water tracing (Forgacs et al., 2004).

The synthetic dyes employed more frequently on industrial scale are the azo, anthraquinone, sulfur, indigoid, triphenylmethyl (trityl), and phthalocyanine derivatives. However, it has to be emphasized that the overwhelming majority of synthetic dyes currently used in the industry are azo derivatives (Forgacs et al., 2004). Synthetic dyes were widely used as compared to natural dyes because it shows several advantages such as high stability to light, oxygen and pH, colour uniformity, low microbiological contamination, relatively lower production costs and others (Alves et al., 2008).

6 1.3.2 Ill Effects of Using Synthetic Dyes

1.3.2.1 Effects on Health

In pharmaceutical industry, the synthetic food dye is added in order to add colour to many medicinal products, as well as to ensure the same colour for all the batches of a given product. Adding a colour makes the medicinal product more attractive, easier to recognise, and in some cases, by forming an opaque layer, it stabilizes the ingredients of the medicine which are light sensitive (Jaworska et al., 2005).

Eventhough synthetic food dyes are more long-lasting and often cheaper than natural ones, but nowadays, using many of these dyes gives rise to serious reservations concerning health. Some of them, such as, tartrazine (E 102), cochineal red (E 124), and sunset yellow (E 110), belonging to the group of azo dyes, can themselves, or in combination with other colourants, provoke allergic or pseudoallergic reactions (PARs), particularly in people allergic to aspirin and other nonsteroidal anti-inflammatory agents, or those suffering from urticaria or asthma (Rowe and Rowe, 1994).

Even supposing the synthetic colourants approved by the Food and Drug Administration (FDA) for use in foods, pharmaceuticals and cosmetic preparations have undergone rigorous scrutiny for their toxicity, surprisingly, a study on the examination of cancer chemopreventive effect of synthetic colourants revealed that, a number of these products were evaluated for their in vitro antitumor promoting effect on EpsteinBarr virus (EBV) antigen induced by tumor promoter 12-Otetradecanoylphorbol-13- actate (TPA). Among these were the azo colourant, tartrazine (FD&C Yellow # 5), and the indigo derivative, indigo carmine (FD&C Blue # 2) (Kapadia et al., 1998). For the external uses of synthetic dyes, several dyes have been withdrawn due to their apparent hazards. For example, the benzidine dyes which can cause bowel cancer while carbon black, the most widely used printing ink pigment, is thought to be a potential carcinogen.

7 1.3.2.2 Effects on Environment

From the environmental point of view, a great variety of synthetic dyes used for textile dyeing and other industrial applications causes serious pollution when part of these dyes penetrates into waste water during these processes. Most of these compounds are toxic, carcinogenic and highly resistant to degradation (Chung et al., 1992).

Azo dyes, which are the largest group of synthetic dyes, are widely used in numerous industries. While textile mills predominantly use them, azo dyes can also be found in the food, pharmaceutical, paper and printing, leather, and cosmetic industries. It is not surprising that these compounds have become a major environmental concern. Many of these dyes find their way into the environment via wastewater facilities. Because these compounds retain their colour and structural integrity under exposure to sunlight, soil, bacteria and sweat, they also exhibit a high resistance to microbial degradation in wastewater treatment systems (Eichlerov et al., 2006).

Effective and economic treatment of a diversity of effluents containing azo has become a problem. No single treatment system is adequate for degrading the various dye structures. Currently, much research has been focused on chemically and physically degrading azo dyes in wastewaters. Despite the existence of a variety of chemical and physical treatment processes, their removal from the environment is very difficult. Adsorption and precipitation methods, chemical degradation or photodegradation are financially and often also methodologically demanding, timeconsuming and mostly not very effective. Some anaerobic bacteria can decolourize several azo dyes, however, under anaerobic conditions, dyes are usually reduced to aromatic amines that are carcinogenic and mostly more toxic than the starting azo dyes (Hu, 2001; Wong and Yuen, 1996).

The synthetic dyes led to the collapse of a huge industry and gave rise to a redistribution of wealth, to the small companies now providing a large proportion of

8 the worlds colouring matters. However, due to German ban on azo dyes, currently there is a move to find renewable sources to supplement the need for safe dye industry and this trend has led to research into the production of natural dyes on a commercial-scale (Vankar et al., 2007).

1.4

Natural Pigments

1.4.1

Importance of Using Natural Pigments

Environmental concerns regarding synthetic dyes saw a revival in the demand for natural dyes as natural dyes are more environmental friendly than their synthetic counterparts. Natural dyes can exhibit better biodegradability and generally have a higher compatibility with the environment (Kamel et al., 2005). Lately, the potential of obtaining natural colour from microbial pigments to be used as natural colourants is being actively investigated (Nagia and EL- Mohamedy, 2007). Table 1.2 shows the microbial production of pigments and their status of development (Liu and Nizet, 2009).

It is interesting to note from Table 1.2 that many of the production of pigments from bacterial sources is still classified as research project or in the development stage. Hence, work on the production of pigments from bacteria should be intensified especially in finding cheap and suitable growth medium which can reduce the cost and applicable for industrial production.

9 Table 1.2: Microbial production of pigments (already in use as natural food colourants or with high potential in this field) (Liu and Nizet, 2009). Microorganism Bacteria Agrobacterium aurantiacum Paracoccus carotinifaciens Bradyrhizobium sp. Streptomyces echinoruber Flavobacterium sp. Paracoccus zeaxanthinifaciens Fungus Monascus sp. Monascus sp. Penicillium oxalicum Blakeslea trispora Fusarium sporotrichioides Cordyceps unilateralis Ashbya gossypi Monascus sp. Blakeslea trispora Fusarium sporotrichioides Mucor circinelloides Neurospora crassa Phycomyces blakesleeanus Penicillium purpurogenum Yeast Saccharomyces neoformans var. nigricans Xanthophyllomyces dendrorhous Rhodotorula sp. Astaxanthin Torularhodin Pink-red Orange-red DS DS Black Melanin RP Ankaflavin Monascorubramin Anthraquinone Lycopene Lycopene Naphtoquinone Riboflavin Rubropunctatin -carotene -carotene -carotene -carotene -carotene Unknown Yellow Red Red Red Red Deep blood-red Yellow Orange Yellow-orange Yellow-orange Yellow-orange Yellow-orange Yellow-orange Red IP IP IP DS RP RP IP IP IP RP DS RP RP DS Astaxhantin Astaxhantin Canthaxhantin Rubrolone Zeaxanthin Zeaxanthin Pink-red Pink-red Dark-red Red Yellow Yellow RP RP RP DS DS RP Pigment Colour Status*

*Industrial production (IP), development stage (DS), research project (RP )

10 1.4.2 Microbial Sources of Natural Pigments

One promising method to produce biopigments by microbes is because of their high growth rate and the feasibility of bioprocess development (Lu et al., 2009). Microbes include bacteria, fungi, yeasts and microalgae.

1.4.2.1 Bacteria

Several intensely coloured compounds have been isolated from certain bacteria which have little resemblance to pigments in other biological systems. Example of pigment from bacteria is violacein, a purple-coloured pigment produced by one of the strains of Chromobacterium violaceum found in the Amazon river, Brazil, which is an indole derivative characterized as 3-(1,2-dihydro-5-(5-hydroxy1H-indol-3-yl)- 2- oxo- 3H-pyrrol- 3-ilydene)-1, 3-dihydro- 2H-indol- 2-one (Duran and Menck, 2001). The biosynthesis and biological properties of this pigment have been extensively studied; in particular, its antitumoral, antibacterial, antiulcerogenic, antileishmanial, and antiviral activities are of interest (Leon et al., 2001, Duran et al., 2003, Melo et al., 2003). The pigment appears to be similar to that of other known species of Chromobacterium and assisted in identification of the genus of the causative organisms (Eugene et al., 1961).

Among the best-recognised bacterial pigments are the carotenoids that impart the eponymous golden colour to the major human pathogen, Staphylococcuc aureus. This organism produces multiple carotenoid pigments via a well described biosynthetic pathway that culminates with golden staphyloxanthin as the major product and yellow 44-diaponeurosporene as a minor product (Wieland, et al., 1994 and Pelz, et al., 2005). Staphyloxanthin consists of a C30 polyene carbon backbone with alternating single and double bonds typical of carotenoid pigments; these alternating bonds are able to absorb excess energy from reactive oxygen species (ROS) (El-Agamey, et al., 2004).

11 Other than that, the most familiar examples of coloured colonies seen in the routine soil, water and medical laboratory are those of pseudomonads such as the blue-green colonies of Pseudomonas aeruginosa or the yellow fluorescent colonies of Ps. fluorescens and related species. An example of a water soluble, nonfluorescent blue-green pigment produced by Ps. aeruginosa is pyocyanin which crystallises as beautiful blue needles and may have a role in respiration. The yellow water soluble fluorescent pigments produced by a number of Pseudomonas species, especially under conditions of iron limitation, are variously known as pyoverdin, pyofluorescein or simply fluorescein (Maurice, 2002).

Actinorhodin, a polyketide antibiotic, is produced by Streptomyces coelicolor A3(2), the best genetically known strain of Streptomyces. Its pigmentation (red at acidic pH and blue at alkaline pH) facilitates visual observation of its produce. Actinorhodin is often used as a model for studying factors regulating the production of antibiotics. Biosynthesis of actinorhodin occurs mainly during the stationary phase in batch cultures, but may be also growth associated according to the medium used (Ozergin-Ulgen and Mavituna, 1994). The pH of the culture medium is important as excretion of actinorhodin appears to occur exclusively at pH values above 67 (Wright and Hopwood, 1976). Moreover, this excretion increases in complex media (Ozergin-Ulgen and Mavituna, 1994) but, according to Bystrykh et al., (1996), the excreted pigment is not actinorhodin but its lactone derivative, -actinorhodin.

Prodigiosin is a secondary metabolite; and numerous factors, including biotic and abiotic factors, have been reported to affect its production in S. marcescens (Omeya et al., 2004). A wide variety of bacterial taxa, including Gram negative rods such as S. rubidaea, Vibrio gazogenes, Alteromonas rubra, Rugamonas rubra, and Gram positive actinomycetes, such as Streptoverticillium rubrireticuli and Streptomyces longisporus ruber form prodigiosin and/or derivatives of this molecule. On some media Rugamonas rubra produces so much prodigiosin that, as the pH drops, it precipitates out within the cells and colonies change from pillar box red to deep maroon, often with a green metallic sheen under reflected light. At this stage most organisms in the colony are no longer viable (Maurice, 2002).

12 1.4.2.2 Fungi

Like plants, the filamentous fungi synthesize the natural products because they have an ecological function and are of value to the producer (Firn and Jones, 2003; Babitha et al., 2004). Depending on the type of the compound, they serve different functionsvarying from a protective action against the lethal photooxidations (carotenoids) to protection against environmental stress (melanins) and acting as cofactors in enzyme catalysis (flavins). Besides providing the functional diversity to the host, these pigments exhibit a unique structural and chemical diversity with an extraordinary range of colours. Several characteristic noncarotenoid pigments are produced by the filamentous fungi, including quinines such as anthraquinones and naphthaquinones (Baker and Tatum, 1998; Medenstev and Akimenko, 1998), dihydroxy naphthalene melanin (a complex aggregate of polyketides) (Butler and Day 1998), and flavin compounds such as riboflavin.

Monascus can produce at least six major related pigments (Wang and Hesseltine, 1979), which may be divided into three groups: two are orange (rubropunctain and monascoubrin), two are yellow (monascin and ankaflavin) and two are red (rubropunctaminea and monascorubramine). Monascus spp. belongs to the group of Ascomycetes and particularly to the family of Monascaceae. The genus Monascus has four species: M. pilosus, M. purpureus, M. ruber and M. froridanus, which account for the majority of strains isolated from traditional oriental food (Sabater-Vilar et al., 1999).

The strain Penicillium oxalicum var. armeniaca CCM 8242, obtained from soil, produces a chromophore of the anthraquinone type. Many toxicological data are available on this red pigment: acute oral toxicity in mice (90-day subchronical toxicological study), acute dermal irritation/corrosion, acute eye irritation/corrosion, anti-tumour effectiveness, micronucleus test in mice, AMES test (Salmonella typhimurium reverse mutation assay), estimation of antibiotic activity, results of estimation of 5 mycotoxins (Dufoss, 2006).

13 1.4.2.3 Yeasts

The carotenoid imparts distinctive orange-red colouration to the animals and contributes to consumers appeal in the market place. Astaxanthin (3,3-dihydroxy,-carotene-4,4-dione) is widely distributed caretenoids in nature and it is the principal pigment in crustaceans and salmonids. Among the few astaxanthin producing microorganisms, Xanthophyllomyces dendrorhous is one of the best candidates for commercial production (Dufoss, 2006).

Yeasts in the genus Rhodotorula synthesize carotenoid pigments. The main compounds produced by these red yeasts are torulene and torularhodin, with minute quantity of -carotene. Feed supplement with a Rhodotorula cell mass has been found to be safe and nontoxic in animals. Its use in the nutrition of laying hens has also been documented. As -carotene content in wild strains of R. glutinis is quite low, efforts have been made to increase it through strain improvement, mutation (Sakaki et al., 2000), medium optimization and manipulation of culture conditions (Tinoi et al., 2005). These studies resulted mainly in an increased yield of torulene and torularhodin, which are of minor interest.

1.4.2.4 Algae

Microalgae are among the fast growing autotrophs on the earth which utilize commonly available material for growth. They produce vast array of natural product including protein, enzymes, bioactive compounds and carotenoids. The unicellular microalgae, Dunaleilla salina is reported to produce - carotene. The pigment was proven to have antioxidant property by quenching excessive free radicals and restoring the physiological balance. In comparison to others, Dunaliella has many advantages such as the disruption of cells is much easier than that in other algae because of its wall-less nature, the growth rate is relatively high and it is resistance to various environmental conditions is higher (Pisal and Lele, 2005).

14 1.4.3 Functions of Microbial Pigments

Pigments can help identify bacteria. Some bacteria produce pigments which can be seen after they grow into colonies. Because colours often provide an easy way of identifying certain microbes, they are often used in names of species. For example, Rosenbach in 1884 named the golden-coloured pathogen Staphylococcus aureus (Latin, golden) to distinguish it from nonpigmented staphylococci of the resident skin microflora that he named Staphylococcus alba (Latin, white). Likewise, the blue-green Pseudomonas species found in the lungs of patients with cystic fibrosis was given the name aeruginosa, which is derived from a Latin word denoting the colour of copper rust. Chromobacterium violaceum not surprisingly elaborates a blue-violet pigment. These hallmark phenotypes not only provide an easy nomenclature for the microorganisms, but continue to be important diagnostic clues in clinical laboratories today for the identification of microbes (Liu and Nizet, 2009).

Pigments have also played a role in the discovery of infectious pathogens. Some natural functions proposed for microbial pigments are, protection against ultraviolet radiation, oxidants, extremes of heat and cold, natural antimicrobial compounds produced by other microbes; antimicrobial activities against other microbes; acquisition of nutrients, such as iron and acquisition of energy by photosynthesis (e.g. cyanobacteria) (Liu and Nizet, 2009).

1.4.4

Advantages of using Bacterial Pigments

One of the limitations on the use of natural dyes or pigments lies in the order of magnitude of their extraction yield factors (a few grams of pigment per kg of dried raw material). This makes their current market price about USD 1/g, thus limiting their application to high-value-added natural- coloured garments only. To overcome this problem, it is suggested to exploit the potential of other biological sources such as fungi, bacteria and cell cultures, since appropriate selection, mutation or genetic

15 engineering techniques are likely to improve significantly the pigment production yields with respect to wild organisms (Mapari et al., 2005).

Nowadays, the potential of obtaining natural colours from microbial pigments to be used as natural colourants is being investigated. Microbial pigments such as from bacterial origins are advantageous, in terms of production, when compared to similar pigments extracted from the vegetables or animals because they have the potential of being exploited using existing culture techniques. The production of pigments by bioprocesses involving microorganisms has relatively high growth velocity (Babitha, 2009). This will cut the production time to a matter of days and the process lends itself to continuous operation (Hendry and Houghton, 1997), which expected to give such a productivity for the processes that brought them industrially competitive (Babitha, 2009). In addition, the production is also flexible and can easily be controlled as compared to plant or animals sources (Hendry and Houghton, 1997).

It is because, the pigments of higher organisms, animal, plant and fungal, may be less accessible to exploitation because of the structural complexity of the pigment-bearing tissue or because the pigment is formed only at critical points of development within a complex life cycle (Hendry and Houghton, 1997). For example, pigment that function as attractants in sexual reproduction may be formed only after completion of other aspect of life cycle. They may not then be amenable to exploitation through manipulation (Hendry and Houghton, 1997).

It is important to note that bacteria also are abundant in nature which can be isolated from normal soil and water habitat as well as not requiring genetic modification. Moreover, for the propagation of bacteria, minimal medium is required because microorganisms have an amazing ability to utilize cheap sources of carbon and nitrogen to overproduce valuable low- and high-molecular-weight metabolites (Demain, 1980).

16 Natural raw materials and by-products of industry have been widely used as culture media in fermentation processes because of their low cost since the medium components can represent from 38 to 73% of the total production cost. This primary product could serve as sustainable raw material for secondary value added product through microbial fermentation (Vidyalakshmi et al., 2009). While, for pigment extraction, it only involves a simple extraction method such as liquid-liquid extraction where the solvent used can be recovered back for subsequent use. These factors can contribute to the low cost for pigment production especially for large scale applications.

1.4.5

Applications of Bacterial Pigments

1.4.5.1 Food Colourants

Streptomyces coelicolor is a kind of actinomyces which can synthesize blue pigments. The pigment is stable to light and heat, and resistant to oxidants and reducers under acidic conditions and to reducers under alkaline conditions. The good characteristics of the pigment makes it a good candidate for the food processing industry as an additive. It can be used to make some colourful beverages and cakes and it is also a safe ingredient in lipstick to colour blue (Zhang et al., 2006). Bradyrhizobium sp. strain was described as a canthaxanthin (4,4-diketo- bcarotene) producer (Lorquin et al., 1997). The carotenoid pigment canthaxanthin produced has been used in aquafeed for many years in order to impart the desired flesh colour in farmed salmonids. While, B. linens (new name: Brevibacterium aurantiacum sp. nov.) is of particular interest as this microorganism is found in the rind of red- smear ripened soft cheeses. These food grade pigments have therefore been consumed by human beings for a long time (Galaup et al., 2005; Dufoss et al., 2005).

Other than that, the yellow pigment known as zeaxanthin or 3,3-dihydroxyb-carotene, produced by Flavobacterium sp. can be used as an additive in poultry

17 feeds to strengthen the yellow colour of the skin of animals of this kind or to accentuate the colour of the yolk of their eggs (Alcantara and Sanchez, 1999). This compound is also suitable for use as a colourant, for example in the cosmetics industry and in the food industry. Other bacterial pigments already in use as natural food colourants or with high potential in this field includes astaxhantin (pink red pigment) from Agrobacterium aurantiacum (Yokoyama et al., 1994) and Paracoccus carotinifaciens (Tsubokura et al., 1999), rubrolone (red pigment) from Streptomyces echinoruber, and zeaxanthin (yellow pigment) from Flavobacterium sp. (Shepherd et al., 1976) and Paracoccus zeaxanthinifaciens (Dufoss, 2006).

1.4.5.2 Pharmaceutical

The genus, Streptomyces or Serratia can produce a red substance of pyrrolylpyromethene skeleton, which is one of following substances: prodigiosin, metacycleprodigiosin, prodigiosen, desmethoxy prodigiosin, and prodigiosin 25-C. These substances have been known to have an antibiotic and anti-malarial effect, especially for prodigiosin 25-C to show immunosuppressing activity (Kim et al., 2003). Immunosuppressive activity of prodigiosins was first described by Nakamura and co-workers in 1989. These researches showed the presence of prodigiosin and metacycloprodigiosin in culture broth of Serratia and observed selective inhibition of polyclonal proliferation of T cells as compared to that of B cells.

Besides that, the cytotoxic potency of prodigiosin has also been investigated in the standard 60 cell line panels of human tumor cells derived from lung, colon, renal, ovarian, brain cancers, melanoma and leukemia. Inhibition of cell proliferation as well as induction of cell death has been observed in these cell lines. In vitro anti cancer activity has also been reported for different prodigiosin analogues and synthetic indole derivative of prodigiosin (Pandey et al., 2007). The antiproliferative and cytotoxic effects of prodigiosin have been observed not only in cultured tumor cell lines but also in human primary cancer cells from B-cell chronic lymphocytic leukemia patients (Campas et al., 2003).

18 The use of prodigiosin for treating diabetes mellitus has also been reported in 2002 (US Patent 6638968) where prodigiosin was found as an active component for preventing and treating diabetes mellitus. The diabetes-suppressing effect of prodigiosin originates from its regulation of immune responses as reported by Kim et al., 2003.

Violacein is produced by several bacterial species, including the Gramnegative species Chromobacterium violaceum, Janthinobacterium lividum,

Pseudoalteromonas luteoviolacea, Ps. sp 520P1 and Ps. sp. 710P1 (Rettori and Duran, 1998; Pantanella et al., 2007; Yada et al., 2007) Violacein has shown antiprotozoan (Matz et al., 2004 ; Leon et al., 2001) , anticancer (Ferreira et al., 2004; Kodach et al., 2006), anti-viral (Andrighetti-Frhner et al., 2003) , antibacterial (both G+ and G-) (Snchez et al., 2006; Lichstein and van De Sand, 1946; Nakamura et al., 2003) and antioxidant activities (Konzen et al., 2006). These characteristics provide the possibility to use violacein for therapeutic purposes, but C. violaceum can also act as an opportunistic pathogen in animals and humans, and cause fatal septicemia accompanied with liver and lung abscesses (Richard, 1993). Therefore, mass production of violacein using C. violaceum was thought to be impractical, and it is essential to search for a substitute of C. violaceum. These characteristics provide the possibility to use violacein for therapeutic purposes (Richard, 1993).

1.4.5.2 Textile Dyeing

Biosynthesis of colourants for textile applications has attracted increased interests in recent years. The currently used colourants are almost exclusively made from nonrenewable resources such as fossil oil. The production of the synthetic colourants is economically efficient and technically advanced with colours covering the whole colour spectrum. However, synthetic colourants are facing the following challenges: dependence on non-renewable oil resources and sustainability of current operation, environmental toxicity, and human health concerns of some synthetic dyes. Thus, biosynthesis of pigments through fermentation can serve as major chromophores for further chemical modifications, which could lead to colourants with a broad spectrum of colours (Hobson and Wales, 2008). Besides, some natural

19 colourants, especially anthraquinone type compounds, have shown remarkable antibacterial activity in addition to providing bright colours (Frandsen et al., 2006), which could serve as functional dyes in producing coloured antimicrobial textiles. Alihosseini et al., (2008) characterized the bright red pigment prodigiosin from Vibrio spp. and suggested that it could be used to dye many fibers including wool, nylon, acrylics and silk.

A study carried out by Yusof (2008) showed the capability of using pigment from Serratia marcescens in textile dyeing. In this study the pigment was used to colour five types of fabric namely acrylic, polyester microfiber, polyester, silk and cotton using tamarind as mordant. From the study, it was found that the pigments were capable of dyeing not only natural fibers but also synthetic fibers. However, the dyeing performances are different, depending on the types of fiber. It is because, different types of textile fiber requires different kinds of dyes, and in general, dyes which are suitable for one type of fiber will not dye other types (Vickerstaff, 1954). From the colourfastness testing, the dyed fabrics show the ability to maintain its colour under several external conditions such as perspiration, washing, and rubbing/crocking.

In Japan, a study conducted by Shirata shows that the pigment from Janthinobacterium lividum which was isolated from wet silk thread could be used to dye not only natural fibers like silk, cotton and wool, but also synthetic fibers like nylon and vinylon, and generally gave a good colour tone. Silk, cotton and wool showed a bluish-purple colour, nylon a dark blue colour, and acetate a purple colour. Dyeing was performed by a simple procedure consisting of either dipping in the pigment extract or boiling with the bacterial cells. The shades of colour varies by changing the dipping time and the temperature of the dye bath. The colour fastness of the dyed material was about the same as that of materials dyed with vegetable dyes, but the colour faded easily when the material was exposed to sunlight. The pigment displayed an antimicrobial activity against phytopathogenic fungi like Rosellinia necatrix which causes white root rot of mulberry. It could also be used as a biofungicide (Shirata et al., 2000).

20 1.5 Objectives of the Study

The objectives of this study include: To isolate and characterize bacterial isolates which have potential to produce coloured pigments. To isolate and characterize the pigment from Chromobacterium violaceum. To optimize the cultural conditions for pigment production by

Chromobacterium violaceum. To apply the pigment from Chromobacterium violaceum in textile dyeing.

1.6

The Scope of Study

In order to achieve the objectives, the pigment producing bacteria will be isolated from wastewater and soil contaminated agricultural waste and treatment system. The coloured bacteria obtained will be sent for identification using 16S rRNA analysis. Then, the pigment will be isolated using solvent extraction method before being characterized using instrumental analysis such as UV/Visible spectroscopy, Fourier-Transform infrared spectroscopy, thin layer chromatography and nuclear magnetic resonance and mass spectroscopy in order to elucidate the structure of pigment. For the optimization of bacterial growth for pigment production, the factors affecting pigment production such as temperature, growth medium and time for pigment production will be studied. In this study, both complex and agriculture waste based medium will be used to compare the production of pigment by bacteria. However, agricultural waste based medium (sugarcane bagasse, pineapple waste, brown sugar and molasses) will be studied in detail so that it can be used as medium to enhance pigment production. Lastly, the pigment will be applied in textile dyeing.

21

CHAPTER 2

ISOLATION AND CHARACTERIZATION OF PIGMENT FROM COLOURED BACTERIA

2.1

Chemistry of Pigments

The pigment in solution owes its colour to the selective absorption by pigment molecule of certain wavelengths of visible lights. The remaining wavelengths of light are transmitted, thus giving rise to the observed colour. The absorption of visible light by the molecules promotes electrons in the molecule from a low energy state, or ground state, to a higher energy state, or excited state. The pigment molecule is therefore said to undergo an electronic transition during this excitation process. Thus there is an inverse relationship between the energy difference between the ground and excited states of pigment and the wavelength of light that it absorbs (Christie, 2001).

The major characteristic of pigments is their insolubility in those media which they are applied, especially in water. Most organic pigments exhibit small solubilities (typically some mg L-1) in one of the following solvents: chloroform, methanol, dimethylformamide, or concentrated sulphuric acid (H2SO4), H2SO4 also being an excellent solvent for inorganic pigments (Christie, 2001). Besides that, other important characteristic of pigments is permanence. Performance (also known as light-fastness) is the ability of a pigment to resist fading due to photochemical deterioration. Pigments with poor light-fastness are said to be fungitive.

22 Photochemical deterioration can produce by-products that remain on the surface. However, most mineral pigments are permanent (Rapp, 2009).

2.1.1

Instrumental Analysis

Instrument analysis is one of the tools that can give the information about physical or chemical characteristics of the compound. The examples of instruments which are widely used are UV/Vis, Fourier-Transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and molecular mass spectrometry (MS).

UV/Vis absorption measurements are widely used for the identification and determination of many different inorganic and organic species. It is one of the most useful tools available for quantitative analysis. Eventhough it may not provide the unambiguous identification of an organic compound, an absorption spectrum in the visible and the ultra violet region is nevertheless useful for detecting the presence of certain functional groups that act as chromophores. The adsorption of UV/Vis radiation generally results from excitation of bonding electron. Because of this, the wavelength of absorption bands can be correlated with the types of bonds in the species under study. Molecular spectroscopy is therefore valuable for identifying functional group in a molecule (Skoog et al., 2007).

Infra red spectrometry is a versatile tool that is applied to the qualitative and quantitative determination of molecular species of all types. It is used for the routine quantitative determination of certain species, such as water, carbon dioxide, sulfur, low-molecular-weight hydrocarbon, amine nitrogen, and many other simple compounds of interest. FTIR is perhaps the most powerful tool for identifying types of chemical bonds (functional groups). By interpreting the infrared absorption spectrum, the chemical bonds in a molecule can be determined (Skoog et al., 2007).

23 NMR spectroscopy is based on the measurement of absorption of electromagnetic radiation in the radio-frequency region of roughly 4 to 900 MHz. In contrast to UV/vis and IR absorption, nuclei of atoms rather than outer electron are involved in the absorption process. Furthermore, to cause nuclei to develop the energy states required for absorption to occur, it is necessary to place the analyte in an intense magnetic field. NMR spectroscopy is one of the most powerful tools available to chemists and biochemists for elucidating the structure of chemical species. The technique is also useful for the quantitative determination of absorbing species (Skoog et al., 2007).

Mass spectrometry (MS) is perhaps the mot widely applicable of all the analytical tools available in the sense that the technique is capable of providing information about (1) the elemental composition of samples of matter; (2) the structure of inorganic and biological molecules; (3) the qualitative and quantitative composition of complex mixture; (4) the structure and composition of solid surface; and isotopic ratios of atoms in samples. Applications of mass spectrometry for quantitative analyses fall into 2 categories. The first involves the quantitative determination of molecular species or types of molecular species in organic, biological and occasionally inorganic samples. The second involve the determination of the concentration of elements in inorganic and less commonly organic and biological sample (Skoog et al., 2007).

2.2

Materials and Methods

2.2.1

Growth Medium

2.2.1.1 Nutrient Broth

Nutrient broth was used as a liquid growth medium for bacteria. To prepare nutrient broth (NB), 8 g of NB powder (MERCK, Germany) was dissolved in 1 L of DDW and sterilized by autoclaving at 121C, 121 kPa for 15 minutes.

24 2.2.1.2 Nutrient Agar

Nutrient agar (NA) was prepared by dissolving 20 g of nutrient agar (MERCK, Germany) in 1 L of deionized water. The medium was sterilized by autoclaving at 121C, 121 kPa for 15 minutes. The molten agar was cooled to about 50C before being poured into sterile Petri dishes. The agar was allowed to harden and then incubated for 24 hours at 30C to ensure that the medium was free from contamination.

2.2.2

Collection of Samples

Liquid and soil samples were obtained aseptically from the Brackishwater Aquaculture Research Centre, Gelang Patah, Johor and an oil refinery in Port Dickson, Seremban, Negeri Sembilan. A total of 16 soil samples were collected behind the wastewater treatment pond of an oil refinery which consists of clay, small sand and mixture of sand and clay. While at Brackishwater Aquaculture Research Centre, a solid sample was collected near the shrimp pond and 8 water samples were collected from recycled water tank for 10 day siakap, recycle tank for tilapia, rotifer breeding tank, fish breeding pond, organic waste and recycle organic waste tank for red talapia.

Soil and liquid samples were collected using sterile 250 mL Schott bottle. For the liquid samples, the sampling was carried out according to Standard Methods for the Examination of Water and Wastewater (Greenberg et al., 1985). The sterile Schott bottles were filled with liquid samples and ample air space (about 2.5 cm) were left to facilitate mixing, aeration and thermal expansion normally encountered during handling and transportation. The soil samples were scooped and placed in sterile sampling bottles. All the bottles were then placed in polystyrene iced-boxed container for transportation purposes to minimize microbial activity and to avoid any unpredictable changes.

25 2.2.3 Cultivation and Isolation of Cultures

2.2.3.1 Liquid Samples

Each of the samples (2.5 mL) was transferred into 250 mL Erlenmeyer flask containing 22.5 mL NB medium and incubated at 30C with agitation at 200 rpm for 24 hours (Certomat-B, B-Braun). When growth was observed, one loopful of each culture was streaked onto NA plates followed by incubation at 30C for 24 hours (Memmert, USA). Subculturings of each plate were made until pure single colonies of bacteria were obtained.

2.2.3.2 Soil Samples

Soil samples of about one gram were first added to a 250 ml flask containing 25 mL of NB and incubated at 30C, shaking at 200 rpm for 24 hours. A serial 10fold dilution was made by mixing sample (1.0 mL) with sterile distilled water (9.0 mL)
while 100-fold (10-2) dilution was made by mixing sample (0.1 mL) with (9.9 mL). The dilutions were repeated to get serial dilutions ranging from 10 to10-6. Then, the aliquots

of bacterial culture (0.1 mL) with dilutions of 10-4, 10-5 and 10-6 were spread over the surface of NA plates using a sterile glass spreader before being incubated at 30C for 24 hours. The pure bacterial colony was then sub-cultured onto fresh NA plates and incubated at 30C for 24 hours.

2.2.4

Characterization of Microorganism

2.2.4.1 Gram Staining

The coloured bacteria obtained was subjected to Gram staining as a preliminary step in the identification of bacteria. The principle behind this simple staining is that bacteria differ chemically from their surrounding and thus can be

26 stained to contrast with their environment. Bacteria also differ from one another chemically and physically, and may react differently to a given staining procedure.

The heat-fixed smear of the bacteria was prepared by withdrawing a loopful of fresh bacterial culture and placed on a slide. A drop of distilled water was placed on the slide containing bacteria followed by slow heating to heat fixed. After cooling off the slide, the smear was flooded with crystal violet and the slide was allowed to stand for 30 seconds on a staining rack. Then, the slide was rinsed with water for 5 seconds to remove excess crystal violet followed by covering the smears with Grams iodine mordant and allowed to stand for 1 minute. The slide was rinsed again and the decolourizer was dropped on the smear for 15 seconds to wash the crystal violet. The slide was washed before and after the smear was counterstained with safranin for about 45 seconds. The slide was then dried with tissue paper and further examined under the microscope (Leica CME) using oil immersion. Under the microscope, Gram positive cells were stained violet while Gram negative cells were stained red. In the Gram stain test, Acinetobacter haemolyticus and Staphylococcus aureus were used as controls for Gram negative and Gram positive bacteria respectively (Harley and Prescott, 1993).

2.2.4.2 Identification of Bacterial Isolates

Prior to identification, a pure single colony of bacteria was inoculated into 25 mL of NB. It was grown at 30C, shaken at 200 rpm for 12 hours. The cultures (1 mL) was diluted to a final concentration of 10-4 to 10-6 followed by spreading 0.1 mL of each diluted culture on NA plates and incubation at 30C for 12 hours. Cultures obtained as pure colonies were sent to Vivantis Technology Sdn. Bhd., Malaysia for 16S rRNA sequence analysis.

27 2.2.5 Maintenance of Stock Culture

2.2.5.1 Short-term Maintenance

The bacteria were streaked on NA plate and incubated at 30C for 24 hours. It can be used directly or stored for a week in a refrigerator.

2.2.5.2 Long-term Maintenance

One of the common medium for long term storage of bacteria is LB-glycerol. Firstly, the LB medium was prepared by adding 10 g of tryptone, 5 g yeast extract and 10 g NaCl in 1 L distilled water. The pH of solution was adjusted to 7.0 using NaOH followed by sterilization using autoclave (HVE-50, Hirayama). Then, 2 mL of active culture was transferred into 5 mL Bijou bottles and added with 2 mL of glycerol, 25 % (v/v) (J. T. Baker, USA). The stock cultures were stored at -20C prior to use.

Another medium used in this study was peptone water (0.1% (w/v). It was prepared by dissolving 0.1 g of peptone in 100 mL of distilled water. Then, 2 mL of peptone water was pipetted into a 5 mL bijou bottle. The bottle containing peptone water was then autoclaved at 121C, 121 kPa for 15 minutes. After that, 2 mL of the fresh culture was added into sterilized peptone water and stored at room temperature prior to use (Gilis and Logan, 2005).

2.2.6

Preparation of Inoculum

Single colonies of C. violaceum on nutrient agar plate were used in all experiments. The strain was cultivated in nutrient broth for 12 hours before inoculation. Unless otherwise stated, the cultivation was carried out using 250 mL Erlenmeyer flasks on a 200 rpm shaker at 30C for 24 hours.

28 2.2.7 Extraction of Pigments

2.2.7.1 Yellow Orange Pigment

The cells of Chryseobacterium sp. (500 mL) grown in NB for 24 hours were harvested by centrifugation (SIGMA 4K-15, B.Braun) at 7500 rpm for 20 minutes and the pellets were frozen at -20C. After thawing, the pigments were extracted from the wet bacteria with 25 mL of acetone (J.T. Baker) at room temperature. The mixture of pigments and acetone were centrifuged (SIGMA 4K-15, B.Braun) at 7500 rpm for 20 minutes. The pellet obtained was discarded and the liquid phase containing the pigments were kept in a bottle for further use (Achenbach and Kohl, 1978).

2.2.7.2 Violet Pigment

The cells of C. violaceum (500 mL) grown in NB for 24 hours were harvested by centrifugation (SIGMA 4K-15, B.Braun) at 7500 rpm for 20 minutes. The cells were washed with distilled water before being extracted with sufficient amount of methanol and centrifuged at 7500 rpm for 20 minutes. After that, the colourless pellet obtained was discarded and the supernatant was extracted using ethyl acetate before kept in a bottle for subsequent use.

2.2.8

Pigment in Powdered Form

The pigment obtained from section 2.2.7.2 was evaporated using a rotary evaporator (BCHI R-210, Switzerland) in order to reduce the solvent content. The extracted pigment (200 mL) was transferred into a 500 mL round bottom flask while the water bath system was set to 50C and chiller set below 10C. The evaporation process was stopped when the solvent content was 1 % (v/v) from the initial volume. The concentrated pigment was transferred to glass Petri dish and placed in an oven

29 for 3 days at 60C to ensure complete dryness. The dried pigments were then scratched using spatula and kept in a bottle, until further use.

2.2.9

Characterization of Crude Violet Pigment

2.2.9.1 Antimicrobial Effect

The agar disc diffusion method was employed for the qualitative determination of antimicrobial activity of crude violet pigment towards bacterial survival. Four types of bacterial species namely Bacillus cereus, Staphylococcus aureus , Pseudomonas aeruginosa and Escherichia coli obtained from

Biotechnology Research Laboratory, Universiti Teknologi Malaysia, Johor were individually grown in NB for 12 hours, at 200 rpm and incubated at their respective optimum growth temperature, 30 and 37C (for E. coli). The tested bacteria were maintained in LB glycerol stock solution stored at -20C prior to use. Subculturing was carried out in NB medium. The agar disc diffusion method was carried out as follows (Barja et al., 1989); 0.1 mL of the culture broth was transferred and spread onto the NA plates. Small filter paper discs (Whatman 113, wet strengthened, 0.5 cm in diameter and thick, 420 m) was previously prepared by punching the filter paper (Whatman 113, wet strengthened, 125mm in diameter and thick, 420 m) using paper puncher. The discs were individually impregnated with 50 L of the crude methanolic - extract was then placed onto the NA plates. Each sample was complimented with two control samples consisting of filter paper discs impregnated with 50 L methanol and filter paper discs only. Inhibition zones were observed after overnight incubation at 30 and 37C respectively.

30 2.2.9.2 UV/Vis Spectroscopy

The pigments were subjected to UV/Vis analysis to check for its maximum wavelength (max). For the analysis, methanol was used as the blank. A full wavelength scan between 800-200 nm was carried out to ascertain the max of the pigments. UV spectrum was recorded using Shimadzu UV 1601PC spectrophotometer.

2.2.9.3 Fourier-Transform Infrared Spectroscopy (FT-IR)

The powdered form of violet pigment was analyzed and interpreted using FTIR spectroscopy. A dried pellet (1 mg) was finely ground with 200 mg KBr (Scharlau). The mixture was pressed under pressure and the disk recovered was immediately recorded using FT-IR spectrophotometer (FT-IR 8300, Shidmadzu) in the range of 4000-400 cm-1.

2.2.9.4 Thin Layer Chromatography (TLC)

Methanolic extracts of the pigment were spotted on 0.20 mm precoated silica gel aluminium sheets (Merck Kieselgel 60F254) and developed with benzene: acetone, 2 : 1 (v/v) (Duran et al., 1994). A small spot of solution containing the pigment was applied to a plate, about one centimeter from the base. The plate was then dipped in the solvent system, and placed in a sealed container. The plate was taken out when the developed solvent reached the solvent front. Spots were visualized with UV light (254 nm and 356 nm) and sprayed with vanillin sulphuric acid. The vanillin sulphuric acid was prepared by mixing vanillin (0.5 g), methanol (80 mL), acetic acid (10 mL) and concentrated sulphuric acid (5 mL).

31 2.2.9.5 Nuclear Magnetic Resonance (NMR) Spectroscopic Analysis

For NMR spectroscopic analysis, the powdered pigment was analyzed for 1H NMR (400 MHz) and 13C NMR (100 MHz) using Bruker Avance 400 spectrometer. Chemical shifts were reported in ppm relative to tetramethylsilane (TMS) in deuterated dimethyl sulfoxide (DMSO) as solvent.

2.2.9.6 Mass Spectrometry (MS) Analysis

The powdered pigment (0.5 g) was dissolved in DMSO before analyzed using the Electron Spray Ionization coupled with mass spectrometry (ESI-MS). The analysis was carried out at the National Metrology Laboratory, Standard and Industrial Research Institute of Malaysia (SIRIM), Malaysia.

2.2.9.7 Spectroscopic Data for Violet Pigment

Violet pigment: violet solid; Rf = 0.43 (benzene: acetone = 2 : 1); IR (KBr) max cm-1: 3700-3000 (OH), 3256.53 (-NH), 1654.93, 1635.13 (C=O amide), 1613.18 (C=C), 1223.69 (C-O phenol), 1543.93, 1223.71 (C-N); UV max (MeOH) nm: 585.72; 1H NMR (DMSO, 400 MHz): 6.79 (1H,dd, J=2.0 Hz and 8.8 Hz, H-7), 6.83 (1H, d, J=7.6 Hz, H-22), 6.94 (1H, t, J=7.6 Hz, H-20), 7.20 (1H, t, J=7.6 Hz, H21), 7.23 (1H, d, J=2.0 Hz, H-5), 7.35 (1H, d, J=8.8 Hz, C-8), 7.55 (1H, d, J=2.0 Hz, H-13), 8.06 (1H, d, J=2.4 Hz, H-2), 8.93 (1H, d, J=7.6 Hz, H-19), 9.32 (s, 6-OH), 10.60 (s, 15-NH), 10.72 (s, 10-NH), 11.87 (s, 1-NH); 13C NMR (DMSO, 100 MHZ): 97.5 (C-13), 105.1 (C-5), 106.2 (C-3), 109.5 (C-22), 113.6 (C-7), 113.9 (C-8), 119.2 (C-17), 121.3 (C-20), 122.9 (C-18), 126.1 (C-4), 126.8 (C-19), 129.9 (C-2), 130.1 (C-21), 132.1 (C-9), 137.5 (C-12), 142.3 (C-23), 148.1 (C-14), 153.4 (C-6), 170.7 (C-16), 172.1 (C-11); ESI-MS m/z (relative intensity): 342.34 [ [M-H]_,

C20H13O3N3]; ESI-MS/MS m/z (m/z 342.34): 298.42, 209.07,157.16.

32 2.2.10 Primary Identification of Yellow - Orange Pigment

2.2.10.1

Simple Test for Flexirubin - Type Pigment

An overnight grown culture of Chryseobacterium sp. Was centrifuged and the pellet was deposited on a glass slide placed on a white background, then flooded with potassium hydroxide (KOH), 20 % (w/v) and the colour shift was observed. The resulting colour may then be compared with the initial colour of the pellet which acted as a control. After removing excess KOH, an acidic solution was flooded to revert their initial colour (Bernardet et al., 2002).

2.3

Results and Discussion

2.3.1

Characteristics of the Soil and Water Sample

A total of 1 soil (S) and 8 water (W) samples were collected from Brackishwater Aquaculture Research Centre (BARC), Gelang Patah, Johor. Soil samples were also obtained from the treatment pond of an oil refinery in Port Dickson, Seremban, Negeri Sembilan. The characteristics of the water and soil samples are shown in Table 2.1 and Table 2.2. Table 2.1 shows the characteristics of water and soil samples collected from Brackishwater Aquaculcure Research Centre, Gelang Patah, Johor. The characteristics of soil samples collected from treatment pond of oil refinery at Port Dickson, Seremban, Negeri Sembilan is shown in Table 2.2.

33 Table 2.1: Characteristics of water and soil samples collected from Brackishwater Aquaculcure Research Centre, Gelang Patah, Johor. Sample W1 W2 W3 W4 W5 W6 W7 W8 S1 Location Recycled water for 10 day siakap Recycle tank for talapia Rotifer breeding Fish breeding pond Water point source (From sea) Shrimp pond Recycled organic waste for red talapia Organic Waste Near shrimp pond Colour Greenish Brown Cloudy grey Clear Clear Cloudy grey Clear Cloudy grey Cloudy grey Brown-orange C 29.7 31.9 28.3 32.1 32.4 28.3 29.5 28.5 pH 6.24 6.24 6.22 6.29 6.21 6.14 6.24 6.18 7

Table 2.2: Characteristics of soil samples collected from treatment pond of oil refinery at Port Dickson, Seremban.

Sample S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16

Type of Soil Clay Small sand Small sand Clay Mixture of sand and clay Clay Small sand Clay Mixture of sand and clay Clay Small sand Mixture of sand and clay Small sand Mixture of sand and clay Small sand Small sand

Colour Orange Grey Grey Yellow Dark green Orange Grey Orange Dark green Orange Black Dark green Black Dark grey Grey Black

pH 7 6 6 5 6 6 7 5 4 6 5 6 5 6 6 5

2.3.2

Isolation and Characterization of Coloured Bacteria

A total of 45 bacterial colonies were isolated from solid and liquid samples from the BARC, Gelang Patah, Johor while 32 isolates were obtained from oil

34 refinery wastewater treatment plant at Seremban, Negeri Sembilan. The features of the coloured bacteria are shown in Table 2.3. Of these, isolate S8b (Figure 2.1) was chosen for further studies as it showed an intense yellow orange colour when grown in NA (Figure 2.1(a)) and NB medium (Figure 2.1(b)). The pure isolated bacterial colonies were sent for identification using 16S rRNA sequencing by Vivantis Technologies Sdn. Bhd.

Table 2.3: Characteristics of bacterial colonies obtained from soil and water isolates grown on NA plate. Bacteria ID W5a S6a S7a S8a # S8b S8c S8d S10a S10c Colony Colour Light Yellow Pale Yellow Yellow Light-yellow Yellow-orange Cream-yellow Yellow-orange Yellow Cream-yellow Colony Characteristics Form Elevation Margin Irregular Flat Erose Punctiform Convex Entire Punctiform Convex Entire Filamentous Raised Fillamentous Punctiform Convex Entire Circular Convex Entire Punctiform Convex Entire Punctiform Convex Entire Punctiform Convex Entire

(a) NA plate

(b) NB medium

Figure 2.1: Morphology of isolate S8b on NA plate (a) and NB medium (b).

From a total of 35 bacterial colonies isolated from the oil refinery in Seremban, 8 were coloured bacterial colonies. Of these, one dark - violet bacterial colony (isolate S1a) was chosen for further study due to its colour intensity (Figure 2.2). Some of the morphological features of the coloured bacterial colonies are

35 shown in Table 2.4. The colour of the bacterial colony experienced gradual colour change from grey to dark violet throughout the incubation period. After several subculturings, the pure single colony of the bacteria was sent for 16S rRNA sequencing.

(a) NA plate

(b) NB medium

Figure 2.2: Morphology of isolate S1a on NA plate (a) and NB medium (b).

Table 2.4: Morphological features of the coloured bacterial colonies on NA plate. Bacteria ID # S1a S1b S2d S5e S6c S7a S10a S11a Colony Colour Violet Opaque-yellow Light-yellow Pale-yellow Pale-yellow Yellow Light-yellow Opaque red Colony Characteristics Form Elevation Margin Punctiform Raised Entire Circular Umbonate Entire Punctiform Raised Entire Punctiform Raised Entire Punctiform Umbonate Undulate Punctiform Convex Entire Circular Raised Entire Circular Convex Entire

For the preliminary identification of microorganism, the Gram stain testing was carried out for isolate S8b and S1a. The isolate appeared pink in colour under the microscope which indicated Gram negative strains.

36 2.3.3 Identification of Bacteria

The identification of bacteria was carried out by Vivantis Technologies Sdn. Bhd. using 16S rRNA sequence analysis. From the analysis, the yellow-orange bacteria was identified as Chryseobacterium sp. and the violet bacteria, Chromobacterium violaceum (C. violaceum).

C. violaceum, a Gram negative bacteria belonging to the Rhizobiaceae family, is a saprophyte found in soil and water in tropical and subtropical areas. In most cases, it is a minor component of the total microflora (Ballows, 1992). Its colonies are slightly convex, not gelatinous, regular and violets, although irregular variants and non-pigmented colonies can also be found (Sneath, 1994). C. violaceum has been reported to produce a violet pigment called violacein. Violacein possesses anti-leishimanial (Leon et al., 2001), anti-viral (Andrighetti-Frhner et al., 2003), antitumoral (Ueda et al., 1994, Melo et al., 2000) and anti-Mycobacterium tuberculosis (De Souza et al., 1999) activities. Other properties of C. violaceum include the production of cyanide (Michaels and Corpe, 1965), the solubilization of gold (Faramarzi et al., 2004), the production of chitinolytic enzymes (Chernin et al., 1998), the synthesis of bioplastics (Steinbchel et al., 1993) and environmental detoxification (Carepo et al., 2004).

The genus Chryseobacterium was created by Vandamme et al., (1994) to accommodate several species formerly classified in the genus Flavobacterium, i.e. Chryseobacterium balustinum, C. gleum, C. indologenes, C. indoltheticum, C. meningosepticum and C. scophthalmum. Six species, Chryseobacterium defluvii (Kmpfer et al., 2003), C. joostei (Hugo et al., 2003), C. miricola (Li et al., 2003), C. daecheongense (Kim et al., 2005), C. formosense (Young et al., 2005) and C. taichungense (Shen et al., 2005), have been added to the genus recently.

Chryseobacterium strains produce translucent colonies, shiny with entire edges, but on prolonged incubation the colonies were not visible as single entities probably due to the profuse production of extracellular substances. On NA, it can produce a bright yellow nondiffusible, nonfluorescent flexirubin pigment. It also was

37 reported to have an ability to produce heat stable metalloproteases and protein deamidating enzymes (Venter, 1987; Yamaguchi and Yokoe, 2000).

2.3.4

Maintenance of Stock Culture

LB-glycerol was used for maintenance of the strains of C. violaceum and Chryseobacterium sp.. However, no growth was observed when C. violaceum was transferred into fresh medium after 14 days of stock culture prepared. Because of this, C. violaceum was then kept in peptone water 0.1 % (v/v) in order to maintain the culture. In this medium, the bacteria was able to grow after it was transferred into a fresh medium event after 2 months of being kept in the refrigerator. According to Gilis and Logan, (2005), organisms may survive for several years in dilute peptone due to the presence of peptic digest of animal tissue found in peptone water which is rich in tryptophan (MacFaddin, 1980). Tryptophan is a precursor molecule in violacein biosynthesis and the production is apparently essential for pigment production in C. violaceum (Vasconcelos et al., 2003). The absence of essential precursors, cofactors and/or accessory proteins in the host organism cause many biosynthetic enzymes to be not functional (August et al., 2000).

2.3.5

Characterizations of Crude Violet Pigments

2.3.5.1

Antimicrobial Effect

The method used to study the antimicrobial action of violet pigment was the agar diffusion method. The antibacterial effect was determined by the existence of a zone of inhibition around the pigment-containing paper disk. The NA plate inoculated with Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus and Escherichia coli were incubated with paper discs containing 50 L of the violet

38 pigment. Table 2.5 shows the antimicrobial effect of the violet pigment on the four different types of bacteria tested.

Table 2.5 : Antimicrobial testing of violet pigments on P. aeruginosa, S. aureus, B.

P. aeruginosa

cereus and E. coli.

*Control

**Control + MeOH

***Control + MeOH + Violet Pigment

** Control + methanol plates inoculated with the respective bacteria with discs containing methanol (medium for the violet pigment)

*** Control + methanol + violet pigment - plates inoculated with the respective bacteria with discs containing methanol (medium for the violet pigment) and violet pigment.

E. coli

* Control plates plates inoculated with the respective bacteria

S. aureus E. coli

E. coli E. coli B. cereus S. B. cereus aureus S. aureusP. aeruginosa

S. aureus

B. cereus

P. aeruginosa

P.B. cereus aeruginosa

39 Visual inspection of the plates showed that the violet pigment exert antimicrobial properties to all the tested organisms. The appearance of the inhibition zone on both the Gram-positive (S. aureus and B. cereus) and the Gram-negative bacteria (P. aeruginosa and E. coli) suggested that the violet pigment had a broadspectrum antibacterial property (August et al., 2000). In this experiment, discs inoculated with methanol were also tested as methanol was used as the solvent for the violet pigment. As expected methanol showed some antibacterial effect but the effect was more pronounced in the presence of the violet pigment. From the size of the zone of inhibition, violet pigment possessed an outstanding inhibitory effect on the growth of Gram-positive bacteria and only a small effect on Gram-negative ones. It seems that the presence and the level of the antibacterial activity of the violet pigment varied significantly with the type of microorganism used. It is because the activity of antibacterial agents against microorganisms depends on two major factors which are the nature of physical environment and the condition of the microorganism (Bloomfield, 1991). Also the antibacterial activity of a material depends on the destruction of the physical structure or the inhibition of the necessary metabolic reaction in a microorganism (Nakamura et al., 2003). This property is advantageous when applied in textile dyeing since it will provide natural coloured fabrics with antibacterial properties.

2.3.5.3

UV/Vis Spectroscopy

UV-Vis analysis was carried out to obtain the maximum absorbance peak of the crude pigment extract in methanol. UV-Vis spectra for the violet pigment in methanol showed a maximum peak with strong absorption at 585.72 nm (Figure 2.3).

40

Figure 2.3:

UV/Vis spectrum for methanolic extract of violet pigment.

This pigment owes their colour due to the presence of chromophore groups (C=C and C=O) which is responsible for electronic absorption. The carbonyl groups absorb intensely at the short wavelength end of the spectrum but carbonyl group has less intense bands at higher wavelength owing to the participation of n electrons (Mohan, 2007). The presences of carbonyl and alkene groups were confirmed by IR data.

The reason why this pigment has stronger absorption at longer wavelength (visible region) is due to the conjugation effect. A conjugated system requires lower energy for the to * transition than an unconjugated system. It is expected that the greater the number of bonding orbital, the lower will be the energy difference, E, between the highest bonding orbitals and the lowest excited * orbital. The obvious extension of this in terms of max is that the greater the number of conjugated double bonds, the longer the wavelength of absorption (bathochromic shift) (Mohan, 2007).

Besides that, the auxochromic or chromophoric substitution of five membered heteroaromatics ring present in this compound (confirmed by IR and NMR data, discussed in section 2.3.5.3 and 2.3.5.4) also causes a bathochromic shift and an increase in the intensity of the bands of the parent molecule. An auxochrome is an auxillary group which interacts with the chromophore which causes a

41 bathochromic shift. The presence of auxochrome group which is a substituted amino groups (NHR) in this compound (further confirmed by IR and NMR data) causes a shift in the UV or visible absorption maximum to a longer wavelength. It happens because of the property of an auxochromic group which has the ability to provide additional opportunity for charge delocalization and thus providing smaller energy increments for transition to excited states. The charge delocalization by the contributing structures is in the presence and absence of electron donating-NHR group as shown in Figure 2.4. From the figure, it is clearly shown that the charge delocalization is greatly enhanced by the presence of electron donating-NHR group (Mohan, 2007).

a) b)

C C C O
NR2 HC C C O H H

C C C O
NR2 HC C C O H H

Figure 2.4: The charge delocalization by the contributing structures in the absence (a) and presence (b) of electron donating-NHR group.

The added opportunity for stabilization of the * excited state brings the lowest excited state closer to the highest ground state and thus permits a lower energy (longer wavelength) for transition (Mohan, 2007).

2.3.5.3

Infrared Spectroscopy

FTIR analysis was carried out to determine the possible functional groups present in the pigment. Figure 2.5 shows the IR spectrum of a crude methanolic extract for violet pigment. From the spectrum, a broad band was observed at 3700 to 3000 cm-1 which corresponds to O-H stretching and it shows overlapping with N-H stretching of a secondary amide detected at 3256.53 cm-1.

42

Figure 2.5: FTIR spectrum for crude methanolic extract of violet pigment.

Secondary amides are associated through hydrogen bonding to form dimers (Figure 2.6 (a)) (cis configuration) or polymers (Figure 2.6 (b)) (trans configuration) resulting in the replacement of free N-H strectching band. The weak band at 3256.53 cm-1 may be due to an overtone of the band at 1543.93 cm-1 (trans secondary amide) or combination of C=O stretching and N-H in-plane bending (cis secondary amide) (Mohan, 2007).

R' O H N R C N H O R'
(a) cis-dimer

R' N

H N

R' R

C R
R

N O H R' R O H

N R'

(b) trans-polymer

Figure 2.6: Structure of secondary amides: ciscoid-ciscoid associations (a) and transoid- transoid polymeric association (b).

43 The C=O stretching of this amide was found as doublet at 1657.07 cm-1 and 1635.13 cm-1. This doublet is highly characteristic of amides where the higher frequency band is predominantly C=O (amide I band) and the lower is predominantly N-H in-plane bending (amide II band) (Mohan, 2007).

The frequency of C=O group for this compound is lower due to the conjugation of the carbonyl group with an aromatic ring results in the delocalization of the electrons of both unsaturated groups and reduces the double bond character of both the bonds causing a lowering of carbonyl frequency from 1718cm -1 (normal absorption frequency for carbonyl) to 1657.07 cm-1 and C=C stretching frequency from 1645 cm-1 (normal absorption frequency for alkene) to 1613.18 cm-1. The lowering of absorption frequencies of both C=C and C=O groups are attributed to the resonance as shown in Figure 2.7 (Mohan, 2007):

C C C O

C C C O

Figure 2.7: Resonance structure of carbonyl containing group in double bond structure.

In addition, the electron releasing groups, i.e. the amino group, attached to the carbonyl group tends to favour the polar contributing form by mesomeric effect and thus lower the force constant of the C=O bond and consequently resulting in a decrease of the carbonyl stretching frequency (Mohan, 2007).

The strong C-O stretching vibration also was observed at 1223.69 cm-1 which highly characterized the C-O stretching vibration of phenol. The C-O stretching vibration provides valuable information in determining the nature of the O-H compound. The distinctions between primary, secondary and tertiary alcohols are determined by examining C-O stretching vibration as shown in Table 2.6 (Mohan, 2007).

44 Table 2.6: OH (free) cm-1 and C-O cm-1 in alcohols and phenols OH (free) cm-1 3640 3630 3620 3610 C-O cm-1 1050 1100 1150 1200

No. Nature of the hydroxyl compound 1. 2. 3. 4. Primary alcohol Secondary alcohol Tertiary alcohol Phenols

The C-N strectching of secondary amide, a strong absorption band was observed at 1543.93 cm-1 and a weaker band at 1223.69 cm-1 due to C-N-H vibration resulting from coupling of N-H in plane bending and C-N stretching vibrations. Nitrogen and hydrogen atoms move in the opposite directions, relative to carbon, in N-H in-plane bending vibrations for the band near 1543.93 cm-1 and in the same direction for the band near 1268.41 cm-1.

2.3.5.6 Thin Layer Chromatography (TLC)

The crude methanolic extract of violet pigment was tested using Silica gel plate developed with benzene: acetone (2:1 (v/v)) to determine the purity of the compound as proposed by Duran et al. (1994). The crude pigment showed one spot with Rf value of 0.43 and after spraying with vanillin sulphuric acid reagent, a small sport with Rf = 0.5 appeared representing violacein and deoxyviolacein respectively, as reported by DeMoss and Evans, (1959).

2.3.5.5 Nuclear Magnetic Resonance (NMR)

In order to elucidate the chemical structure of the violet pigment produced by the C. violaceum, the analysis was carried out by recording and analyzing the 1H NMR and 13C NMR spectra on a Bruker Avance 400 spectrometer.

The 1H NMR spectrum (Figure 2.8) showed the presence of thirteen protons. Three singlet signals were observed at down-field region which corresponds to indole NH at 11.87 ppm, lactam NH at 10.72 ppm and isatin NH at 10.60 ppm.

45

Figure 2.8:

H NMR spectrum of violet pigment

Low-field signals from indole and pyrrole NH protons have been observed due to the presence of electron-withdrawing group substituents attached to the pyrrole ring and attributed to hydrogen bonding with the solvent resulting in three sharp peaks in low-field signal (Laatsch et al., 1984). These three sharp NH signals were the most striking features of violacein as reported by Laatsch et al., 1984, Hoshino et al., (1987) and Yada et al., (2007). The numbering of the structure was cited from Nakamura et al., (2002) (Figure 2.9).

Indole nucleus

Lactam ring Isatin nucleus

Figure 2.9: The structure of violacein.

46 Three aromatic protons in the indole skeleton which corresponds to metacouple signal and ortho-couple signal were detected at 6.79 ppm (dd, J=2.0 Hz and 8.8 Hz) and 7.23 ppm (d, J=2.0 Hz) which were attributed to H-7 and H-5 respectively and 7.35 ppm (d, J=8.8 Hz) attributed to H-8. A doublet peak at 8.06 ppm with J=2.4 Hz correspond to H-2. The hydroxyl group was observed at 9.32 ppm at singlet peak. The chemical shift observed at lower-field region might be due to the hydrogen bonding occurring between phenolic hydroxyl group with an ortho group (H-5 and H-7) which exists in the indole skeleton (Lambert and Mazzola, 2004). The presence of OH group was confirmed by IR spectrum which showed a broad absorption band at 3300 to 2900 cm-1.

Four protons were observed in aromatic isatin skeleton. These protons appeared at 8.93 ppm (d, J=7.6 Hz), 6.94 ppm (t, J=7.6 Hz), 7.20 ppm (t, J=7.6 Hz) and 6.83 ppm (d, J=7.6 Hz) which was assigned as H-19, H-20, H-21 and H-22 respectively. A doublet peak at 7.55 ppm with J= 2.0 Hz was assigned to H-13 in the lactam skeleton.

The

13

C NMR spectrum (Figure 2.10) exhibited twenty signals, which

corresponded to twenty carbons. The presence of carbonyl carbons were observed at 170.7 ppm (C-16) and 172.1 ppm (C-11) and absorption bands at 1654.33 cm-1 and 1637.86 cm-1 in IR spectrum confirmed the lactam and isatin skeleton.

47

Figure 2.10: 13C NMR spectrum of violet pigment.

Eighteen other carbons were observed at 97.5 (C-13), 105.1 (C-5), 106.2 (C-3), 109.5 (C-22), 113.6 (C-7), 113.9 (C-8), 119.2 (C-17), 121.3 (C-20), 122.9 (C18), 126.1 (C-4), 126.8 (C-19), 129.9 (C-2), 130.1 (C-21), 132.1 (C-9), 137.5 (C-12), 142.3 (C-23), 148.1 (C-14), and 153.4 (C-6). The signal at 153.4 indicates that the pigment was violacein, since the signal for corresponding carbon of deoxyviolacein which lacks hydroxyl residue appears in the higher magnetic field (Hoshino et al., 1987). Full assignments of 1H and Table 2.7.
13

C NMR data of violet pigment are tabulated in

48 Table 2.7: 1H and 13C data of violet pigment.

1

Position 1-NH 2 3 4 5 6 7 8 9 10-NH 11 12 13 14 15-NH 16 17 18 19 20 21 22 23 6-OH

H NMR

13

(ppm) 11.87 8.06 7.23 6.79 7.35 10.72 7.55 10.60 8.93 6.94 7.20 6.83 9.32

Multiplicity s d, J=2.4 Hz d, J=2.0 Hz dd, J=2.0 Hz, 8.8 Hz d, J=8.8 Hz s d, J=2.0 Hz s d, J=7.6 Hz t, J=7.6 Hz t, J=7.6 Hz d, J=7.6 Hz s

C NMR

(ppm) 129.9 106.2 126.1 105.1 153.4 113.6 113.9 132.1 172.1 137.5 97.5 148.1 170.7 119.2 122.9 126.8 121.3 130.1 109.5 142.3 -

2.3.5.6 Mass Spectrometry (MS)

In the negative - ion ESI-MS spectrum of the pigment (Figure 2.11), a quasimolecular ion peak was observed at m/z 342.34 [MH]_. The MS / MS spectrum (Figure 2.12) of the ion at m/z 342.34 showed the fragment ions at m/z 298.42, m/z 209.07 and m/z 157.16. The molecular ion peak and their fragment ions correspond to the molecular formula of C20H13O3N3. Together with the NMR and FTIR analysis, this confirms that the violet pigment is violacein. Similar conclusion was made for violet pigment isolated from other bacteria such as Janthinobacterium lividium (Lu et al., 2009), Duganella sp. B2 (Wang et al., 2009), Pseudoalteromonas luteoviolacea (Yada et al., 2007) and Alteromonas luteoviolacea (Laatsch et al., 1984).

49

Figure 2.11: The ESI - MS spectrum of violet pigment

Figure 2.12: The ESI MS / MS spectrum of violet pigment (m/z = 342.34)

50 2.3.6 Primary Identification of Yellow - Orange Pigment

2.3.6.1 Simple Test for Flexirubin - Type Pigment

The Flavobacteriaceae group was reported to produce yellow to orange (or rarely red) pigmentation. As Chryseobacterium sp. falls into this group and it produces yellow-orange pigment, a simple test for flexirubin type pigment was done to diagnose the type of pigment. In this test, a plate containing the Chryseobacterium sp. was scrapped and flooded with 20 % (w/v) KOH. From the observation, the colour of the colony changes from yellow-orange to red-brown. The resulting colour was then compared with the bacteria which was not in contact with KOH. After removing the excess KOH, an acidic solution was flooded to revert their initial colour. It was suggested that flexirubin reaction was shown in the presence of acid and base condition because it is one diagnostic feature of flexirubin type pigment (Ballows et al., 1992). Similar finding was obtained Kim et al., 2005, where the Flexirubin-type pigment was detected in Chryseobacterium daecheongense sp. nov. according to this method.

According to Bernardet et al., 2002, since the colour shift may pass unnoticed when the KOH solution is poured directly over a thin colony on an agar plate, it is strongly recommended that the test be performed on a small mass of bacterial cells collected with a loop and deposited on a glass slide placed on a white background. Another similar mass of bacteria on which no KOH is poured may be used as a control. The colour changed induced by KOH is not absolutely specific for the flexirubin type of pigment (Fautz and Reichenbach, 1980), but it is still helpful when combined with the results of other tests.

51 2.3.7 Conclusion

A total of 17 coloured bacteria were isolated from soil and water samples of BARC and oil refinery wastewater. Two of these bacteria which produced violet and yellow orange pigments were identified as Chromobacterium violaceum and Chryseobacterium sp. using 16S rRNA analysis. The violet pigment showed a broad spectrum of antimicrobial property. From the spectroscopic analysis of pigment, it was confirmed that the violet pigment corresponded to violacein. Also, the yellow orange pigment was suspected to be a flexirubin type pigment based on the change in colour with 20 % (w/v) KOH.

52

CHAPTER 3

OPTIMIZATION OF CULTURE CONDITIONS FOR PIGMENT PRODUCTION BY Chromobacterium violaceum

3.1

Utilization of a Cheap Growth Medium for Pigment Production

Due to high cost of currently used technology for pigment production on an industrial scale, there is a need for developing low cost process for the production of pigments that could replace the synthetic ones. In recent years, considerable research in converting agricultural waste, which is a renewable and abundantly available material, into value-added products has been carried out. Most literature reports that the utilization of a cheaply available substrate can attain the objective of pigment production in an economically feasible way (Pandey, 1992).

For example, apple pomace, a by-product of apple juice processing industry comprising peel, seed and remaining solid parts - a rich source of carbohydrates, dietary fibres, minerals and vitamin C has been used as a base medium for the production of pigment by Micrococcus species (Attri and Joshi, 2005). Other than that, a study by Babitha et al., (2006) shows the feasibility of using jackfruit seed powder as substrate for the production of pigments using a fungal culture of Monascus purpureus. Surprisingly, due to the buffering nature of jackfruit seed powder, the colour of pigments produced was stable over a wide range of initial pH of the substrate (Babitha et al., 2006). Besides that, Chiu and Chan (1992) also reported on the production of pigments by Monascus purpurea using sugar cane bagasse in roller bottle cultures.

53 The application of agro-industrial residues in bioprocesses would provide a profitable means of reducing substrate cost. It also provides an active way of protecting the environment by treating the waste and helps in solving pollution problems, which their disposal may otherwise cause. With the advanced of biotechnological innovations, mainly in the area of enzyme and fermentation technology, many new avenues have been opened for their utilization (Pandey et al., 2000).

3.1.1

Immobilized Cell Systems

Immobilization cell systems create exciting new opportunities for commercial development and profits in a wide range of industrial sectors including, healthcare and medicine, agriculture and forestry, fine and bulk chemicals production, food technology, fuel and energy production, pollution control and resource recovery (Mahmoud and Helmy, 2009).

Immobilized cell system/aggregate is composed of 3 components namely; the cells, the support material (or carrier/matrix), and the solution that fills the remainder of the space (interstitial solution), which is also known as microenvironment. On the basis of physical localization and the nature of microenvironment, immobilized cells systems can be classified into 4 categories which are surface attachment of cells, entrapment within porous matrices, containment behind a barrier and selfaggregation (Willaert, 2007).

It has been traditionally considered as an alternative to increase the process productivity and minimize the production costs, while offering advantages over free cell fermentation systems (Carvalho et al., 2003; Santos et al., 2005; Sarrouh et al., 2007) such as high biomass, high metabolic activity, and strong resistance to toxic chemicals (Zhou et al., 2008). Besides that, immobilization of biocatalysts helps in their economic reuse and in the development of continuous bioprocesses (Mahmoud and Helmy, 2009).

54 3.2 Materials and Methods

3.2.1

Preparation of Inoculum

Single colony of C. violaceum on nutrient agar was used in all experiments. The strain was cultivated in nutrient broth for 16 hours before inoculation. Unless otherwise stated, the cultivation was carried out using a 250 mL Erlenmeyer flasks at a 200 rpm agitation speed and at 30C for 24 hours.

3.2.2

Preparation of L-tryptophan Stock Solution

L-tryptophan stock solution was prepared by dissolving 1 g of L-tryptophan powder in 1 L of distilled water. To dissolve the powder, 0.1 M NaOH was added. The solution was than neutralized using 0.1 M HCl and sterilized using a hydrophobic-edge 0.45 m Whatman filter paper.

3.2.3

Growth Profile of C. violaceum

A 12 hours-grown culture of bacteria (20 mL) was inoculated into a 2 L Enlenmeyer flask containing 180 mL of NB followed by incubation at 200 rpm, 30oC for 24 hours. Culture turbidity (OD600) was measured at regular intervals using a spectrophotometer (Genesys 20, ThermoSpectronic). Pigment production by C. violaceum was also recorded. Similar experimental setup without the bacterial cells acted as a control.

3.2.4

Effect of Temperature on Pigment Production

Active cultures of bacteria (10 mL) were inoculated into a series of 1 L Erlenmeyer flasks containing 90 mL NB. The mixtures were shaken at 200 rpm for

55 24 hours at 25, 30 and 37C. These temperatures were selected so that large scale production of pigments can be carried out at room temperature, hence save cost. At the end of the bacterial growth cycle, the cell dry weight and pigment production were determined. The cell dry weight was determined as follows; the culture broth was centrifuged at 7000 rpm for 20 min where the pellet obtained was suspended in a small volume of distilled water. Cell suspension (5 mL) was then filtered using a hydrophobic-edge 0.45 m Whatman filter paper prior to drying at 50C for 48 hours. The violet pigment produced by C. violaceum grown at 25, 30 and 37oC were extracted with 6 mL of ethyl acetate. The three extracts were then analysed using UV-vis spectrophotometer (Perkin Elmer).

3.2.5

Production Profile of Violacein by C. violaceum in Complex Media

The production profile of crude violacein was obtained using a 1 L flask containing 200 mL of four kinds of complex media such as luria-bertani (LB) [tryptone 10 g/l, yeast extract 5 g/l, sodium chloride 10 g/l], nutrient broth (NB) [peptone 10 g/l, sodium chloride 5 g/l, yeast extract 3 g/l], tryptic soy broth (TSB) [peptone from casein 17 g/l, peptone from soymeal 3 g/l; D(+) glucose 2.5 g/l; sodium chloride 5 g/l; dipotassium hydrogen phosphate 2.5 g/l] and peptone glycerol broth (PGB) [meat extract 10 g/l, peptone 10 g/l, glycerol 10 % (v/v)]. The pH of all the above media were maintained at 7.0. The various media were autoclaved at 121C for 15 minutes. Fermentations were carried out at 30C for 24 h with an inoculum size of 10 % (v/v) of a 16-hours-old culture. Samples were withdrawn for measurements of cell concentration (OD600), pH, and violet pigment concentration. The optical densities of the cells at 600 nm were measured by using a spectrophotometer (Genesys 20, ThermoSpectronic), and the pH of the culture was measured by a pH meter (Cyberscan pH 510).

56 3.2.6 Evaluation of Various Agricultural Waste Substrates for Pigment Production

The use of a cheap carbon source is important to reduce the production cost in large scale pigment production. In view of this, a study was carried out to produce the pigment in a submerged culture using locally available agricultural wastes. For this purpose, four agricultural wastes namely, solid pineapple waste (SPW), molasses (M), brown sugar (BS) and sugarcane bagasse (SCB) were used as nutrient source for pigment production. Molasses and brown sugar were purchased from the local sundry shop while, both solid and liquid pineapple wastes were obtained from the downstream process at Lee Pineapple Manufacturing Industry, Tampoi.

SCB was first dried at 30oC and cut into 1.2-1.5 cm pieces in length. The SPW collected consists of pineapple peals and core that was previously ground yielding small pieces of solid waste. Prior to use, SPW and SCB were kept in the refrigerator.

To prepare the BS stock solution, 40 g of BS was dissolved in 1 L of distilled water. The solution was heated, stirred and then filtered using filter paper (Whatman No. 1, UK) to remove insoluble materials. The solution was sterilized by autoclaving at 105oC, 101.3 kPa for 15 minutes (HVE-50, Hirayama). Sterilization was carried out at 105C in order to prevent caramelization hence supporting bacterial growth (Nordin, 2009). Prior to use, the sterilized BS was diluted accordingly with sterilized DDW. The molasses used was obtained from a supplier in Skudai, Johor. The molasses stock solution was prepared by dissolving 0.5 mL of molasses in 1 L of distilled water. The solution was neutralized with 0.1 M NaOH solution and the pH was adjusted to 7.0. The neutralized solution was filtered and autoclaved at 105C, 101.3 kPa for 15min.

Experiments involving solid substrate such as SPW and SCB were conducted in a 500 mL Erlenmeyer flask with 10 % (v/v) working volume. The substrates were weighed (1 %, 3 % and 5 % (w/v)) and transferred into the respective Erlenmeyer

57 flasks. After that, L-trypthophan 10 % (v/v) was added with distilled water to obtain a final working volume of 50 mL. L- tryptophan was used as a precursor for pigment production in C. violaceum (Vasconcelos et al., 2003). After thorough mixing, the pH was adjusted to 7 using 0.1 M NaOH (QRC). The respective flask was than inoculated with 10 % (v/v) active culture of C. violaceum, shaken at 200 rpm, 30oC for 24 hours. For liquid substrate, various concentrations of BS (1 %, 5 % and 10 % (v/v)) and M (0.1 %, 0.5 % and 1 % (v/v)) were transferred into 250 mL Erlenmeyer flasks containing tryptophan, 5 % (v/v), active culture of C. violaceum, 10% (v/v) and topped up with distilled water to a total volume of 25 mL respectively. The flasks were shaken at 200 rpm, 30C for 24 hours.

3.2.7

Laboratory Scale Column System

A glass column with inner diameter (i.d.) of 1.5 cm, outer diameter (o.d.) 2.0 cm and height of 20 cm was used. Inlet and outlet points were set at 2 cm from the bottom and top of column, respectively. Silicone tubing with i.d of 2.0 mm and o.d. of 4.0 mm was fitted to the inlet and outlet points, respectively. Inert stones were packed to 3 cm3 at the bottom of the column to ensure good flow distribution inside the column and to retain the column content. Following this, SCB was packed into the column to a volume of 27 cm3. This volume is considered as the working volume of the column. A headspace of around 9 cm3 was allowed in the column. The total volume of the column is therefore 35 cm3 (Zakaria et al., 2007).

3.2.8

Immobilization of C. violaceum onto SCB

SCB was used as support material during the immobilization of C.violaceum in column system. To allow the SCB surface material to acquire necessary charge for bacterial attachment, the column was first rinsed with distilled water using a peristaltic pump (Eyela MP-1000 D) (Volesky, 1990). An active culture of C. violaceum (100 mL) was pumped at flow rate of 3.5 mL min-1 for 24 hours to ensure

58 initial bacterial attachment. A mixture of 10% (v/v) L-tryptophan in 100 mL of distilled water (final pH of mixture 7.00) was pumped continuously for 24 h, using the same flow rate to provide nutrient for bacterial growth and pigment production.

For recovery of pigment from SCB, the same column set up was used. Prior to recovery of pigment, the medium was completely drained out from the column. After that, 100 mL of methanol was recycled through the column to elute the pigment from SCB. When the solution was darker in colour, the solution was replaced with fresh methanol. The process was repeated until the pigment was completely eluted from SCB. The methanolic extract was collected and dried at room temperature and the concentration was determined gravimetrically.

3.2.9

Determination of Pigment Concentration

The extraction of pigment from bacteria grown in NB, LB, M and BS was carried out as follows; ethyl acetate (1 part) was added to 5 parts of the growth medium. The ethyl acetate fractions were then placed into a preweighed vial, left to evaporate at room temperature until constant weight was achieved. The concentration of the pigment was recorded as g L-1. The extraction of pigments from bacteria grown in SCB and SPW are as follows; to 3 g of SCB or SPW, 200 mL of 99 % (v/v) methanol were added. The methanolic extract of the pigment was subjected to drying as stated above. The concentration of pigment was expressed in g L-1.

3.3

Results and Discussion

3.3.1

Growth Profile of Bacteria

Growth of C. violaceum in NB was monitored for 24 hours (Figure 3.1). The bacteria showed a typical growth curve with distinct log, stationary and death phase. Lag phase was not observed as exponential growing culture was used to inoculate the

59 medium causing cells to commence growth immediately. Optical density reading decreased slightly but increased and remained constant till 24 hours. This might be due to the formation of precipitates after 5 hours which might interfere with the optical density readings.

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 5 10 15 20 25 Time (Hours)

Optical Density, OD 600

Figure 3.1: Growth profile of C. violaceum in NB.

It is interesting to note that pigment production by bacteria was observed 4 hours after growth i.e. at the late exponential phase (Figure 3.2). This seems to suggest that the pigments produced by bacteria are secondary metabolites as the colour of the pigment was produced after the active stage of growth (Lu et al., 2009).

Intensity of the violacein produced by C. violaceum was maximum at 4 hours of growth and decreased with time due to precipitation of the pigments. As violacein is reported to be poorly water soluble (De Azevedo et al., 2000) this might explain the decreased intensity of the violet colour with time.

0.5 hr

1 hr

2 hr

4 hr

8 hr

12 hr

16 hr

24 hr

Figure 3.2: Effect of time on pigment production on C. violaceum.

60 3.3.2 Effect of Temperature on Bacterial Growth and Pigment Production

C. violaceum showed good growth and high pigment production at 25oC and 30oC as compared to 37oC (Figure 3.3).

2.2 2

Optical Density, OD 600

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 5 10 15 20 25C 30C 37C

Time (Hours)

Figure 3.3: Growth profile of C.violaceum in NB at 25, 30 and 37oC.

Temperature is an important factor as it influences metabolic activities and microbial growth. At 37oC, the metabolic activities of the bacteria decreases which leads to no pigment production by the bacteria. This is in contrast with bacterial growth at 25oC and 30oC, where C. violaceum showed the ability to produce pigment after 4 hours of growth at 30C.

On the other hand, the pigment only appeared after 8 hours of growth when the temperature was lowered to 25C. However, the pigment production increased with time at both growth temperatures. UV-vis analysis of the pigment produced by bacteria at 30C gave higher absorbance than at other temperatures (Figure 3.4). This could be due to 30oC being the optimum temperature for bacterial growth hence, the higher bacterial number present in the solution. This is substantiated by the higher cell dry weight obtained at 30C (1.03 mg mL-1) compared to at 25C (0.97 mg mL1

) and 30C (0.48 mg m L-1).

61
Dat e: 5/8/ 2009 Ti me: 10: 27:29 AM

0. 700 0. 65 0. 60 0. 55 0. 50 0. 45 0. 40 0. 35 A 0. 30 0. 25 0. 20 0. 15 0. 10 0. 05 0. 000 400.0 450 500

30 C
562.63,0.67280

563.61,0.31598

25 C

37C
550 600 nm 650 700 750 800.0

P37.SP - 5/8/2009 P25.SP - 5/8/2009

Figure 3.4: The absorption spectrum of the violet pigment obtained at different
P30.SP - 5/8/2009

incubation temperatures.

3.3.3

Violacein Production on Various Complex Media

Figure 3.5 shows a representative time course of cell growth and pH value during the cultivation of C. violaceum in 4 different complex medium namely, NB, LB, TSB and PGB. The OD600 reached a maximum value of 1.83 after 24 hours in LB medium followed by 1.81 in TSB medium, 1.66 in NB medium and 1.15 in PGB medium. The pH of the culture increased during bacterial growth in all medium except for PGB medium. Increase in pH was probably related to initiation of pigment production.

62

10 9

10 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours)

Optical Density (OD 600)

8 7 6 5 4 3 2 1 0

Figure 3.5: Time course of cell growth and pH profile of C. violaceum cultivated in NB, TSB, PGB and LB. Symbols: ( OD600, ( TSB pH. ) PGB pH, ( ) LB OD600, ( ) NB OD600, ( ) LB pH, ( ) NB pH, ( ) PGB )

) TSB OD600 and (

The use of different growth media directly affects the production of violacein by C. violaceum as depicted in Figure 3.6. Maximum production of violacein was observed in NB medium (0.15 g L-1) whereas the lowest concentration was observed in TSB medium (0.05 g L-1). No pigment production was observed in PGB medium. Although good bacterial growth was observed in TSB medium (Figure 3.5), it did not yield high pigment production. This could imply that violacein is not required for growth and survival of C. violaceum (Sivendra and Lo, 1975; Durn and FaljoniAlario, 1980).

Pigment Production (gL -1)

0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 NB LB TSB Medium PGB

Figure 3.6: Violacein production by C. violaceum in complex media.

pH

63 3.3.4 Utilization of Agricultural Wastes as an Alternative Medium for Violacein Production

In this study, 2 agricultural wastes i.e. SCB and SPW and 2 agricultural based products, M and BS were used as substrates for pigment production. From Figure 3.7, C. violaceum capable of producing pigment in both solid and liquid substrates (Figure 3.7). Highest pigment production i.e. 0.822 g L-1 was achieved when C. violaceum was grown in the SCB3 medium which consists of a mixture of distilled water, 3 g of SCB and 10 % (v/v) L tryptophan (Figure 3.8). The pigment production was observed both on the solid support and in the aqueous solution. After 24 hours, the original pale yellow appearance of SCB and green colour of SPW turned to dark purple due to the formation of pigment.

M0.1

M0.5

M1

BS1

BS5

BS10

a) Liquid Substrate

SCB1

SCB3

SCB5

SPW1

SPW3

SPW5

b) Solid Substrate

Figure 3.7: Colour intensity of pigments in a) liquid substrate and b) solid substrate.

Lowest pigment production (0.19 g L-1) was observed when the bacterium was grown in the SCB1 medium. This is because the pigment was produced only in the support material and not excreted in the solution. Other medium formulations

64 showed negligible (SCB5) or poor yields of pigment production (in g L-1); SPW1 0.01, SPW3 - 0.07, SPW5 - 0.03, BS1 - 0.02, BS5 - 0.02, BS10 - 0.08, M0.1 - 0.05, M0.5 - 0.03 and M1 - 0.03 (Figure 3.8).

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Pigment Yield (g/L)

W 1

W 3

W 5

10

0. 1

0. 5 M

SC B

SC B

SC B

BS

BS

BS

SP

SP

SP

Substrates

Figure 3.8: Pigment production by C. violaceum grown in SCB, SPW, M and BS supplemented with 10% (v/v) L- tryptophan, in distilled water; SCB sugar cane bagasse, SPW - solid pineapple waste, brown sugar - BS; M molasses.

Since sugarcane bagasse contains cellulose, hemicellulose and lignin, it might provide a good source of carbon and adhesion site for bacterial growth and pigment production. However, higher amount of SCB impedes pigment production which was due to poor aeration. This promotes anaerobic condition which in turn retarded the pigment formation since C. violaceum is known to produce violacein only in aerobic condition (DeMoss and Evans, 1959).

3.3.5

Preliminary Study on the Immobilization of C. violaceum on SCB

This study was carried out in order to test the ability of C.violaceum to produce pigments in a continuous system. It was observed that, the C.violaceum was able to produce pigments up to 0.15 g L-1 of pigment in a column system (Figure 3.9). The

65 advantage of a column system is that the pigments are adsorbed onto the solid support and easy recovery of pigment can be made by eluting with a solvent. Immobilized cells offer many advantages and it will become a promising technique for pigment production since it provides more stable and uniform environment. The viability of cells can be maintained without stimulating cell divisions when the cells are adhered on an inert matrix provided with the medium. Besides that, the immobilized cells can be maintained under these conditions for a long period without the need for frequent subculturings (Lindsey and Yoeman, 1984). Therefore, extensive research will be carried out on the different support systems and the viability and biosynthetic performance of cell within them.

Violacein production in SCB

Figure 3.9: Violacein production in sugarcane bagasse.

3.4 Conclusion

From the study, violacein was found to be a secondary metabolite product since it was produced after 4 hours of active stage of growth. UV-vis analysis on the pigment revealed that the pigment produced by bacteria at 30C gave higher absorbance which correlated well with the highest cell dry weight obtained. C. violaceum not only can produce pigments in complex medium but also in agricultural

66 waste materials in the presence of L tryptophan. The highest pigment yield (0.82 g L-1) was achieved when C. violaceum was grown in the presence of 3 g of SCB supplemented with 10 % (v/v) L - tryptophan in distilled water. Up to 0.15 g L-1 of violacein can be obtained using the SCB immobilized cells system.

67

CHAPTER 4

APPLICATION OF PIGMENT ISOLATED FROM C. violaceum IN TEXTILE DYEING

4.1

Natural Colourants in Textile Dyeing

Natural dyes and pigments have been used rigorously for millennia, up to the middle of the 19th century. The invention of the first synthetic dyes changed the situation and were substituted for natural colourants almost completely. However, in some niche segments or specialty segments of the market, natural dyes survived. Recently the awareness for the environment as well as increasing disputes about the risks of synthetic dyes resulted in growing interest in natural resources, environmentally friendly products and new strategies. The application of natural sources as the origin for colourants seems to be a promising approach (Mussak and Bechtold, 2009).

In the last decade, investigations about possible use of natural dyes in textile dyeing processes have been carried out by various research groups. Deo and Desai (1999) studied the dyeing of cotton and jute with tea as a natural dye using alum, copper sulfate, or ferrous sulfate mordants. Shanker and Vankar (2006) investigated the dyeing of cotton, wool and silk using Hibiscus mutabilis (Gulzuba). Other than that, Onal et al., (2005) reported the use of ellagic acid extracted from gallnut (Quercus infectoria) in dyeing of woolen strips, feathered-leather and cotton using three types of mordanting methods at various pH values. The study on dyeing properties and colour fastness of cotton and silk fabrics dyed with Cassia tora L. extract was reported by Lee and Kim (2004).

68 Studies on techniques of natural dyeing were attempted using both conventional and non-conventional methods. In conventional dyeing, it can be carried out in an alkaline bath, acidic bath or in a neutral bath, depending on the chemical nature of natural dyes. For the non-conventional dyeing, the use of sophisticated instruments such as ultrasonic, microwave, sonicator, supercritical carbon dioxide fluids and many more were attempted to meet customers demand for ecofriendly textiles and dyes with the newer energy efficient dyeing process and more reproducible shade developing processes (Samanta and Agarwal, 2009).

4.1.1

Reasons for Natural Colouration

The use of natural dyes on textile materials has been widely studied due to several reasons such as their wide ability and huge potential to be used as an alternative to synthetic dyes. Besides that, the available experimental evidence for allergic and toxic effects of some synthetic dyes, and non-toxic and non-allergic effects of natural dyes also attract consumer attention to choose the natural product (Samanta and Agarwal, 2009).

Natural dyes also produce special colour tones and effects as well as providing promising fastness properties. The availability of knowledge base and database on application of natural dyes on different textile and availability of scientific information on chemical characterizations of different natural colourants including their purification and extraction also made them look interesting to be explored (Samanta and Agarwal, 2009).

69 4.2 Materials and Methods

4.2.1

Fabrics

A total of 7 types of fabrics were used in fabric dyeing. The natural fibers used were pure cotton, pure silk, pure rayon, jacquard rayon, cotton and silk satin while the synthetic fiber used was polyester. The fabrics were obtained from the school of Textile Technology, Faculty of Applied Sciences, Universiti Teknologi Mara (UiTM), Shah Alam and Malaysian Craft Development Corporation (Kelantan).

4.2.2

Soap Solution

Soap solution was prepared by mixing 5 g of standard soap (SDC enterprises limited, UK) with 2 g of anhydrous sodium carbonate (Hamburg Chemicals) in 1 L of distilled water. The solution was heated at 60 oC for 15 minutes to homogenize the mixture (Salmiah, 2006).

4.2.3

Perspiration Solution

Perspiration solution was prepared by dissolving 0.5 g L-histidine monohydrochloride monohydrate (Merck), 5.0 g of sodium chloride (Merck), and 5.0 g of crystallized disodium hydrogen orthophosphate (Merck) in 1.0 L of distilled water. Then, 0.1 M sodium hydroxide (Merck) and hydrochloric acid (Merck) were added to increase the pH to 5.5 and 8.0 respectively (Salmiah, 2006).

70 4.2.4 Mordants Both natural products and chemicals were employed as mordants in fabric dyeing with bacterial pigment. The mordants used are as follows: alum [KAl(SO4)2.12H2O], ferric sulphate [Fe2(SO4)3], copper sulphate (CuSO4), sodium silicate (Na2SiO3), and slaked lime [Ca(OH)2]. To prepare the mordant solutions from alum, 70 g of alum powder was dissolved in 1 L of water. The mixture was stirred for 5 min and the precipitates were left to settle until a clear solution was obtained. The clear solution was used as a mordant. The same procedure was used to prepare slaked lime, 50 g in 1 L of water. The mordant solution of ferric sulphate and copper sulphate was prepared by dissolving 5 g each of the salts in 1 L of distilled water respectively.

4.2.5

Dye Material

Two types of dyes were used in this study i.e. natural dye from bacteria and synthetic dye. Prior to use, the natural dye from bacteria was prepared by growing the bacteria in 2 L Enlenmeyer flask containing NB for 24 hours, shaken at 200 rpm at 30 C. The synthetic dye was prepared by dissolving 0.35 g of reactive remazol blue and 0.15 g of reactive remazol violet in 1 L of distilled water. The solution was stirred before use.

4.2.6

Mordanting Procedure

The method of mordanting used in this study was post-mordanting where the material was first dyed followed by addition of mordant. The mordanting technique was carried out for 15 min at room temperature followed by washing and drying at room temperature. However, for the mordanting procedure using sodium silicate, the dyed fabrics were immersed in this solution for 1 hour 30 minutes. The fabrics were

71 then washed with tap water to remove excess mordant on the fabric before it was dried at room temperature.

4.2.7

Method of Dyeing

In this study, two methods of dyeing were attempted; boiling of fabric with bacterial cells and dyeing of fabric with reactive dye using brushed technique. For the dyeing procedure involving boiling with bacterial cells, the fabric (1 g) as described in section 4.2.1 was immersed in bacterial cells (20 mL). Mixtures containing natural and synthetic fibres were heated at 80 C and 130 oC for 1 hour and 1 hour 30 minutes respectively. Finally, the dyed fabrics were washed with cold water and mordanting was carried out as described in section 4.2.6. In dyeing using reactive dye, the dye was applied using a brush to make sure the dye was distributed properly on the fabrics before the mordant (sodium silicate) layer was applied on the fabrics. The fabrics were than left to dry at room temperature.

4.2.8

Dyeability of Pigment on Different Fibers

In order to study the dyeability of pigment on different fabrics, a total of 7 types of fabrics were used namely pure cotton, pure silk, pure rayon, jacquard rayon, silk satin, cotton and polyester. The fabrics were dyed by boiling with bacterial cells and alum was used as the mordant.

4.2.9

Dyeing of Silk Satin and Cotton Fibres

In the study on dyeing of silk satin and cotton fibres, four parameters affecting the colour performance and their fastness were studied which were types of mordants, mordant concentration, pre-treatment of fabrics and comparison of

72 fastness properties of natural dyes with reactive dyes. For the study on the effect of types of mordant, 4 types of mordants were used namely, alum, ferrous sulphate, copper sulphate, sodium silicate and calcium hydroxide. The study on the effect of mordants concentration was carried out using ferrous sulphate and copper sulphate. The concentrations used were 5 g L-1 and 10 g L-1.

Pre-treatment of fabric was carried out to remove pectic substances and cotton wax contained in cotton woven fabrics and substance present on the surface of silk fabrics. For the pre-treatment of cotton fabrics, the solution containing alum and sodium carbonate was prepared by dissolving 0.2 g of alum and 0.06 g of sodium carbonate (Na2CO3) in 33 mL of tap water followed by heating and homogenizing it by stirring.

Prior to pre-treatment process, 1g of fabric was wetted before it was immersed in alum and sodium carbonate. The solution together with fabrics was boiled for 2 hours to ensure the complete treatment of fabrics. The heating was stopped and the fabrics were left in solution for 24 hours. After that, the fabric was washed and cooled at room temperature. A similar procedure was applied for the pretreatment of silk, without the addition of sodium carbonate.

4.2.10 Colourfastness Standard Tests

The dyed samples was tested according to ISO standard methods; MS ISO 105-X12 , colour fastness to rubbing/crocking; MS ISO 105-C01, colour fastness to washing; MS ISO 105-E01, colour fastness to water; MS ISO 105-E04, colour fastness to perspiration; and MS ISO 105-B02, colour fastness to light (carbon arc).

73 4.2.11 Preparation of Fabric for Testing

4.2.11.1

Colourfastness to Washing

A 10 cm x 4 cm of dyed fabric was prepared and weighed. The dyed fabric was attached with undyed fabrics and undyed cotton fabric. The sample was placed inside a jar filled with standard soap solution (section 4.2.2). The ratio of liquor is 1g of fabrics to 50 mL of soap solution. Then, the jar was placed inside the Auto-Wash (Labtec) and allowed to wash for 30 minutes at 60 oC. After that, the sample was washed and dried under mild sunlight. Evaluation was carried out by comparing the fabric sample for the change in colour and staining of the white fabrics with the respective Gray Scale Standard (BSI Standards, SDC Standard methods) of 1 to 5 (Salmiah, 2006).

4.2.11.2

Colourfastness to Light

A 114 mm x 50 mm of cardboard was prepared. The dyed fabrics were cut into 50 mm x 10 mm and placed (stapled) horizontally, one above the other on the same cardboard. Blue wool standards (No.1 to No.8) were cut into strips of 50 mm x 10 mm and were placed one above the other on a separate cardboard. Both specimens were placed in the sample holder. The sample holder allowed both ends of the standards or samples to be exposed to light. The central area was covered by the framework, thus, the area was not exposed to light. Both samples and standards were exposed to light in Light Fastness Tester (Model No. 225, Halifax, England) for 24 hours (Salmiah, 2006).

4.2.11.3

Colourfastness to Rubbing/Crocking

Four rubbing cotton cloths (50 mm x 50 mm) were prepared for each fabric. Each cloth was fixed to the rubbing finger. After that, two warp and two weft of dyed fabrics for each sample was prepared (200 mm x 110 mm), one warp and weft sample for dry rubbing, and one warp and one weft for wet rubbing. For dry rubbing,

74 the cotton cloths were rubbed using a crockmeter on the dry dyed fabric sample ten times forward and backward in a straight line along a track of 100 mm in length. The same procedure was applied for wet rubbing except, the cotton cloth was wetted using distilled water before the rubbing on the dyed fabric sample. Lastly, the result was evaluated by comparing the staining on the white rubbing cotton cloth with the Gray Scale Standards of 1 to 5 (Salmiah, 2006).

4.2.11.4

Colourfastness to Perspiration

A 60 mm x 60 mm for each dyed fabric was prepared and weighed. The perspiration solution in section 4.2.3 was poured into a beaker containing the dyed fabric. The liquor ratio is 1 g of fabric to 50 mL of perspiration solution. Then, the sample was stirred occasionally for 30 minutes before being removed from the solution and wringed in the perspirometer. The perspirometer was left in the oven at 37 C for 4 hours. After 4 hours the results were evaluated by comparing the change in colour of the fabrics and staining of the undyed fabric with the respective Gray Scale Standards of 1 to 5 (Salmiah, 2006).

4.2.11.5

Colourfastness to Water

One sample (60 mm x 60 mm) for each dyed fabric was prepared and weighed. Distilled water was dropped on the dyed fabric. The dyed fabric was left in the oven at 37C for 4 hours. After 4 hours the results were evaluated by comparing the change in colour of the fabrics and staining of the undyed fabric with the respective Gray Scale Standards of 1 to 5 (Salmiah, 2006).

75 4.3 Results and Discussion

4.3.1

Dyeability of Violacein on Different Fabrics

The pigments were used to dye seven fabrics consisting of natural and synthetic fibres namely, pure cotton (PC), pure silk (PS), pure rayon (PR), jacquard rayon (JR), cotton (C), silk satin (SS) and polyester (P). From the study, violacein was found capable of dyeing not only natural fibers but synthetic fibers as well. However, the dyeing performances and shades are different, depending on the types of fiber. The dyeing performance of 7 different fibers is shown in Figure 4.1.

Figure 4.1 : Dyeability of violacein on different fabrics using alum as mordant; a) pure cotton (PC), b) pure silk (PS), c) pure rayon (PR), d) rayon jacquard (RJ), e) silk satin (SS), f) cotton (C) and g) polyester (P).

Intense colourations on pure rayon (Figure 4.1c), rayon jacquard (Figure 4.1d) and silk satin (Figure 4.1e) indicated its suitability to be dyed with violacein compared to other fabrics. Cotton (Figure 4.1f), pure cotton (Figure 4.1a), pure silk (Figure 4.1b) and polyester (Figure 4.1g) showed moderate violacein dyeing ability. The shades of colour differs depending on the types of fiber used where for example, rayon showed a bluish - purple appearance while polyester, light purple.

Different colour shades obtained during dyeing were due to different rates of adsorption between dye - dye and fiber - fiber. In the fabric dyeing process, the chemical nature of different textile materials is important as this will determine the

76 exact mechanism by which dye is adsorbed onto particular reactive groups on the fabrics. This condition prevails due to the following:- when the dye molecules enter the fiber, it has to be transported from the aqueous phase onto the solid phase (fiber) so that their places can be taken by fresh dye from the outside liquid, leading to a gradual exhaustion of the dye - bath or accumulation of dye on the fiber (Vickerstaff, 1954).

For example, rayon (pure and jacquard) and cotton (pure and normal) showed different dyeability although both are made up of cellulosic materials. It is because, in the manufacturing of viscose rayon, dissolution of the natural cellulose corresponds to a very intensive swelling and leads to a much greater micellar surface in the finished rayon than in the original cellulose. At the same time oxidative degradation occurs, leading to an increase number of carboxyl groups in the rayon. Since the carboxyl group forms negative charges in water, the pigments bind to the COO- group by hydrogen bond, resulting in good colouration of the rayon based fabric (Vickerstaff, 1954). It is well known that a hydrogen atom can act as an electron acceptor particularly when it is directly attached to nitrogen or oxygen. With greater number of electron-donating and electron - accepting groups between the fiber and pigment, more pigments will be attached to the fiber, hence increasing the dyeability (Vickerstaff, 1954).

The fastness properties of all dyed samples are shown in Table 4.1. Overall, the fastness properties of all fabrics are rated from 3 (fair) to 5 (excellent) except for lightfastness.

77 Table 4.1: Shades and fastness properties of different fabrics dyed with natural pigment.
Colourfastness Mordant Washing Light AC S wf 1 2 1 Alum 1 2 1 3 4 3 4 3/4 4 4 3 4/5 4 4 3 4 4 4/5 4 4/5 4 4 4/5 4 4/5 wp 4/5 4/5 4 4 4/5 4 4/5 wf 4/5 4/5 4/5 4/5 4/5 4/5 4/5 wp 4/5 4 4/5 4/5 4/5 4/5 4 CC 4/5 4 4/5 3/4 3/4 5 4 AC 4/5 4/5 4/5 4/5 4/5 5 4/5 CC 4/5 4 4/5 3/4 3 5 4/5 AC 4/5 4/5 4/5 4/5 4/5 5 4/5 4/5 4/5 5 4 4/5 5 4/5 4/5 4/5 5 4/5 4/5 5 4/5 Rubbing/crocking Dry Wet A Perspiration B CC AC Water

PC PS PR JR SS C P

PC- pure cotton, PS- pure silk, PR- pure rayon, RJ- rayon jacquard, SS- silk satin, C- cotton, Ppolyester, AC- assessing colour, S- staining on white fabric, wf- weft, wp- warp, A- acidic condition, B- basic condition, CC- colour change, 1 very poor, 2 poor, 3 fair, 4 good, 5 excellent.

4.3.2

Fabric

Effect of Types of Mordant on Cotton and Silk Satin

Natural dyes often require a metallic mordant to increase affinity between the fibre and the dye, and different mordants also help to prevent the colour from either fading after exposure to light or being washed out (Chairat et al., 2007). In this study, the dyed fabrics were mordanted using alum, ferrous sulphate, copper sulphate and slake lime. The control samples were not mordanted.

Figure 4.2 shows the colour of cotton and silk satin fabrics dyed with four different metals as mordant. The use of slake lime mordant resulted in darker colour compared to the control fabrics for both cotton and silk satin. Fe2(SO4)3 resulted in a gold coloured fiber while CuSO4 turned the dyed fiber into grey. However, alum did not show any significant differences between colour formed on the dyed fabrics and

78 on the control samples. As shown in Table 4.2, Fe2(SO4)3 and CuSO4 managed to increase the lightfastness rating from 1 (control) to 2 and 2 / 3 respectively for cotton (but not on silk satin). On silk satin, alum and slaked lime was found to increase the lightfastness rating from 1 (control) to 2.

Cotton

Silk Satin

Figure 4.2: Colour of dyed cotton and silk satin control (without mordant) (a) and mordanted using (b) alum, (c) Fe2(SO4)3, (d) CuSO4 and (e) Ca(OH)2.

The colour change observed for the dyed fabric after applying with mordant (Fe2(SO4)3 and CuSO4) was due to the strong colour of the mordant itself which is from the transition elements group. Transition elements produce colour when it complexes with suitable ligands such as water for this study (Rodger, 1994).

Because of its structure, transition metals form many different coloured ions and complexes, for example, Fe2(SO4)3 is orange while CuSO4 is royal blue. Transition metals complexed with coloured organic ligand exhibit better light fastness than that of the ligand only. This may be due to the coordination with transition metal ion that reduces the electron density of the chromophore, which in turn leads to improved resistance to photochemical oxidation (Christie, 2001).

79 In this study, good colourfastness of fabric to washing was obtained when alum was used as the mordant. This is because Al3+ can act as a good electron acceptor to form coordinate bonds with dye molecule, followed by the formation of an insoluble complex, hence increasing the binding affinity of pigment to the fibers (Lee and Kim, 2004).

Other colourfastness tests showed that cotton and silk satin have a good (4) to excellent (5) colourfastness properties to perspiration, rubbing / crocking and water in the absence of mordant. This result might be attributed to the high affinity of the pigment with cellulose and protein.

Table 4.2: Shades and fastness properties of dyed cotton and silk satin in the presence of various mordants.
Colourfastness Fabric Light Mordant Washing AC 2/3 4 2 2/3 3 3/4 4 2 3 S 3/4 4 4/5 4 3 3 4 4/5 4 Rubbing/crocking Dry wf 4 4 4 4/5 4 4/5 4/5 4 4/5 wp 4/5 4 4 4/5 4 4/5 4/5 4 4/5 Wet wf 4/5 4/5 4 4/5 4/5 4/5 4/5 4/5 4/5 wp 4/5 4/5 4 4 4/5 4/5 4/5 4 4/5 CC 5 5 3/4 3 5 5 3/4 3 3/4 Perspiration A AC 5 5 4/5 4/5 4/5 5 4/5 4/5 4/5 CC 5 5 3 3 5 5 3 3/4 4 B AC 5 5 4/5 4/5 4/5 5 4/5 4/5 4/5 Water CC 5 5 4/5 4/5 5 5 4/5 4/5 4/5 AC 5 5 4/5 4/5 4/5 5 4/5 4/5 4/5

No Cotton Alum Fe2(SO4)3 CuSO4 Ca(OH)2 No Silk Satin Alum Fe2(SO4)3 CuSO4

1 1 2 2/3 1 1 2 1 1

2 3/4 3/4 4/5 4/5 4/5 4/5 5 5 5 5 5 5 Ca(OH)2 AC- assessing colour, S- staining on white fabric, wf- weft, wp- warp, A- acidic condition, B- basic condition, CC- colour change, 1 very poor, 2 poor, 3 fair, 4 good, 5 excellence.

80 From this study, it was shown that, the pigments can act both as direct dye and mordant dye since the pigments can adhere to the fabric molecules without help from other chemicals (in case of un-mordanted fabrics) and it can also work in the presence of chemical intermediate, known as a mordant, to attach themselves to the fabric. However, the colourfastness result of dyed fabric mordanted using alum is better compared to un-mordanted fabric. A possible reason for this observation is that the auxochromic groups (-OH and NH2) present in the pigment are in a favorable position resulting in easy formation of metal-complexes with the fabrics and tendency to form quite strong bonds with the dye and fibers.

4.3.3

Effect of Mordant Concentrations on Cotton and Silk Satin

The effect of mordant concentration on cotton and silk satin is shown in Table 4.3. Better colouring of the dyed fabrics was observed with increasing mordant concentration. At 10 g L-1 of mordant, the colour of dyed fabric becomes darker compared to dyed fabric applied with lower mordant concentration i.e. 5 g L-1.

Table 4.3: Colour of dyed fabrics mordanted using different concentration of mordant. Fe2(SO4)3 5 g L-1 Cotton Fe2(SO4)3 10 g L-1 CuSO4 5 g L-1 CuSO4 10 g L-1

Silk Satin

Increased intensity of fabric colour at high mordant concentrations was due to the increase in the number of complex formed between fiber and dye molecules from the high concentration of metal ions present in the solution. However, the fastness properties between dyed fabrics applied with and without mordant did not show any

81 significant difference (Table 4.4). This may be due to reactive sites present which is responsible to bind metal mordants. When the site is occupied by a metal mordant, it is no longer available to attract more metal mordant, so that only a monomolecular layer on the fiber surface can be obtained. The excess metal mordant may leach out during the fastness test since it does not have any interaction with the fabrics (Vickerstaff, 1954).

Table 4.4: Shade and fastness properties of dyed cotton and silk satin mordanted using different concentration of mordants.

Concentration

Colourfastness Washing Light AC 2 2 2/3 3 3 3 3/4 3/4 S wf 2 4/5 4/5 4 4 4 4 4/5 4 4 4 4/5 4/5 4 4 4/5 4/5 wp 4 4 4/5 4/5 4 4 4/5 4/5 wf 4 4/5 4/5 4/5 4 4/5 4 4 wp 4 4 4 4/5 4/5 4 4 4 CC 3/4 3 3 3/4 4 3/4 4 3 AC 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 CC 3 3/4 3 4 3/4 3 4/5 3/4 AC 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4 4/5 4/5 4/5 Rubbing/crocking Dry Wet Perspiration A B CC AC Water

C SS C SS C SS C SS

Fe2(SO4)3

Mordants

Fabric

5g L-1 10g L-1

1 2/3 1 1 2/3 1 2/3

C- cotton, SS- silk satin, AC- assessing colour, S- staining on white fabric, wf- weft, wp- warp, Aacidic condition, B- basic condition, CC- colour change, 1 very poor, 2 poor, 3 fair, 4 good, 5 excellence.

4.3.4

to remove natural impurities present on the cotton and silk fabrics. The examples of

CuSO4

Fe2(SO4)3

CuSO4

Effect of Pre-Treatment on Dyeing Performance

The pre-treatment of fabric was carried out before the dyeing process in order

82 impurities that be may present in those fabrics are fats and waxes, pectins and related substances, minerals and heavy metals, amino acids or proteins and lubricants or knitting oils. The method of impurities removal is different depending on the types of impurities present (Karmakar, 1999).

In this study, the pre-treatment involves scouring of cotton in alkaline agents (mixture of sodium carbonate and alum) and degumming of silk with alkali (alum solution). The pre-treatment process was found to increase the pigment performance on the fabric but the fastness properties are not improved. This can be clearly seen in Figure 4.3, where the pigment stain fabrics well and gave darker colour for both fabrics compared to fabrics dyed without pre-treatment (section 4.3.2). The pigment stains fabrics well because the pre-treatment increases fibre swelling, thus contributing to the release of impurities from the fibre. When the impurities are removed, more pigment will replace and occupy the space between the fibres, hence increasing the colour of fabrics (Karmakar, 1999).

Cotton

Silk Satin

Figure 4.3: Colour of dyed cotton and silk satin fabric after pretreatment process; (a) unmordanted and mordanted using (b) alum, (c) Fe2(SO4)3, (d) CuSO4 and (e) Ca(OH)2. The fastness properties of dyed fabrics after the pre-treatment process is shown in Table 4.5. When comparing the fastness properties results of dyed fabrics which undergo pre-treatment and dyed fabric without pre-treatment (section 4.3.2), no

83 significant difference was observed. This could be due to limited number of reactive sites on the fiber to bind with dye molecules. Since the sites are fully occupied by dye molecules, no attraction is available for other dyes and only a monomolecular layer of dye molecule on the fibre surface can be obtained. The excess dye may wash out during the fastness testing since it does not have any attraction to the fabrics (Vickerstaff, 1954).

Table 4.5: Shades and fastness properties of fabrics after pre-treatment process.
Colourfastness Fabric Light Mordant Washing AC 2/3 3 3/4 4 3/4 3/4 4 3 3/4 S 2 3 4 3/4 2/3 3/4 3/4 4 3/4 Rubbing/crocking Dry wf 4 4 4 4 4 4 4 4 4/5 wp 4 4 4/5 4 4 4/5 4/5 4 4/5 Wet wf 4 4/5 4/5 4 4/5 4/5 5 4 4/5 wp 4/5 4 4/5 4/5 4/5 4/5 4/5 4/5 4/5 CC 4/5 5 4 3/4 4/5 4/5 4 3 3 Perspiration A AC 4/5 5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 CC 4/5 5 4 3/4 4/5 4/5 4/5 3/4 3 B AC 4/5 5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 Water CC 5 5 4/5 4 5 4/5 4/5 4/5 4/5 AC 5 5 4/5 4/5 5 4/5 4/5 4/5 4/5

No Alum Cotton Fe2(SO4)3 CuSO4 Ca(OH)2 No Alum Silk Satin Fe2(SO4)3 CuSO4

1 1 1 2 1 1 1 2 2

1 4 3/4 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 Ca(OH)2 AC- assessing colour, S- staining on white fabric, wf- weft, wp- warp, A- acidic condition, B- basic condition, CC- colour change, 1 very poor, 2 poor, 3 fair, 4 good, 5 excellence.

4.3.5

Comparison of Fastness Properties of Violacein with Reactive Dyes

The colour of fabric dyed using violacein and reactive dyes is shown in Figure 4.4. The reactive dye showed intense colouration on both fabrics compared to violacein. This clearly indicates the higher affinity of reactive dye towards cotton and

84 silk satin fabrics compared to violacein as well as strengthening the notion that natural pigment would produce a mild shade of colour (Samanta and Agarwal, 2009).

a
Na2SiO3

b
Na2SiO3

a
Ca(OH)2

b
Alum

Reactive Dye

Violacein

Figure 4.4: Colour performance of fabrics dyed with reactive dye and violacein, a) silk satin and b) cotton.

The colourfastness data for fabric dyed with violacein and reactive dye is shown in Table 4.6. From the washfastness data, fabric dyed with violacein and applied with alum (silk) and slaked lime (cotton) as mordant, gave a fair (3/4) to good (4) ratings which was slightly lower than the good (4) ratings for shades developed using reactive dye. The good wash - fastness of fabrics dyed with reactive dye was due to the stronger covalent bonds between the reactive dye molecules and the fabric (Ali et al., 2009) compared to the bonds formed with the violacein molecules.

85 Table 4.6: Comparison of fastness properties of natural dyes with reactive dyes
Colourfastness Mordant Fabric Dye Washing Light AC S wf Cotton VIO RD VIO RD Alum SS Ca(OH)2 SS 1 4 2 3 4 4 3/4 4 4 4 3/4 4 4 4 4/5 4/5 wp 4 4 4/5 4/5 wf 4/5 4 4/5 4 wp 4/5 4/5 4/5 4 CC 5 5 5 5 AC 5 4/5 5 4/5 CC 5 5 5 5 AC 5 4/5 5 4/5 5 5 5 5 5 4/5 5 4/5 Rubbing/crocking Dry Wet Perspiration Acid Alkaline CC AC Water

VIO- violacein, RD- reactive dye, AC- assessing colour, S- staining on white fabric, wf- weft, wpwarp, A- acidic condition, B- basic condition, CC- colour change, 1 very poor, 2 poor, 3 fair, 4 good, 5 excellence.

Silk Satin

Surprisingly, for all of the other fastness tests (except for light), fabrics dyed with violacein showed fair (3/4) to excellent (5) ratings compared to the reactive dye. The higher light fastness properties for reactive dyes can be attributed to the strong intramolecular H-bonding which exists in the form of six membered rings. This enhances the stability of the compound by a reduction in electron density at the chromophore. As a result, sensitivity of dye towards photochemical oxidation was reduced (Ali et al., 2009).

4.4 Conclusion

From the study, violacein was found to be capable of dyeing not only natural fibers but synthetic fibers as well. The use of slake lime mordant resulted in darker colour compared to the control fabrics for both cotton and silk satin. The use of Fe2(SO4)3 resulted in a gold coloured fiber while CuSO4 turned the dyed fiber into grey. Fe2(SO4)3 and CuSO4 managed to increase the lightfastness rating from 1 (control) to 2 and 2 / 3 respectively for cotton (but not on silk satin). On silk satin, alum and slaked lime were responsible for increasing the lightfastness rating from 1

86 (control) to 2. At 10 g L-1 of mordant, the colour of dyed fabric becomes darker compared to dyed fabric applied with lower mordant concentration i.e. 5 g L-1. Pretreatment of fabric increase the pigment performance on the fabric. The reactive dye showed intense colouration on both fabrics compared to violacein. From the washfastness data, the good (4) rating for shades developed using reactive dye was observed. Surprisingly, for all of the other fastness tests (except for light), fabrics dyed with violacein showed fair (3/4) to excellent (5) ratings comparable to the reactive dye. This study demonstrated that violacein was successfully applied in textile dyeing and it can be used as an alternative to synthetic dye.

87

CHAPTER 5

CONCLUSION

5.1

Conclusion

A total of 17 bacterial isolates from 2 sampling ports were screened for the production of pigment. Of these, two bacteria which produced violet and yelloworange pigments were identified as Chromobacterium violaceum and

Chryseobacterium sp. using 16S rRNA analysis, respectively. The yellow-orange pigment shows change in colour with 20 % (w/v) KOH which indicated the presence of flexirubin type pigment. Whilst, the violet pigment having broad spectrum antimicrobial properties was confirmed as violacein using UV/Vis, FTIR, NMR and MS analysis.

A cheaper way to produce the pigment using sugarcane bagasse (SCB) in the presence of L- tryptophan was carried out in batch system and immobilized system

Finally, the violet pigment showed potential application in textile dyeing with comparable performance to some common reactive dyes.

5.2

Suggestions for Future Work

Based on the study carried out in this research, C. violaceum was able to utilize agricultural waste through submerged fermentation. However, solid state

88 fermentation offer many advantages such as higher fermentation productivity, higher end-concentration of products, lower cost of down-stream processing and lower demand on sterility due to the low water activity used in solid state fermentation. Thus, studies to investigate the potential use of agricultural waste for pigment production by C. violaceum via solid-state fermentation needs to be carried out.

Violacein also exhibit poor lightfastness compared to synthetic dye. Hence, attempts to improve the lightfastness of violacein needs to be studied in order to be competitive in the market.

Since violacein was reported to have many distinct biological properties such as anticancer, antioxidant, and antibiotic properties, the possibility of applying violacein as colourants in food and pharmaceutical product would provide new field of application for the natural pigments.

89

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108 APPENDIX

List of publications (journal/article), awards and seminar/ paper presentation during MSc study period (July 2008- June 2010).

Publications

Nur Zulaikha Yusof

and Wan Azlina Ahmad. (2010).

Extraction and

Characterization of Violacein from Chromobacterium violaceum Grown in Agricultural Waste Material. Carbohydrate Polymer (Correction in progress).

Nur Zulaikha Yusof and Wan Azlina Ahmad. (2010). Production of Pigments from Bacteria Grown in Solid and Liquid Pineapple Waste. 7th International Pineapple Simposium (IPS 2010). Oral Presentation.

Patent A patent entitled Organic Dye Isolated from Bacteria. IP: PI 20092217.

Trademark ColorBac - Environmental- Friendly Pigments from Bacteria for Textile and Related Industry.

Awards and Recognitions

Awarded the National Science Fellowship scholarship to pursue MSc programme in UTM for a period of 2 years (July 2008- June 2010).

Bronze Medal- BioMalaysia 2009, KLCC, Kuala Lumpur with invention entitled ColorBac- Environmental- Friendly Pigments from Bacteria for Textile and Related Industry (November 2009).

109

Treasurer for Postgraduate Association of Faculty of Science (PoSAFS) for 2008/2009 session.

Committee member in the Industrial Wastewater Treatment Workshop jointly organized by UTMBacTec and Department of Environment Malaysia, Johor Branch on 2nd June 2010.

Grant/Fund TechnoFund Environmental Friendly Bacterial Pigments - Large Scale Production Utilizing Agricultural Wastes And Its Application In Biodegradable Plastic And Food Packaging Approved RM 1.4 Million. Cradle Fund (U-chip) Bacterial Pigments Extraction and Application (Approved, October 2010).