Nur Zulaikha Yusof Ms083038

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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 :

iv

bahagian a – PENGESAHAN KERJASAMA*

Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui

kerjasama antara _______________________ dengan _______________________

Disahkan oleh:

Tandatangan : Tarikh :

Nama :

Jawatan :

(Cop rasmi)

* 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 : Tarikh :

Nama :

v

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 : Nur Zulaikha Yusof

Date :

iii

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.

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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 pre-

treatment 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 L-

1). 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.

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION

DEDICATION

ACKNOWLEDGEMENTS

ABSTRACT

ABSTRAK

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

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1

GENERAL INTRODUCTION

1.1 Definition of Pigments

1.2 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

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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 Objectives of the Study

1.6 The Scope of Study

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

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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 Identification of Bacteria


2.3.5 Characterizations of Crude Violet Pigments



2.3.5.3 Infrared Spectroscopy


2.3.5.5 Nuclear Magnetic Resonance

(NMR)

2.3.5.6 Mass Spectrometry (MS)

2.3.6 Primary Identification of Yellow - Orange

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Pigment

2.3.6.1 Simple Test for Flexirubin – Type

Pigment

2.3.7 Conclusion

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



3.2.2 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


3.3.1 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

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C. violaceum on SCB

3.4 Conclusion

APPLICATION OF PIGMENT ISOLATED FROM

C. violaceum IN TEXTILE DYEING

4.1 Natural Colourants in Textile Dyeing

4.1.1 Reason for Natural Colouration


4.2.1 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 Colourfastness to Washing

4.2.11.2 Colourfastness to Light

4.2.11.3 Colourfastness to

Rubbing/Crocking

4.2.11.4 Colourfastness to

Perspiration

4.2.11.5 Colourfastness to Water


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

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REFERENCES 89

APPENDIX 108

5

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

CONCLUSION

5.1 Conclusion

5.2 Suggestions for Future Work

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LIST OF TABLES

TABLE NO. TITLE PAGE

1.1

1.2

2.1

2.2

2.3

2.4

2.5

2.6

2.7

4.1

4.2

4.3

4.4

Naturally occuring pigments in biology (Hendry and

Houghton, 1997).

Microbial production of pigments (already in use as natural

food colourants or with high potential in this field) (Liu

and Nizet, 2009).

Characteristics of water and soil samples collected from

Brackishwater Aquaculcure Research Centre, Gelang

Patah, Johor.

Characteristics of soil samples collected from treatment

pond of oil refinery at Port Dickson, Seremban.

Characteristics of bacterial colonies obtained from soil and

water isolates grown on NA plate.

Morphological features of the coloured bacterial colonies

on NA plate.

Antimicrobial testing of violet pigments on P. aeruginosa,

S. aureus, B. cereus and E. coli.

ν OH (free) cm-1

and ν C-O cm-1

in alcohols and phenols

1H and

13C data of violet pigment.

Shades and fastness properties of different fabrics dyed

with natural pigment.

Shades and fastness properties of dyed cotton and silk satin

in the presence of various mordants.

Colour of dyed fabrics mordanted using different

concentration of mordant.

Shade and fastness properties of dyed cotton and silk satin

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4.5

4.6

mordanted using difference concentration of mordants.

Shades and fastness properties of fabrics after pre-

treatment process.

Comparison of fastness properties of natural dyes with

reactive dyes.

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LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

2.10

2.11

2.12

3.1

3.2

3.3

Morphology of isolate S8b on NA plate (a) and NB

medium (b).

Morphology of isolate S1a on NA plate (a) and NB

medium (b).

UV/Vis spectrum for methanolic extract of violet

pigment.

The charge delocalization by the contributing structures

in the absence (a) and presence (b) of electron donating-

NHR group.

FTIR spectrum for crude methanolic extract of violet

pigment.

Structure of secondary amides: ciscoid-ciscoid

associations (a) and transoid-transoid polymeric

association (b).

Resonance structure of carbonyl containing group in

double bond structure.

1H NMR spectrum of violet pigment.

The structure of violacein.

13C NMR spectrum of violet pigment.

The ESI - MS spectrum of violet pigment.

The ESI – MS / MS spectrum of violet pigment

(m/z = 342.34).

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.

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3.4

3.5

3.6

3.7

3.8

3.9

4.1

4.2

4.3

4.4

The absorption spectrum of the violet pigment obtained

at different incubation temperatures.

Time course of cell growth and pH profile of C.

violaceum cultivated in NB, TSB, PGB and LB.

Symbols: ( ) NB OD600, ( ) NB pH, ( ) PGB

OD600, ( ) PGB pH, ( ) LB OD600, ( ) LB pH,

( ) TSB OD600 and ( ) TSB pH.

Violacein production by C. violaceum in complex media.

Colour intensity of pigments in a) liquid substrate and b)

solid substrate.

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.

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).

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.

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.

Colour performance of fabrics dyed with reactive dye

and violacein, a) silk satin and b) cotton.

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LIST OF ABBREVIATIONS

AATCC - American Association of Textile Chemists and Colorists

Abs - Absorbance

AMES - Salmonella typhimurium reverse mutation assay

EBV - Epstein–Barr virus

13C - Carbon-13

ESI-MS - Electronspray Ionization Mass Spectrometry

ESI-MS/MS - Electrospray Ionization Tandem Mass Spectrometry

1H - Proton

Hz - Hertz

FDA - Food and Drug Administration

FT-IR - Fourier-Transform Infrared Spectroscopy

MHz - Megahertz

MS - Molecular Mass Spectrometry

NMR - Nuclear Magnetic Resonance

PARs - Provoke allergic or pseudo-allergic reactions

Rf - Retention factor

ROS - Reactive oxygen species

rRNA - Ribosomal ribonucleic acid

SIRIM - Standard and Industrial Research Institute of Malaysia

SCB - Sugarcane bagasse

SPW - Solid pineapple waste

TPA - Tumor promoter 12-O-tetradecanoylphorbol-13- actate

TLC - Thin layer chromatography

USD - United States dollar

UV/Vis - Ultraviolet/visible

Π - Pi

xviii

J - Coupling constant

γ - Gamma

δ - Chemical shift

d - Doublet

s - Singlet

t - Triplet

dd - Doublet of doublet

µm - Micrometer

m/z - Mass-to-charge ion

λmax - Wavelength at an absorption maximum

υmax - Maximum absorption frequency

kPa - Kilo pascal

1

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,

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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 Alternative or

familiar name

Major example Predominant

colour

Tetrapyrroles Porphyrins and

porphyrin

derivatives

Chlorophylls

Heams (hemes)

Bilins (Bile

pigments)

Green

Red

Blue-green-

yellow-red

Tetraterpenoids Carotenoids Carotenes

Xanthophylls

Yellow-red

Yellow

O-heterocyclic

compounds

Flavonoids Anthocyanins

Flavonols

Blue-red

Yellow-white

Quinines

Phenolics

compounds

Naphtaquinones

Anthraquinones

Red-blue-

green

Re-purple

N-heterocyclic

compounds

Indigoids and

indole derivatives

Substituted

pyrimidines

Betalaines

Eumelanins

Pterins

Purines

Yellow-red

Black-brown

White-yellow

Opaque white

Metalloproteins Cu-proteins

Haemerythrin

Blue-green

Red

Blue-green

Red

miscellaneous Lipofuscins

Fungal pigments

Brown-grey

Various but

commonly

yellow

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

artist‘s 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 pseudo-

allergic reactions (PARs), particularly in people allergic to aspirin and other non-

steroidal 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 Epstein–Barr virus (EBV) antigen induced by tumor promoter 12-O-

tetradecanoylphorbol-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 (Eichlerová 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, time-

consuming 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 world‘s 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 Pigment Colour Status*

Bacteria

Agrobacterium aurantiacum Astaxhantin Pink-red RP

Paracoccus carotinifaciens Astaxhantin Pink-red RP

Bradyrhizobium sp. Canthaxhantin Dark-red RP

Streptomyces echinoruber Rubrolone Red DS

Flavobacterium sp. Zeaxanthin Yellow DS

Paracoccus zeaxanthinifaciens Zeaxanthin Yellow RP

Fungus

Monascus sp. Ankaflavin Yellow IP

Monascus sp. Monascorubramin Red IP

Penicillium oxalicum Anthraquinone Red IP

Blakeslea trispora Lycopene Red DS

Fusarium sporotrichioides Lycopene Red RP

Cordyceps unilateralis Naphtoquinone Deep blood-red RP

Ashbya gossypi Riboflavin Yellow IP

Monascus sp. Rubropunctatin Orange IP

Blakeslea trispora ß-carotene Yellow-orange IP

Fusarium sporotrichioides ß-carotene Yellow-orange RP

Mucor circinelloides ß-carotene Yellow-orange DS

Neurospora crassa ß-carotene Yellow-orange RP

Phycomyces blakesleeanus ß-carotene Yellow-orange RP

Penicillium purpurogenum Unknown Red DS

Yeast

Saccharomyces neoformans

var. nigricans

Black Melanin RP

Xanthophyllomyces dendrorhous Astaxanthin Pink-red DS

Rhodotorula sp. Torularhodin Orange-red DS

*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-hydroxy-

1H-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 4‘4‘-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, non-

fluorescent 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 6·7

(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, γ-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 functions—varying from a protective action against the lethal photo-

oxidations (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 non-

carotenoid 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 (Dufossé, 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-

β,β-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 (Dufossé, 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 β-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 β-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 ß- 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- b-

carotene) 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; Dufossé et al.,

2005).

Other than that, the yellow pigment known as zeaxanthin or 3,3‘-dihydroxy-

b-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 (Dufossé, 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 Gram-

negative 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 anti-

protozoan (Matz et al., 2004 ; Leon et al., 2001) , anticancer (Ferreira et al., 2004;

Kodach et al., 2006), anti-viral (Andrighetti-Fröhner et al., 2003) , antibacterial (both

G+ and G-) (Sánchez 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 bio-

fungicide (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 (FT-

IR), 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).


2.2.1 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 121°C, 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 121ºC, 121 kPa for 15 minutes. The molten agar was cooled to about

50ºC before being poured into sterile Petri dishes. The agar was allowed to harden

and then incubated for 24 hours at 30ºC 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 30°C 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 30°C 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 30°C, shaking at 200 rpm for 24 hours. A serial 10-

fold 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 30°C for

24 hours. The pure bacterial colony was then sub-cultured onto fresh NA plates and

incubated at 30°C 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

Gram‘s 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 30°C, 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 30°C for 12 hours. Cultures

obtained as pure colonies were sent to Vivantis Technology Sdn. Bhd., Malaysia for

16S rRNA sequence analysis.

27


2.2.5.1 Short-term Maintenance

The bacteria were streaked on NA plate and incubated at 30°C 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 -20°C

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 121°C, 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).


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 30°C 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 -20°C. 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 (BÜCHI 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 50°C and chiller set below 10°C. 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 60°C 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


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 37°C (for E. coli). The tested bacteria were

maintained in LB – glycerol stock solution stored at -20°C 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 µm) was previously prepared by punching the filter paper

(Whatman 113, wet strengthened, 125mm Ø in diameter and thick, 420 µm) using

paper puncher. The discs were individually impregnated with 50 µL 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 µL methanol and filter paper discs only. Inhibition zones were observed after

overnight incubation at 30 and 37ºC respectively.

30


The pigments were subjected to UV/Vis analysis to check for its maximum

wavelength (λmax). For the analysis, methanol was used as the blank. A full

wavelength scan between 800-200 nm was carried out to ascertain the λmax 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 FT-

IR 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

.


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 13

C 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)

υmax 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 λmax (MeOH) nm:

585.72; 1H NMR (DMSO, 400 MHz): δ 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, H-

21), 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); 13

C NMR (DMSO, 100 MHZ):

δ 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).


2.3.1 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 Location Colour °C pH

W1 Recycled water for 10 day

siakap Greenish Brown 29.7 6.24

W2 Recycle tank for talapia Cloudy grey 31.9 6.24

W3 Rotifer breeding Clear 28.3 6.22

W4 Fish breeding pond Clear 32.1 6.29

W5 Water point source (From sea) Cloudy grey 32.4 6.21

W6 Shrimp pond Clear 28.3 6.14

W7 Recycled organic waste for red

talapia Cloudy grey 29.5 6.24

W8 Organic Waste Cloudy grey 28.5 6.18

S1 Near shrimp pond Brown-orange - 7

Table 2.2: Characteristics of soil samples collected from treatment pond of oil

refinery at Port Dickson, Seremban.

Sample Type of Soil Colour pH

S1 Clay Orange 7

S2 Small sand Grey 6


S4 Clay Yellow 5

S5 Mixture of sand and clay Dark green 6

S6 Clay Orange 6


S8 Clay Orange 5


S10 Clay Orange 6

S11 Small sand Black 5



S14 Mixture of sand and clay Dark grey 6



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 Colony Colour Colony Characteristics

Form Elevation Margin

W5a Light Yellow Irregular Flat Erose

S6a Pale Yellow Punctiform Convex Entire

S7a Yellow Punctiform Convex Entire

S8a Light-yellow Filamentous Raised Fillamentous

# S8b Yellow-orange Punctiform Convex Entire

S8c Cream-yellow Circular Convex Entire

S8d Yellow-orange Punctiform Convex Entire


S10c Cream-yellow 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.

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.

Bacteria

ID Colony Colour

Colony Characteristics

Form Elevation Margin

# S1a Violet Punctiform Raised Entire

S1b Opaque-yellow Circular Umbonate Entire

S2d Light-yellow Punctiform Raised Entire

S5e Pale-yellow Punctiform Raised Entire

S6c Pale-yellow Punctiform Umbonate Undulate


S10a Light-yellow Circular Raised Entire

S11a Opaque red Circular Convex Entire

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-Fröhner 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 (Steinbüchel 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

(Kämpfer 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).


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


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 µL 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.

cereus and E. coli.

* Control plates – plates inoculated with the respective bacteria

** 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.

**Control + MeOH ***Control + MeOH +

Violet Pigment

P. a

erug

inos

aB

. cer

eus

E. c

oli

S. a

ureu

sP

. ae

rugi

nosa

B.

cere

usE

. co

liS.

aur

eus

P.

aeru

gino

saB

. ce

reus

E.

coli

S. a

ureu

sP

. ae

rugi

nosa

B.

cere

usE

. co

liS.

aur

eus

*Control

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 broad-

spectrum 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.


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 Π to Π* transition than an unconjugated system. It is expected that the

greater the number of bonding Π orbital, the lower will be the energy difference, ΔE,

between the highest bonding Π orbitals and the lowest excited Π* orbital. The

obvious extension of this in terms of λmax 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).

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 Π* 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

.

C C C O C C C O

HC CH

CH

ONR2HC C

HCH

ONR2

a)

b)

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).

(a) cis-dimer (b) trans-polymer

Figure 2.6: Structure of secondary amides: ciscoid-ciscoid associations (a) and

transoid- transoid polymeric association (b).

CR

O

N

H

H

R'O

N

R'

C RN

R

H R'

O H

N

R'

R O H

N

R'

R O H

N

R'

R O

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):

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).

C C C O C C C O

44

Table 2.6: ν OH (free) cm-1

and ν C-O cm-1

in alcohols and phenols

No. Nature of the hydroxyl compound ν OH (free) cm-1

ν C-O cm-1

1. Primary alcohol 3640 1050

2. Secondary alcohol 3630 1100

3. Tertiary alcohol 3620 1150

4. Phenols 3610 1200

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

.


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 13

C 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 δ 11.87 ppm, lactam NH at δ 10.72 ppm and isatin NH at δ 10.60 ppm.

45

Figure 2.8: 1H 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).

Figure 2.9: The structure of violacein.

Indole nucleus Lactam ring

Isatin nucleus

46

Three aromatic protons in the indole skeleton which corresponds to meta-

couple signal and ortho-couple signal were detected at δ 6.79 ppm (dd, J=2.0 Hz and

8.8 Hz) and δ 7.23 ppm (d, J=2.0 Hz) which were attributed to H-7 and H-5

respectively and δ 7.35 ppm (d, J=8.8 Hz) attributed to H-8. A doublet peak at δ 8.06

ppm with J=2.4 Hz correspond to H-2. The hydroxyl group was observed at δ 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 δ 8.93 ppm (d, J=7.6 Hz), δ 6.94 ppm (t, J=7.6 Hz), δ 7.20 ppm (t, J=7.6

Hz) and δ 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 δ 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

δ 170.7 ppm (C-16) and δ 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: 13

C NMR spectrum of violet pigment.

Eighteen other carbons were observed at δ 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), and 153.4 (C-6). The signal at δ 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

13C NMR data of violet pigment are tabulated in

Table 2.7.

48

Table 2.7: 1H and

13C data of violet pigment.

Position

1H NMR

δ (ppm) Multiplicity

13C NMR

δ (ppm)

1-NH 11.87 s -

2 8.06 d, J=2.4 Hz 129.9

3 - - 106.2

4 - - 126.1

5 7.23 d, J=2.0 Hz 105.1

6 - - 153.4

7 6.79 dd, J=2.0 Hz, 8.8 Hz 113.6

8 7.35 d, J=8.8 Hz 113.9

9 - - 132.1

10-NH 10.72 s -

11 - - 172.1

12 - - 137.5

13 7.55 d, J=2.0 Hz 97.5

14 - - 148.1

15-NH 10.60 s -

16 - - 170.7

17 - - 119.2

18 - - 122.9

19 8.93 d, J=7.6 Hz 126.8

20 6.94 t, J=7.6 Hz 121.3

21 7.20 t, J=7.6 Hz 130.1

22 6.83 d, J=7.6 Hz 109.5

23 - - 142.3

6-OH 9.32 s -

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 [M–H]_. 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 self-

aggregation (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



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 30°C 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 µm 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 37°C. 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 µm Whatman filter paper prior to drying at 50°C 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

121°C for 15 minutes. Fermentations were carried out at 30°C 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 105°C 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 105°C,

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 (QRëC). 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, 30°C 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

.


3.3.1 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.

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.

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

12 hr 0.5 hr 1 hr 2 hr 4 hr 8 hr 16 hr 24 hr

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 5 10 15 20 25

Time (Hours)

Op

tical

Den

sit

y,

OD

600

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 37

oC (Figure 3.3).

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 30

oC, where C. violaceum showed the ability to produce pigment

after 4 hours of growth at 30°C.

On the other hand, the pigment only appeared after 8 hours of growth when

the temperature was lowered to 25°C. However, the pigment production increased

with time at both growth temperatures. UV-vis analysis of the pigment produced by

bacteria at 30°C 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 30°C (1.03 mg mL-1

) compared to at 25°C (0.97 mg mL-

1) and 30°C (0.48 mg m L

-1).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

0 5 10 15 20

Time (Hours)

Op

tic

al D

en

sit

y, O

D600

25°C

30°C

37°C

61

Figure 3.4: The absorption spectrum of the violet pigment obtained at different

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.

Time: 10:27:29 AMDate: 5/8/2009

P37.SP - 5/8/2009

P25.SP - 5/8/2009

P30.SP - 5/8/2009

400.0 450 500 550 600 650 700 750 800.0

0.000

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.700

nm

A

562.63,0.67280

563.61,0.31598

37°C

25°

C

30°

C

62

Figure 3.5: Time course of cell growth and pH profile of C. violaceum cultivated in

NB, TSB, PGB and LB. Symbols: ( ) NB OD600, ( ) NB pH, ( ) PGB

OD600, ( ) PGB pH, ( ) LB OD600, ( ) LB pH, ( ) TSB OD600 and ( )

TSB pH.

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; Durán and Faljoni-

Alario, 1980).

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

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

NB LB TSB PGB

Medium

Pig

me

nt

Pro

du

cti

on

(g

L-1

)

0

1

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (hours)

Op

tical

Den

sit

y (

OD

600)

0

1

2

3

4

5

6

7

8

9

10

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.

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

SCB1 SCB3 SCB5 SPW1 SPW3 SPW5

BS1 BS10 BS5 M0.1 M1 M0.5

a) Liquid Substrate

b) Solid Substrate

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).

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

SCB1

SCB3

SCB5

SPW

1

SPW

3

SPW

5BS

1BS

5

BS10

M0.1

M0.5 M

1

Substrates

Pig

me

nt

Yie

ld (

g/L

)

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.

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 30°C 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

Violacein

production

in SCB

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 customer‘s 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


4.2.1 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 pre-

treatment 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 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).

75


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

a b c d

e f g

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.

Fab

ric

Mo

rdan

t

Colourfastness

Lig

ht

Washing Rubbing/crocking Perspiration Water

AC S

Dry Wet A B

CC AC wf wp wf wp CC AC CC AC

PC

Alu

m

1 4 4/5 4 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5

PS 2 3 4 4/5 4/5 4/5 4 4 4/5 4 4/5 4/5 4/5

PR 1 4 4 4 4 4/5 4/5 4/5 4/5 4/5 4/5 5 5

JR 1 3/4 3 4 4 4/5 4/5 3/4 4/5 3/4 4/5 4 4/5

SS 2 4 4 4/5 4/5 4/5 4/5 3/4 4/5 3 4/5 4/5 4/5

C 1 4 4 4 4 4/5 4/5 5 5 5 5 5 5

P 3 3 4/5 4/5 4/5 4/5 4 4 4/5 4/5 4/5 4/5 4/5

PC- pure cotton, PS- pure silk, PR- pure rayon, RJ- rayon jacquard, SS- silk satin, C- cotton, P-

polyester, 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 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).

a b c d e

a b c d e

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.

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.

Fab

ric

Mordant

Colourfastness

Lig

ht


AC S Dry Wet A B


Co

tto

n

No 1 2/3 3/4 4 4/5 4/5 4/5 5 5 5 5 5 5

Alum 1 4 4 4 4 4/5 4/5 5 5 5 5 5 5

Fe2(SO4)3 2 2 4/5 4 4 4 4 3/4 4/5 3 4/5 4/5 4/5

CuSO4 2/3 2/3 4 4/5 4/5 4/5 4 3 4/5 3 4/5 4/5 4/5

Ca(OH)2 1 3 3 4 4 4/5 4/5 5 4/5 5 4/5 5 4/5

Sil

k S

atin

No 1 3/4 3 4/5 4/5 4/5 4/5 5 5 5 5 5 5

Alum 2 4 4 4/5 4/5 4/5 4/5 3/4 4/5 3 4/5 4/5 4/5

Fe2(SO4)3 1 2 4/5 4 4 4/5 4 3 4/5 3/4 4/5 4/5 4/5

CuSO4 1 3 4 4/5 4/5 4/5 4/5 3/4 4/5 4 4/5 4/5 4/5

Ca(OH)2 2 3/4 3/4 4/5 4/5 4/5 4/5 5 5 5 5 5 5

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

Fe2(SO4)3

10 g L-1

CuSO4

5 g L-1

CuSO4

10 g L-1

Cotton

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.

Fab

ric

Mo

rdan

ts

Co

nce

ntr

atio

n

Colourfastness

Lig

ht


AC S Dry Wet A B


C

Fe 2

(SO

4) 3

5g

L-1

2 2 4/5 4 4 4 4 3/4 4/5 3 4/5 4/5 4/5

SS 1 2 4/5 4 4 4/5 4 3 4/5 3/4 4/5 4/5 4/5

C

Cu

SO

4 2/3 2/3 4 4/5 4/5 4/5 4 3 4/5 3 4/5 4/5 4/5

SS 1 3 4 4/5 4/5 4/5 4/5 3/4 4/5 4 4/5 4/5 4/5

C

Fe 2

(SO

4) 3

10

g L

-1

1 3 4 4 4 4 4/5 4 4/5 3/4 4/5 4 4

SS 2/3 3 4 4 4 4/5 4 3/4 4/5 3 4/5 4/5 4/5

C

Cu

SO

4 1 3/4 4/5 4/5 4/5 4 4 4 4/5 4/5 4/5 4/5 4/5

SS 2/3 3/4 4 4/5 4/5 4 4 3 4/5 3/4 4/5 4/5 4/5

C- cotton, SS- silk satin, 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.

4.3.4 Effect of Pre-Treatment on Dyeing Performance

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

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

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

a c b d e

e d c b a

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.

Fab

ric

Mordant

Colourfastness

Lig

ht Washing Rubbing/crocking Perspiration Water

AC S Dry Wet A B


Co

tto

n

No 1 2/3 2 4 4 4 4/5 4/5 4/5 4/5 4/5 5 5

Alum 1 3 3 4 4 4/5 4 5 5 5 5 5 5

Fe2(SO4)3 1 3/4 4 4 4/5 4/5 4/5 4 4/5 4 4/5 4/5 4/5

CuSO4 2 4 3/4 4 4 4 4/5 3/4 4/5 3/4 4/5 4 4/5

Ca(OH)2 1 3/4 2/3 4 4 4/5 4/5 4/5 4/5 4/5 4/5 5 5

Sil

k S

atin

No 1 3/4 3/4 4 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5 4/5

Alum 1 4 3/4 4 4/5 5 4/5 4 4/5 4/5 4/5 4/5 4/5

Fe2(SO4)3 2 3 4 4 4 4 4/5 3 4/5 3/4 4/5 4/5 4/5

CuSO4 2 3/4 3/4 4/5 4/5 4/5 4/5 3 4/5 3 4/5 4/5 4/5

Ca(OH)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

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).

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.

Reactive Dye Violacein

Na2SiO3

a

Ca(OH)2

a

Na2SiO3

b

Alum

b

85

Table 4.6: Comparison of fastness properties of natural dyes with reactive dyes

Fab

ric

Dy

e

Mo

rdan

t

Colourfastness

Lig

ht


AC S Dry Wet Acid Alkaline


Co

tto

n VIO Alum 1 4 4 4 4 4/5 4/5 5 5 5 5 5 5

RD SS 4 4 4 4 4 4 4/5 5 4/5 5 4/5 5 4/5

Sil

k S

atin

VIO Ca(OH)2 2 3/4 3/4 4/5 4/5 4/5 4/5 5 5 5 5 5 5

RD SS 3 4 4 4/5 4/5 4 4 5 4/5 5 4/5 5 4/5

VIO- violacein, RD- reactive dye, 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.

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

. Pre-

treatment 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 yellow-

orange 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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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).

Nur Zulaikha Yusof Ms083038

Documents

16s rrna sequencing

universiti

vivantis technologies

wan azlina

nur zulaikha

nuclear magnetic

universiti

vanillin sulphuric