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«AKRAM NESHATI A Dissertation Submitted To The Faculty Of Science In Partial Fulfillment Of The Requirement For The Award Of The Degree In Masters of ...»

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FROM Chromobacterium violaceum GROWN IN



A Dissertation Submitted To The Faculty Of Science In Partial Fulfillment Of The

Requirement For The Award Of The Degree In Masters of Science (Chemistry)

Faculty of Science

Universiti Teknologi Malaysia APRIL 2010


Chromobacterium violaceum GROWN IN



iii To my Beloved Mother and Father iv


I would like to express my deep and sincere gratitude to my supervisor Prof. Dr. Wan Azlina Ahmad. Her wide knowledge and patience have been of great value for me. Her understanding, encouraging and personal guidance have provided a good basis for the present thesis.

I also want to acknowledge all my friends from Biotechnology laboratory specially: Nur Zulaikha, Nordiana and Lee Jin Kuang for their guidance, advices encouragements. They have contributed toward my understanding which without that, this thesis have not been the same as it is present here.

My sincere appreciation also extends to all my friends and others who have provided assistance at various occasions. Unfortunately it is not possible to list down all of them in this limited space.

Lastly, I would like to thank my family for their support and encourage all along this project.



There has been an increasing trend towards replacement of synthetic colorants with natural pigments in last decades because of the strong consumer demand for more natural products. Among three groups of main natural pigments, bacterial pigments are considered as an alternative to synthesized dye. Production and extraction of violet pigment of Chromobacterium violaceum grown on agricultural waste such as solid pineapple waste (SPW) and brown sugar (BS) was studied. From the study, the optimum growth temperature of C. violaceum and pigment production is at 25°C and optimum pH is 7. The pigment was extracted from the growth media using two solvents which were methanol and ethyl acetate.

Characterization of the purple pigment was carried out using UV-VIS spectrophotometer, FTIR and 1H and 13C-NMR. UV-VIS analysis of the purple pigment samples from nutrient broth (NB), BS and SPW media shows λmax at 566.50,

567.50 and 571.94 nm respectively. FTIR spectrum of purple pigment pellet from BS growth medium showed a broad peak at 3430.10 cm-1 assigned to OHstretching, overlapping of N-H bond with O-H stretching observed at 3330.1 cm-1, two stretching bonds at 1640 cm-1 and 1723.5 cm-1 assigned to the C=O amide groups and C=C peak at 1615.92 cm-1. 1H-CNMR and 13C-NMR spectra were recorded in DMSO-d6 and 20 carbon peaks and also 13 proton peaks appeared in the result to confirm the present of violacein in the samples. Lastly, stability of the produced pigment towards changes of the pH was examined. The pigment shows different colors at different pH.



Terdapat peningkatan hala tuju dalam beberapa dekad ini terhadap penggantian pewarna sintetik dengan pigmen asli disebabkan peningkatan permintaan pengguna terhadap produk-produk asli. Di antara tiga kumpulan utama pigmen asli, pigmen daripada bakteria dianggap sebagai alternatif kepada pewarna sintetik. Penghasilan dan pengekstrakan pigmen ungu oleh Chromobacterium violaceum yang dikulturkan di atas sisa pertanian seperti sisa pepejal nenas (SPW) dan gula perang telah (BS) telah di kaji. Daripada kajian ini, suhu optimum untuk pertumbuhan dan penghasilan pigmen oleh C. violaceum ialah pada 25°C dan pH optimum ialah 7. Pigmen tersebut diekstrak daripada media pertumbuhan menggunakan dua pelarut iaitu metanol dan etil asetat. Pencirian pigmen ungu ini dilakukan dengan menggunakan spektrofotometer UV-VIS, FTIR dan 1H dan 13CNMR. Analisis UV-VIS ke atas sampel pigmen ungu yang diperolehi daripada kaldu nutrien (NB), BS dan SPW masing-masing memberikan λmax pada 566.50, 567.50 dan 571.94 nm. Spektrum FTIR untuk pelet pigmen ungu daripada media pertumbuhan gula perang menunjukkan jalur yang lebar pada 3430.10 cm-1 mewakili regangan O-H, pertindihan jalur regangan ikatan N-H dan O-H pada 3330.1 cm-1, dua jalur regangan pada 1640 cm-1 dan 1723.5 cm-1 mewakili kumpulan amida C=O dan jalur ikatan C=C pada 1615.92 cm-1. Spektra 1H -NMR dan 13C-NMR telah direkodkan menggunakan pelarut DMSO-d6 dan didapati 20 puncak karbon dan 13 puncak proton muncul, mengesahkan kehadiran violacein tulen di dalam sampel. Akhir sekali, kestabilan pigmen yang dihasilkan terhadap perubahan pH turut dikaji. Pigmen ungu memberikan warna yang berbeza dalam pH yang berbeza.


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2.9 General structure of violacein (3-(1,2-dihydro-5-(5- 17 hydroxy-1-H-indol-3-yl)-2-oxo-3H-pyrrol-3-ilydene)-1.3dihydro-2H-indol-2-one)

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4.1 Growth of C. violaceum and production of violet pigment 39 at different temperatures

4.2 Growth of C. violaceum and production of violet pigment 40 at different temperatures

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4.5 Growth of C. violaceum and production of violet pigment 42 on different temperatures in BS.

4.6 Absorbance of violacein extracted from BS samples with 43 different concentration.

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4.8 Spectrophotometric cubes showing production of pigment 45 in BS medium after 6 hours of inoculation

4.9 Vitality test of preservation method for C. violaceum 46

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1.1 Background of Study For decades, both natural pigments and synthetic dyes have been extensively used in various fields of everyday life such as food production, textile industries, paper production, agricultural practices and researches, water science and technology (Tibor, 2007).

According to green technology curriculum, less toxic products and more natural starting material is favorable for today’s production lines. In case of dyes, it is well known that some synthesized dye’s manufacturing is prohibited due to the carcinogenicity of the precursor or product and also because of the effects of disposal of their industrial wastes on the ecosystem. The wastewater generated from dye and dye intermediate industries mainly have intense color having various shades of red, blue green, brown and black through the production of different color containing dyes and usually have high level of COD, BOD, acidity, chlorides, sulphates, phenolic compounds and various heavy metals like copper, cadmium and chromium (Yogendra, 2008).

2 Dyes, as they are intensively colored, cause special problems in effluent discharge (even small amount is noticeable). The effect is aesthetically more displeasing rather than hazardous, and can prevent sunlight penetration decreasing photosynthetic activity in aquatic environment. Although, some azo dyes that causes the effluent color have been implicated as being mutagenic/carcinogenic as well as toxic to aquatic life (Yogendra, 2008).

Thus, extensive research has been conducted to find alternative dyes whose production and use would meet high environmental and safety requirements (Georgeta et al, 2004).

Increasingly, with the improvements in fermentation and other biotechnological techniques, bacteria, single-celled fungi and protozoa offer considerable scope for the commercial production of many pigments. There are many source of natural pigments which are derived from plants, animal, fungi and bacteria. Several intensely colored compounds have been isolated from certain bacteria which have resemblance to pigments in other biological systems (Britton, 1983).

Indigoidine or bacterial indigo, a dimeric pyridine structurally unrelated to the indigo of plants, is found in Pseudomonas indigofera. The highly pigmented Chromobacterium has also yielded the dark antibiotic prodigiosin with almost uncommon structure, a trimeric pyrrole (Hendry and Houghton, 1996).

The same genus also produces dimeric indoles such as the purple violacein pigment, although this one has, at least, some resemblance to the indole derivatives of higher plants (Hendry and Houghton, 1996).

Natural pigments not only have the capacity to increase the marketability of products, they also display advantageous biological activities as antioxidants and anticancer agents. Synthetic pigments, on the other hand, cause considerably environmental pollution and adverse toxicological side effects. Both classes of pigment exhibit considerable structural diversity (Tibor, 2007).


1.2 Statement of Problem The use of synthetic dye has several disadvantages amongst them are carcinogenicity, ambient pollution possibility and increase of the cutaneous allergies for the user of the product.

Green technology is leading all producers to go towards ecological and less polluted products with fewer by-products; in the case of synthesized dye, natural pigments can be considered as an ideal alternative.

The most important issue regarding natural pigment is the price of final product which is more expensive than cheap synthesized dye. In this research possibility of using cheap growth media (agricultural wastes) such as Solid Pineapple Waste (SPW) and Brown Sugar (BS) which leads to inexpensive and competitive product, have been studied.

1.2 Objectives and Scope of Study

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1.3 Significance of Study This study aims at introducing bacterial pigments as an alternative to synthetic dye. In this study cheap medium were employed for bacterial growth and the simplest method for bacterial pigment extraction was developed to overcome the higher price of natural pigments compare to synthesized dye.


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2.1 Pigment Pigment is defined as the coloring agent in substances which can be produced either by living organisms or chemical reagents. The history of pigment application dates back to prehistoric cave painting, which gives evidence of the use of ocher, hematite, brown iron ore and other mineral-based pigments more than 30,000 years ago (Daniel, 1986).

It is certain that the art of using plant and animal pigments to extend the spectral range of available inorganic colorants by a selection of more brilliant shades had been practiced thousands of years ago.

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2.1.1 Natural Pigment Biological pigments, known simply as pigments or biochromes are substances produced by living organisms that have a color resulting from selective color absorption. Biological pigment includes plant pigments and animal pigments.

Many biological structures, such as skin, eyes, fur and hair contain pigments such as melanin in specialized cells called chromatophores (Ball, 2002). Pigments in Plants

Among the most important molecules for plant function are pigments. In plants the major pigments are the carotenes (reddish orange to yellow), the anthocyanins (red, blue, and violet), and the chlorophylls (green). The red and yellow colors of autumn foliage are due to the exposure of the anthocyanins after the green chlorophyll pigments, which usually mask them, have decomposed and faded. All biological pigments selectively absorb certain wavelengths of light while reflecting others. The light that is absorbed may be used by the plant to power chemical reactions, while the reflected wavelengths of light determine the color the pigment will appear to the eye. Pigments also serve to attract pollinators.

Chlorophyll is the primary pigment in plants; it is a porphyrin that absorbs yellow and blue wavelengths of light while reflecting green. It is the presence and relative abundance of chlorophyll that gives plants their green color. All land plants and green algae possess two forms of this pigment: chlorophyll a and chlorophyll b.

Kelps, diatoms, and other photosynthetic heterokonts contain chlorophyll c instead of b, while red algae possess only chlorophyll a. All chlorophylls serve as the primary means plants use to intercept light in order to fuel photosynthesis and the reason why most plants are green (Goodwin, 2002).

7 Carotenoids are red, orange, or yellow tetraterpenoids. They function as accessory pigments in plants, helping to fuel photosynthesis by gathering wavelengths of light not readily absorbed by chlorophyll. The most familiar carotenoids are carotene (an orange pigment found in carrots), lutein (a yellow pigment found in fruits and vegetables), and lycopene (the red pigment responsible for the color of tomatoes). Carotenoids have been shown to act as antioxidants and to promote healthy eyesight in humans (Ball, 2002).

Anthocyanins are water-soluble flavonoid pigments that appear red to blue, according to pH. They occur in all tissues of higher plants, providing color in leaves, stems, roots, flowers, and fruits, though not always in sufficient quantities to be noticeable. Anthocyanins are most visible in the petals of flowers, where they may make up as much as 30% of the dry weight of the tissue. They are also responsible for the purple color seen on the underside of tropical shade plants such as Tradescantia zebrina; in these plants, the anthocyanin catches light that has passed through the leaf and reflects it back towards regions bearing chlorophyll, in order to maximize the use of available light (Goodwin, 2002).

Betalains are red or yellow pigments. Like anthocyanins they are watersoluble, but unlike anthocyanins they are indole-derived compounds synthesized from tyrosine. This class of pigments is found only in the Caryophyllales (including cactus and amaranth), and never co-occur in plants with anthocyanins. Betalains are responsible for the deep red color of beets, and are used commercially as foodcoloring agents (Daniel, 1986).

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