EXTRACTION AND CHARACTERIZATION OF PURPLE PIGMENT
FROM Chromobacterium violaceum GROWN IN
AGRICULTURAL WASTES
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 Science (Chemistry)
Faculty of Science
Universiti Teknologi Malaysia
APRIL 2010
EXTRACTION AND CHARACTERIZATION OF PURPLE PIGMENT FROM
Chromobacterium violaceum GROWN IN
AGRICULTURAL WASTES
AKRAM NESHATI
iii
To my Beloved Mother and Father
iv
ACKNOWLEDGEMENT
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.
v
ABSTRACT
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 OH stretching,
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.
vi
ABSTRAK
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.
vii
TABLE OF CONTENTS
CHAPTER
TITLE
PAGE
TITLE OF THESIS
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENT
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xi
LIST OF FIGURES
xii
LIST OF ABBREVIATIONS
xv
LIST OF APPENDICES
xvi
1 INTRODUCTION
1.1
Background of Problem
1
1.2
Statement of the Problem
3
1.4
Objectives and Scope of Study
3
1.5
Significance of study
4
2 LITERATURE REVIEW
2.1
2.2
2.3
Pigment
5
2.1.1 Natural Pigment
6
2.1.1.1 Pigments in Plants
6
2.1.1.2 Pigments in Animals
10
2.1.1.3 Bacterial Pigments
11
2.1.2 Synthetic Pigments
13
2.1.2.1 Azo Dyes
13
2.1.2.2 Indigoid
14
2.1.2.3 Azobenzene
15
2.1.2.4 Phthalocyanine
15
Chromobacterium violaceum
16
2.2.1 Application of Violacein
20
Violacein Production
21
2.3.1
Growth Profile
23
2.4
Violacein Extraction
24
2.5
Violacein Characterization
24
2.5.1 UV-VIS
25
2.5.2 FTIR
25
2.5.3 NMR
26
3 EXPERIMENTAL
3.1
Materials
27
3.1.1 Bacteria
27
3.1.2 Growth Media
28
3.1.2.1 Nutrient Broth
28
3.1.2.2 Nutrient Agar
28
3.1.2.3 Solid Pineapple Waste (SPW)
28
3.1.2.4 Brown Sugar (BS) Stock Solution
29
ix
3.1.3
3.2
Tryptophan solution
29
3.1.4 Active Culture
29
Methods
30
3.2.1
Growth of Chromobacterium violaceum
30
3.2.1.1 Cultivation of C .violaceum in NB at different
30
temperatures
3.2.1.2 Cultivation of C .violaceum on SPW at different
30
temperatures
3.2.1.2.1 Effect of tryptophan on Growth of
31
C. violaceum
3.2.1.3 Cultivation of C .violaceum in BS at different
33
temperatures
3.2.2
Growth Profile of C. violaceum
33
3.2.3
Extraction of Violet Pigment
34
3.2.3.1 Extraction of Violet Pigment from SPW
34
3.2.3.2 Extraction Preliminary Purification of Violet
34
Pigment from BS
3.2.4 Characterization of Violacein
3.2.4.1 Characterization of Violacein using UV-VIS
36
3.2.4.2 Characterization of Violacein using FTIR
36
3.2.4.2
Characterization of Violacein using 1H-NMR and
13
3.3
36
36
C-NMR
3.2.5
Stability Test of Pigment Towards pH Changes
37
3.2.6
Column Chromatography Preparation
37
Bacterial Preservation
37
4 RESULTS AND DISCUSSION
4.1
Effect of Growth Parameters on Chromobacterium violaceum
39
4.1.1 Effect of Temperature on growth of C. violaceum in NB
39
4.1.2 Effect of Temperature on growth of C. violaceum in SPW
40
4.1.3 Effect of Temperature on growth of C. violaceum in BS
42
4.1.4 Effect of Time on Growth of C. violaceum in NB and BS
44
x
4.1.5
4.2
4.3
Bacterial Preservation
46
Characterization of Violet Pigment
47
4.2.1
Characterization of Violet Pigment from SPW
47
4.2.2
Characterization of Violet Pigment from BS
48
4.2.2.1
49
Column Chromatography
4.2.2.2 UV-VIS Spectrophotometer Analysis of violacein
50
4.2.2.3
FTIR Spectroscopic Analysis
51
4.2.2.4
NMR analysis of violacein
53
pH Test
58
5 CONCLUSION
5.1
Conclusion
59
5.2
Future Study
60
REFERENCES
61
APPENDIX
66
xi
LIST OF TABLES
TABLE NO.
TITLE
PAGE
2.1
Examples of different bacteria and their pigment.
12
2.2
Application of violacein
20
2.3
Possible growth media for production of C .violaceum.
22
3.1
Sample preparation with addition of DL-tryptophan
31
3.2
Preparation of samples in the presence of L-tryptophan
32
3.3
Preparation of controls for SPW culture in the presence
32
of L-tryptophan
4.1
λmax of violacein using UV-VIS spectrophotometer
50
4.2
Data of FTIR (Gregor and Wolfgang, 2001; Lara et
51
al, 2005)
4.3
13
C-NMR peaks of violacein and its related carbons
54
(Gregor and Will, 2001; Ruhul and Tsutomu, 1999)
4.4
Collected data from 1H-NMR in comparison with
reference (Yoshitoshi et al, 2002; Hartmut and
Ronald, 1984)
56
xii
LIST OF FIGURES
FIGURE NO
TITLE
PAGE
2.1
Crocin C44H64O26 (mw: 1,008.97)
8
2.2
Crocetin C20H24O4 (mw : 328.41)
8
2.3
Molecular structure of melanoidin (C27H31N4O31)
9
2.4
Structure of Azo dye
13
2.5
Structure of Indigoid dye
14
2.6
The general structure of Azobenzene dyes
15
2.7
Chemical structure of Phthalocyanine
15
2.8
Purple colonies of Chromobacterium violaceum
16
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)
2.10
3D structure of violacein
17
2.11
Conversion of two modified tryptophan molecule into one
18
violacein molecule.
2.12
Structures of Violacein 1 and Deoxyviolacein 2. The left
side
denotes
5-hydroxyindole
and
half
of
19
the
2-pyrrolidone ring.
2.13
A typical bacterial growth profile
23
xiii
3.1
Two separated phases during extraction of violacein with
34
ethyl acetate
3.2
C. violaceum preserved on agar slant in the presence of
38
paraffin oil
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
4.3
Effect of incubation period on growth of C. violaceum
41
4.4
Plate assay for checking growth of C. violaceum in the
41
mixture of SPW and BS.
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.
4.7
Growth profile of C. violaceum in NB and BS
44
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
4.10
Pigment extracted from SPW
47
4.11
UV-VIS spectrum of violacein obtained from SPW at 25
47
and 30°C
4.12
TLC result of violacein extracted with 1) methanol and 2)
48
ethyl acetate
4.13
Brownish fraction of melanoidin, pigment from BS
49
4.14
TLC
column
49
UV-VIS Absorption spectrum of violacein extracted from
50
results
of
violacein
fractions
from
chromatography
4.15
C. violaceum grown in BS showed a linear increase in
xiv
response to maximum centered at 567 nm
4.16
FTIR spectrum obtained from KBr pellet of violacein
52
from BS
4.17
13
C NMR spectrum of preliminary purified violacein
53
running by DMSO and adequate amount of CDCl3
4.18
1
H-NMR spectrum of preliminary purified violacein from
55
BS
4.19
Violacein at pH 5.85
57
4.20
Colors resulting from pH test on violacein in extreme
57
acidic and alkaline pH
xv
LIST OF ABREVIATIONS
IR
Infrared
ml
Milliliter
NMR
Nuclear Magnetic Resonance
ppm
part per million
s
singlet
d
doublet
TLC
Thin Layer Chromatography
xvi
TABLE OF APPENDIX
APENDIX
1
TITLE
UV-VIS spectrum of samples from growth media with
PAGE
66
different concentrations (1-10)
2
UV-VIS spectrum of samples from growth media with
67
different concentrations (11-20)
3
FTIR spectrum obtained from KBr pellet of violacein
from NB
68
CHAPTER 1
INTRODUCTION
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).
3
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
The objective of this study is to extract the purple pigment, violacein, from
Chromobacterium violaceum which was grown on SPW and BS. The
characterization of the pigment was made using UV-VIS spectrophotometer, IR, and
NMR.
4
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.
CHAPTER 2
LITERATURE REVIEW
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.
The most satisfactory way to classify pigment is according to its source,
because most of the significant properties which any pigment groups may have in
common can be attributed to their composition. They are divided into two major
groups which are natural (biological pigments) and synthetic pigments. Synthetic
pigment is divided into two main groups which are known as organic and inorganic
pigments (Daniel, 1986).
6
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).
2.1.1.1
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).
Saffron, known also as CI natural yellow 6, safran, crocin, crocetin, and
crous, is the dried stigma of Crocus sativus, a plant indigenous to the orient but also
grown in North Africa, Spain, Iran and France. It is a reddish, brown or golden
yellow odoriferous powder with a slightly bitter taste. The stigma of approximately
165,000 blossoms is required to make 1 kg of colorant (Daniel, 1983). The coloring
principles of saffron are crocin (Figure 2.1) and crocetin (Figure 2.2).
8
Figure 2.1: Crocin C44H64O26 (MW: 1,008.97)
Figure 2.2: Crocetin C20H24O4 (MW: 328.41)
Crocin is a yellow-orange glycoside that is freely soluble in hot water,
slightly soluble in absolute alcohol, glycerine, and propylene glycol and insoluble in
vegetable oils. Crocetin is a dicarboxylic acid that forms brick-red rhombs from
acetic anhydride that melts with decomposition at about 285°C. It is very sparingly
soluble in water and most organic solvents (Daniel, 1986).
As a food colorant, saffron shows good overall performance. In general it is
stable toward light, oxidation, microbiological attack, and changes in pH. Its
tinctorial strength is relatively high, resulting in use levels of 1-260 ppm (Daniel,
1986).
Daily, human being ingests melanoidins (Figure 2.3) from brown processed
food. Nutritional and physiological effects of melanoidins have been widely
investigated. When rats were fed nondialyzable melanoidins prepared from Dglucose and glycine, the melanoidin was difficult to excrete and was partly
transformed into metabolizable compound (Hiromichi and Fumitaka, 2002).
9
When D-xylose and glycine were reacted at low temperature (2-26.5°C), the
reaction mixture produced yellow, red and blue pigment. The isolated blue pigment
reveals its novel chemical structure as shown in Figure 2.3. Blue melanoidin has two
pyrrolopyrrole ring coupled with Methane Bridge. The UV-VIS spectrum of blue
melanoidin shows a large peak at 625 nm and a small peak at 283, 322 and 365 nm.
COOH
COOH
CH2
CH2
NH
N
HOHC
C
CH2OH
CHOH
HC
N
CHOH
CH2
CH2OH
COOH
Figure 2.3: Molecular structure of melanoidin (C27H31N4O31)
10
2.1.1.2
Pigments in Animals
The infinite variety of animal colors certainly suggests that coloration plays a
significant role in the life of animals. Humans use animal colors as a way of
differentiating one species from another, and this also happens among the animals
themselves. In closely related species, coloration may be the initial signal for species
identification (Martha, 2002).
Color also provides a way for animals to determine the sex of another
individual. In Ruby-throated Hummingbirds, for example, only the adult male has
throat feathers that form a red gorget; females and young males have a white throat.
When a territorial male ruby-throat encounters another hummer, he can quickly
determine if the intruder is an adult male that he needs to chase away, or a female
that he might like to woo (Martha, 2002).
A third function of animal coloration is evident in the juvenile stage, or eft
(small lizard), of the Red-spotted Newt (a common South Carolina salamander). The
juvenile's skin is fire-orange in color, with two rows of small red dots down its back.
In its eft stage, this newt wanders the forest floor pursuing earthworms; its striking
color is a warning to predators that its skin is loaded with toxins. As an adult, the
newt retains its spots but the rest of its skin becomes dark green just at the time when
it returns to the safe haven of a small pond to mate and live out the rest of its days as
an aquatic organism (Martha and Daniel. 2003).
Pigmentation is used by many animals for protection, by means of
camouflage, mimicry, or warning coloration. Chameleons use pigments to blend into
their surroundings by controlling the absorption levels of the electromagnetic
spectrum (Ball, 2002).
11
2.1.1.3
Bacterial Pigments
If someone leaves a plate of nutrient agar exposed to the air for about 30 min,
or makes a spread plate of an appropriate dilution of river water, and incubates at
25°C for a few days, a number of colored colonies of bacteria will usually appear
(Austin and Moss, 1986).
In general, bacteria contain many pigments that are similar, if not identical, to
those of more complex organisms, particularly plants. Bacterial chlorophylls differ
from plant chlorophylls in the reduction of one double bond (Chapman and Hall,
1996).
The yellow and pink colonies from the air exposed plate will usually be
Gram-positive micrococci whereas the much wider range of colors from the river
water will often be Gram-negative rods such as Flavobacterium, Cytophaga,
chromobacteria, Serratia and pseudomonads (Logan, 1994).
Perhaps the most familiar examples of colored 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
Pseodomonas fluorescens and related species. An example of a water soluble, nonfluorescent blue-green pigment produced by Pseodomonas aeruginosa is pyocyanin
which crystallises as beautiful blue needles and may have a role in respiration
(Austin and Moss, 1986).
One group of pigments apparently confined to bacteria is the phenazines
based on dibenzopyrazine skeleton. Among these often intensely colored
compounds, are the purple iodinin from species of Chromobacterium and the dark
blue (in acid solution) pyocyanine isolated from Pseudomonas aeruginosa. Many of
the several dozen phenazines so far described have potential commercial intrest
particularly as antibiotics (Chapman and hall. 1996).
12
Pigment can be produced either as primary or secondary metabolites of
bacterial growth. Primary metabolite is the one which forms pigment during the
growth phase of the microorganism. The production of pigments is not significant
and pigments are not essential for growth and reproduction of bacteria because cell
still can maintain normal growth rate after all the pigment have been removed (Nur
Zulaikha, 2006).
Numerous microorganisms synthesize small molecular weight compounds
that have no verified function in the cell. Maximum production generally occurs after
cellular multiplication has finished. Since the substances are not required to the
primary metabolism of cellular growth and multiplication, they are called secondary
metabolites. Secondary metabolites are the ones forming near the end of the growth
phase, frequently at or near the stationary phase (Nur Zulaikha, 2006).
Bacterial pigments can be extracted from the bacterial cells and be used in
industries as drug or dye etc. Table 2.1 shows some examples of pigmented bacteria
and the pigment they produce:
Table 2.1: Examples of different bacteria and their pigment.
Name
Pigment color
Rugamonas ruba
Green
Chromobacterium violacein
Violet
Serratia marcescens
Red
Image
13
2.1.2
Synthetic pigments
A huge number of dyes have been synthesized and used mainly for dying
textiles. According to their chemical structure they are generally classified into six
classes : Azo, indigoid, anthracene, azobenzene, phtalocyanine, triphenylmethan
(trityl).
However the structural characteristic of dye sometimes overlaps, uniting in
the molecule more than one structural element, making impossible the unambiguous
classification. Besides their use in textile industry, various dyes have found
application in a wide variety of other fields of up-to-date research and industrial
activity (Heinrich, 2003).
2.1.2.1
Azo dyes
Azo colors (Figure 2.4) comprise the largest group of certified colorants. The
compounds bear the functional group R-N=N-R', in which R and R' can be either aryl
or alkyl. The N=N group is called an azo group, although the parent compound,
HNNH, is called diimide. The more stable derivatives contain two aryl groups. The
name azo comes from azote, the French name of nitrogen that is derived from the
Greek a (not) zoe (to live) (Daniel, 1986).
Figure 2.4 : Structure of Azo dye
14
Azo pigments are important in a variety of paints including artist's paints.
They have excellent coloring properties, again mainly in the yellow to red range, as
well as lightfastness. The lightfastness depends not only on the properties of the
organic azo compound, but also on the way they have been adsorbed on the pigment
carrier. Many azo pigments are non-toxic (Heinrich, 2003).
2.1.2.2
Indigoid
A series of water-soluble sulfonated indoxyl derivatives have been prepared,
including their base salts with pharmacologically acceptable cations. These particular
compounds are useful as food dyes or as cosmetic colorants.
Figure
2.5
(2,2'-(1,4-Phenylenedimethylidyne)-bis(2,3-dihydro-3-oxo-1H-
indole-5-sulfonic acid)) represents a typical and preferred member compound.
Methods for preparing these compounds are provided.
O
H
N
N
H
O
Figure 2.5: Structure of Indigoid dye
15
2.1.2.3
Azobenzene
Azobenzene compound (Figure 2.6) is an important and valuable multifunctional dye with pure chromophoric properties, high molar extinction coefficient,
and fine staining qualities.
Figure 2.6: The general structure of Azobenzene dyes
2.1.2.4
Phthalocyanine
Phtalocyanine or copper phthalocyanine is a blue synthesized pigment with
the following chemical structure (Figure 2.7).
Figure 2.7: Chemical structure of Phthalocyanine
16
2.2
Chromobacterium violaceum
Chromobacterium violaceum (C. violaceum), (Figure 2.8), a bacteria
belonging to the Rhizobiaceae (Soilborne Phytopathogen) family is found in soil and
water in tropical and subtropical areas (Natalia and Nelson, 2001). Chromobacterium
violaceum is a Gram negative, facultatively anaerobic, rod-shaped bacterium that is
generally considered being non-pathogenic (Rettori and Duran, 1998).
Figure 2.8: Purple colonies of Chromobacterium violaceum
Its colonies are lightly convex, not gelatinous, regular and violet, although
irregular and non-pigmented colonies can also be found (in anaerobic conditions as
violacein is produced only in the presence of oxygen (Marlon et al., 2006).
As in all chemoheterotrophic bacteria, C. violaceum is able to grow in
minimal medium that includes simple sugars, such as glucose, fructose, galactose, or
ribose. However, C. violaceum is not able to synthesize glucose through
gluconogenesis, since, based on genome analysis, it lacks the gene that codes for
glucose-6-phosphatase (Tania and Regina, 2004).
Violacein (Figure 2.9 and Figure 2.10), the main pigment produced by
Chromobacterium violaceum, is a bactericide, trypanocide, tumoricide pigment and
in addition it has anti-viral activities (DeMoss and Happle, 1958).
17
Violacein consist of three structural units: 5-hydroxyindol, 2-oxoindol, and 2pyrolidone which are poorly water soluble and during formation rapidly precipitates
either as discrete particles or on cells or cell clumps (DeMoss and Happle, 1958).
Figure 2.9: General structure of violacein (3-(1,2-dihydro-5-(5-hydroxy-1H-indol-3yl)-2-oxo-3H-pyrrol-3-ilydene)-1.3-dihydro-2H-indol-2-one)
Figure 2.10: 3D structure of violacein
Experiments have been carried out by researchers to find the significant
carbon source for production of violacein. Labeled substrates such as: Succinate,
Ribose, Glucose, DL-alanine and L-tryptophan have been tested for this purpose
(DeMoss and Evans, 1959).
It is apparent from the collected data that of the several substrates tested;
tryptophan was the only significant added source of pigment carbon.
18
The low 14C dilution observed with tryptophan-2- 14C suggests that at least a
protein of the tryptophan side chain enters pigment directly and without dilution
from other carbon sources. Since alanine, lactate and acetate do not contribute either
directly or indirectly to pigment synthesis, it is probable that the tryptophan
molecule, with the possible carboxyl carbon, is incorporated intact into pigment
(DeMoss and Evans, 1959).
It is clear that the carboxyl carbon of tryptophan is eliminated during pigment
synthesis, and it is quite probable that all other carbon atom of the tryptophan
molecule is incorporated as a unit (Figure 2.11). These results may be expected from
a consideration of the pigment’s structure, although no consideration can be formed
concerning the synthetic pathway (DeMoss and Evans, 1959).
Figure 2.11: Conversion of two modified tryptophan molecule into one violacein
molecule.
Ruhul and Tsutomu have shown that the production of violacein yielded a
minor product which is Deoxyviolacein that has the same structure as violacein with
just one less OH group in 6th carbon of the left side ring (Figure 2.12).
19
Figure 2.12: Structures of Violacein 1 and Deoxyviolacein 2. The left side denotes
5-hydroxyindole and half of the 2-pyrrolidone ring.
20
2.2.1
Application of Violacein
Violacein has attracted much attention in literatures lately due to its broad
applications in various industries such as pharmaceutical industries. Some activities
of violacein are as follows:
Table 2.2: Application of violacein
NO
APPLICATION
REFERENCE
1
Antioxidant activity
(Lauro et al., 2000)
2
Antimalarial activity
(Stefanie et al, 2009)
Antibacterial, trypanocide,
3
antimycobacterial, tumoricide, anti-viral and
(Bromberg and Duran, 2001)
cytoxic activities
4
Quorum sensing
(Yi et al, 2008)
Antibiotic activity against Staphylococuos
5
aureus, Neisseria meningitides,
(John et al, 1991)
Streptococcus spp and Bacillus
6
7
Antioxidant and genotoxic Activities
(Marlon et al., 2006)
Quantitative bioassay for N-acyl homoserine
(Renee and Kendall, 2000)
lactone autoinducers
Pigmentation has been utilized in problems
8
of taxonomy as a secondary or even as a
(DeMoss and Happel, 1958)
primary factor in identification
9
10
Used as dye not only for natural fibers but
also for synthetic fibers
Pigment production can help identify
Chromobacterium violaceum
Violacein (with10 percent of
11
deoxyviolacein) showed activity against
herpes and polioviruses
(Brumberg and Duran, 2001)
(Inniss and Mayfield, 1979)
(Antonisamy and Ignacimuthu,
2009)
21
2.3
Violacein Production
Due to the vast applications of violacein, researches have been working on
growth of C. violaceum in order to ease the process and enhance production of
pigment. It is well known that C. violaceum is a very selective bacterium in terms of
conditions of growth (Common growth culture for C. violaceum is Nutrient broth and
Nutrient agar). It has been reported that the optimum growth conditions should be
obtained to achieve the desired product.
Growth of bacteria using soil extract agar (SEA) was reported by Innis and
Mayfield at 20°C and 0°C. The results showed that the colonies which developed at
20°C were totally pigmented where as colonies grown at 0°C were non-pigmented.
When colonies grown at 0°C were incubated at 20°C pigments started to appear
indicating optimum temperature for pigment production was at 20°C (Inniss and
Mayfield, 1979).
The same experiment was carried out with using soil extract broth (SEB), at
0°C, 15°C, 20°C, 25°C. Growth was measured spectrophotometrically at 650 nm.
Since results show that violacein production was lacking in SEB at 0°C, experiment
were performed in which various concentration of tryptophan, a known precursor of
violacein, were added to SEB in flasks and SEA in plates, growth and pigment
production determined. The optimum and suitable pH for this experiment reported
around 7.4 where the temperature of medium was 20°C (Inniss and Mayfield, 1979).
22
A summary of the growth media used for production of violacein is shown in
Table 2.3.
Table 2.3: Possible growth media for production of C .violaceum.
Growth Medium
Liquid medium consist of D-glucose,
peptone, yeast extract
Temp.
30°C
Reference
(Rettori and Duran, 1998)
Lactose broth
-
(Walter, 1934)
Wakimoto medium
-
(Yuang, 2008)
Nutrient broth, Nutrient agar
25°C
Soil Extract Agar (SEA), Soil
15, 20,
Extract broth (SEB)
TY medium (containes: tryptone,
yeast extract and NaCl)
RPMI1640 medium containing
25°C
30°C
(Haisheng et al, 2008)
(Iniss and Mayfield, 1979)
(Rene´e and Kendall,
2000)
-
(Carmen et al, 2004)
Terrific Broth
37°C
(Marlon et al, 2006)
Nutrient Agar
30°C
(DeMoss and Happel, 1958)
Luria broth
28°C
(Leonid et al, 1998)
glutamine and antibiotics.
23
2.3.1
Growth Profile
A spectrophotometer is an instrument that measures the amount of light that
is able to pass through a bacterial culture. It shines a constant beam of light on the
sample that is being tested. If the light hits the bacterial cell, then it will bend and
bounce off the cell.
The more cloudy a culture is, the more bacterial cells are present within the
culture allowing less light to penetrate through and more light is bounced back to the
register within the spectrophotometer. This is the instrument used for measuring
bacterial production which aims to draw the growth profile of bacteria. Growth
profile consists of four phases (Figure 2.13):
Figure 2.13: A typical bacterial growth profile.
Lag phase is an adjustment period when the bacteria are switching on or off
different machinery necessary to break down the energy source within the immediate
environment. Log phase is the rapid growth of bacteria at an accelerated pace.
Stationary phase is when rate of growth and death is equal so that overall
bacterial numbers stay the same. Finally, death phase is rapid cell death that leads to
cells bursting open, also known as cell lysis.
24
2.4
Violacein Extraction
Natalia and Nelson, (2001), extracted the violacein from reaction mixture
with ethyl acetate and evaporated the solvent under reduced pressure while Walter,
(1934), filtered out the pigment, dried it and extracted it with alcohol.
Centrifugation is another possible way to extract the pigment as Yuan et al
(2008) centrifuged the cells first at 8000 g for 5 minutes and the supernatant was
discarded. The cell pallets were then rinsed with deionized water, followed by
centrifugation to recover the cells by discharging the supernatant again. The
recovered cells were fully mixed with ethanol. The mixture of the cells and ethanol
was treated by ultra-sonication until the cells were completely bleached. With this
protocol, it was confirmed that there was no residual pigment in the cell pellets after
the extraction.
2.5
Violacein Characterization
The most widely used and in general the most conclusive procedure for
identifying pigments is instrumental in nature. Characterization of pigment, such as
violacein, can be carried out by several methods such as UV/Vis, FTIR, NMR, MS,
and HPLC.
25
2.5.1 UV-VIS
UV and visible spectrometry are usually the simplest analysis method to
perform and require the least amount of sample, often as little as 0.1 mg. Where
possible, spectra of the unknown compound should be compared with those of
known compound in several solvents. UV-VIS spectrometry is fast, reliable and
relatively simple procedures for identifying dyestuffs and should be used whenever
possible. Their use required only a modest amount of training whereas the necessary
equipment is moderate to expensive in price (Daniel, 1986).
2.5.2 FTIR
For pigment analysis, FTIR is considered as a complementary technique that
can provide the molecular and structural information of organic and inorganic
materials (Douglas et al, 2003).
Solid samples can be prepared for IR examination by making KBr pellet
which is prepared by grinding the solid sample with solid potassium bromide (KBr)
and applying great pressure to the dry mixture. Again, KBr is chosen because it is
transparent to infrared radiation. If the pellet is prepared properly, one can actually
see through it, as through a pane of glass (Douglas et al, 2003).
26
2.5.3 NMR
Recently proton nuclear magnetic resonance (NMR) has been used to identify
both primary and secondary pigments. Good spectra of the certified water-soluble
colors have already been obtained and published using a mixed, deuterated solvent
(water : dimethylsulfoxide; D2O:DMSO-d6, 2:1v/v) at 100-105°C. NMR is one of the
least sensitive, most complicated of the spectral techniques in use today but it is an
excellent tool for identification purposes also for studying the structure of organic
compounds (Daniel, 1986).
Much like using infrared spectroscopy (IR) to identify functional groups,
analysis of a NMR spectrum provides information on the number and type of
chemical entities in a molecule.
CHAPTER 3
EXPERIMENTAL
3.1
Materials
All the glassware used during this experiment were washed with distilled
water and sealed and sterilized by autoclaving at 121°C for 15 minutes (HVE-50,
Hirayama). All reagents and chemicals were of analytical grade.
3.1.1
Bacteria
The bacteria used in this project were isolated from the soil of an oil refinery
company in Port Dickson, Seremban, Malaysia.
28
3.1.2
Growth Media
3.1.2.1 Nutrient Broth
Nutrient broth (NB) was used as a liquid growth medium for preparing active
culture. NB powder (8 g) (MERCK, Germany) was dissolved in 1 litter of distilled
water and sterilized by autoclaving at 121°C for 15 minutes.
3.1.2.2 Nutrient Agar (NA)
Nutrient agar (NA) was prepared by dissolving 20 g of nutrient agar
(MERCK, Germany) powder in 1 litter of distilled water. The medium was sterilized
by autoclaving at 121°C for 15 minutes. The molten agar was cooled to about 50°C
before being poured into sterile Petri dishes. The agar was allowed to harden and
then incubated for 24 hours at 30°C to ensure that the medium was free from
contamination.
3.1.2.3 Solid Pineapple Waste (SPW)
The pineapple waste was obtained from the downstream process at Lee
Pineapple Manufacturing Industry, Tampoi.
29
3.1.2.4 Brown Sugar (BS) Stock Solution
To prepare the Brown Sugar (BS) stock solution, 40 g BS was dissolved in 1
L distilled water. The solution was heated, stirred and filtered using filter paper
(Advantec, Japan) 125 mm to remove insoluble particles. pH of solution was set at 8
using 0.1 M NaOH before autoclaving at 105°C for 15 minutes.
3.1.3 Tryptophan Solution
Solutions of DL-tryptophan and L-tryptophan were prepared by dissolving
0.1 g of the amino acid in 100 mL of distilled water. The solution was sterilized
using 0.45 µm cellulose acetate membrane.
3.1.4
Active Culture
Active culture of Chromobacterum violaceum was prepared by incubating a
loopful of bacterial cell from NA plate or agar slant into 25 mL of NB. The culture
was incubated at 25°C with agitation at 200 rpm using an orbital shaker (Certomat
®R, B. Braun) for 12 hours.
30
3.2
Methods
3.2.1
Growth of Chromobacterium violaceum
3.2.1.1 Cultivation of C . violaceum in Nutrient Broth at Different Temperatures
NB (22.5 mL) was poured into a sterile 250 mL conical flask under laminar
flow followed by the addition of 2.5 mL of active culture. The mixture was shaken
for 24 hours at 200 rpm in an orbital shaker at room temperature. Similar flask were
prepared but grown at 30°C and 37°C.
3.2.1.2 Cultivation of C. violaceum on SPW at Different Temperatures
SPW (10 g) was added to 22.5 mL of distilled water. Ethanol (1.25 mL) (5%
of total volume) was added to the mixture in order to kill other available
microorganisms. pH of the mixture was adjusted to 7 with the addition of adequate
amount of 1M NaOH.
The mixture was then transferred to sterilized 250 mL flask under laminar
flow followed by the addition of 2.5 mL of active culture. The mixture was shaken
for 24 hours at 200 rpm in an orbital shaker at room temperature. Similar flasks were
prepared but grown at 30°C and 37°C.
For large scale production, 40 g of SPW was added to 90 mL distilled water
plus 10 mL of active culture and 5 mL ethanol (pH adjusted to 7 prior to cultivation)
followed by incubation at 30°C, 37°C at room temperature.
31
Addition of BS solution, DL-tryptophan, L-tryptophan as a supplement of
growth was also studied in this project. Prepared BS solution added to the flask
containing 5 g SPW in different ratio as follow: 1mL, 5 mL, and 10 mL. A control
solution was prepared with addition of 2.5 mL active culture to 22.5 mL BS. Growth
of bacteria was observed only in the control solution.
3.2.1.2.1 Effect of Tryptophan on Growth of C. violaceum
Study of effects of tryptophan initiated by addition of DL-tryptophan solution
into seven 250 mL flask containing 10 g SPW, 2.5 mL active culture and 1.25 mL
ethanol. Distilled water was added to each flask and the pH adjusted to 7. The
amount of DL-tryptophan added is as shown in Table 3.1. (The control flask contains
22.5 mL DL-Tryptophan solution and 2.5 mL active culture).
Table 3.1: Sample preparation with addition of DL-tryptophan
Sample
DL-tryptophan
Distilled water
1
1 mL
21.5 mL
2
2 mL
20.5 mL
3
3 mL
19.5 mL
4
5 mL
17.5 mL
5
7 mL
15.5 mL
6
10 mL
12.5 mL
32
Growth of C. violaceum was also studied in the presence of L-tryptophan.
Table 3.2 shows sample’s ingredients while that of the control is shown in Table 3.3.
Table 3.2: Preparation of samples in the presence of L-tryptophan
SPW
L-tryptophan
Ethanol
Active Culture
5g
22.5 mL (10 pH)
1.25 mL
2.5 mL
5 g (pH 7)
22.5 mL
1.25 mL
2.5 mL
Table 3.3: Preparation of controls for SPW culture in the presence of L-tryptophan
SPW L-tryptophan
Ethanol
Active Culture
-
22.5 mL
-
2.5 mL
5g
-
1.25 mL
-
5g
-
1.25 mL
2.5 mL
Comment
Control of Ltryptophan
Study presence of
other bacteria
pH set at 7
33
3.2.1.3 Effect of Temperature on Growth of C. violaceum
The effect of temperature on growth of C. violaceum was also studied
samples were prepared by addition of 22.5 mL of BS solution and 2.5 mL of active
culture into three flasks. The mixture was shaken at 200 rpm for 24 hours at 3
different temperatures (25°C, 30°C and 37°C).
After 24 hours of incubation, pigments were extracted from the samples and
UV-VIS analyses of the samples were taken.
3.2.2
Growth Profile of C. violaceum Grown in NB and BS
Growth of C. violaceum was also monitored in NB and BS. Active culture of
C. violaceum (30 mL) was inoculated into NB (300 mL) and BS (300 mL) in
Erlenmeyer flask. Each of the samples were complemented with a cell-free control
set. The culture and control were shaken at 200 rpm and 25°C for 24 hours. At
regular intervals, the turbidity of the culture was determined using a
spectrophotometer (Spectronic 21D, Milton Roy) at 600 nm. The growth profile was
obtained by plotting OD600 versus time.
34
3.2.3
Extraction of Violet Pigment
3.2.3.1 Extraction of Violet Pigment from SPW
To extract violacein from SPW, firstly, the mixture was filtered with a filter
paper (0.45 µm). After filtration, the filter paper was rinsed with distilled water
several times. The filtered pineapple pieces were placed into a clean conical flask and
20 mL of methanol added to it. The mixture was left to shake for 15 minutes at 200
rpm to leach the pigments into solution.
Pigment was extracted using ethyl acetate (Figure 3.1) (MeMoss and Evans,
1959). Supernatant (30 mL) was poured into a separatory funnel and 10 mL ethyl
acetate added to it. The mixture was then shaken and after releasing its gas the funnel
was placed into the holder in order to separate the organic and aqueous phases. The
organic layer which contains the pigments was kept in a sealed container.
Figure 3.1: Two separated phases during extraction of violacein with ethyl acetate.
35
3.2.3.2 Extraction and Preliminary Purification of Violet Pigment from BS
Two methods were employed to extract the violet pigment from BS samples.
The first method is as stated in 3.2.3.1. The first step was filtering the sample and
then rinsing it with distilled water. Precipitated pigment on the filter paper was then
washed with methanol till the filter paper becomes white in color. The pigment was
then extracted using ethyl acetate as described in 3.2.3.1.
To check for purity of the sample, the ethyl acetate and methanolic fractions
were spotted on a TLC plate and developed using n-hexane : ethyl acetate (4:6)
solvent system.
The second method is as follows; sample (30 mL) was poured into a
separatory funnel and added with 6 mL of ethyl acetate (5:1). The funnel was shaken
to allow separation of the organic and aqueous phase. The organic layer was poured
into a plate and left in the fume cupboard to evaporate the solvent.
After complete evaporation the violacein was dried in the oven for 1 hour at
50°C. The dried pigment was scrapped from the plate and placed into a dry clean
bottle and sent for
13
C-NMR and 1H-NMR analysis. Another set of sample was
extracted by the same method, but before the evaporation stage, sample was filtered
using filter paper (0.45 µm).
36
3.2.4
Characterization of Violacein
3.2.4.1 Characterization of Violacein using UV-VIS
UV-VIS analysis was carried out for samples extracted from NB, SPW, and
BS media. UV-VIS samples were prepared from the extracted pigments from NB
and BS using the following ratio 2:2:1:1 (violacein from filter paper, violacein from
supernatant, methanol ethyl acetate). A similar procedure was applied to the purple
pigments extracted from SPW.
3.2.4.2
Characterization of Violacein using FTIR
Dried samples obtained from 3.2.3.2 were analyzed using FTIR analysis.
KBr pellets were prepared out of each sample with grinding dry violacein with KBr
powder to get a fine powder. The concentration of the sample in KBr was in the
range of 0.2% to 1%.
3.2.4.3
Characterization of Violacein using NMR
Dried samples obtained from 3.2.3.2 were subjected to 1H-NMR and
NMR analysis. DMSO-d6 was used as the solvent for the analysis.
13
C-
37
3.2.5
Stability Test of Pigment Towards pH Changes
The effect of pH on the stability of the pigment extracted from BS was
carried out by adjusting the pH at extreme acidic pH (2) and extreme alkaline (13)
using and 0.1 M HCl and 0.1 M NaOH respectively.
3.2.6
Column Chromatography Preparation
Column chromatography was carried out using a column with the following
diameter: 12 cm height and 18 mm width. The column was then packed with silica
gel (column chromatography grade) and washed twice with n-hexane. The
concentrated sample obtained in methanol was placed on the top of the column and
separation initiated with the addition of solvent. Different fractions consisting of
different colors ranging from dark violet to bright pink were collected.
3.3
Bacterial Preservation
Agar slants were prepared in order to preserve the bacteria (Figure 3.2). To
make the slant, glass bottle was sealed and autoclaved at 121°C for 15 minutes.
Nutrient agar was poured into the sterilized bottles and left to harden. Paraffin oil
was added to the slant and autoclaved at 121°C for 15 minutes (Morton and Pulski,
1938).
38
Figure 3.2: C. violaceum preserved on agar slant in the presence of paraffin oil.
C. violaceum was successfully maintained throughout this study by a very
simple method of preservation in paraffin oil. Maintenance was carried out by
streaking the bacteria on agar slant, followed by 48 hours of incubation at 30°C in
order to obtain higher amounts of colonies (Harstel, 1952).
Final step of preservation was pouring sterile paraffin oil into agar slant to a
height of one centimeter above the top of the slant’s surface. Prepared slant was kept
in the fridge for almost four months.
To check for viability of the preserved bacteria, a sterile loop was used to
remove a colony from the agar slant and placed into NB or streaked onto agar plate.
CHAPTER 4
RESULTS AND DISCUSSION
4.1
Effect of Growth Parameters on Chromobacterium violaceum
4.1.1
Effect of Temperature on growth of C. violaceum in NB
The optimum growth temperature of C. violaceum and production of pigment
was monitored in nutrient broth (NB). The results are shown in Figure 4.1.
a) 25⁰C
b) 30⁰C
c) 37⁰C
Figure 4.1: Growth of C. violaceum and production of violet pigment at different
temperatures
40
At 25°C, the color of the medium was dark purple 6 hours after inoculation
(Figure 4.1(a)). However, the color of the culture medium was bright violet at 30°C
and majority of pigment precipitated in the medium (Figure 4.1(b)) and at 37°C no
violet pigment was observed.
4.1.2
Effect of Temperature on growth of C. violaceum in SPW
Figure 4.2 shows the growth of C. violaceum in SPW at different
temperatures and intensity of produced pigments.
a) 25⁰C
b) 30⁰C
c) 37⁰C
Figure 4.2: Growth of C. violaceum and production of violet pigment at different
temperatures.
Out of three flasks, pigment production was only observed at 25°C (Figure
4.2 (a)) and 30°C (Figure 4.3 (b)). However, when this experiment was repeated, no
pigment production was observed. This could be due to a different batch of SPW
used to carry out the experiment.
Another important factor effecting growth of bacteria was incubation time.
Figure 4.3 shows the effect of incubation time on the growth of C. violaceum on
SPW.
41
After three days, no colonies of C. violaceum were observed on the plate.
C. violaceum has been replaced by other microorganism present in the SPW and
compete for the available nutrients that lead to insufficient carbon source to support
bacterial growth (Nordiana Nordin, 2006). Fermentation process also leads to the
decrease in the pH (5.89) of the medium and makes it unsuitable for bacterial growth
(Chua and David, 1995).
Figure 4.3: Effect of incubation period on growth of C. violaceum. 1) Streaking right
after inoculation; 2) after 1 day; 3) after 3 days.
The effect of supplementation on growth of C. violaceum in SPW was also
looked into. Addition of BS did not have a positive effect on growth of C. violacein
in the SPW culture (Figure 4.4).
Figure 4.4: Plate assay for checking growth of C. violaceum in the mixture of SPW
and BS
42
Among all samples prepared with addition of DL-tryptophan and
L-tryptophan (section 3.2.1.2.1), only control cultures and samples number 3 and 5
from Table 3.1, showed production of pigment after a day of cultivation.
Reproducibility of obtained result from growth of C. violaceum in SPW studied by
repeating the experiment. This time, none of the prepared growth media showed
growth of bacteria.
4.1.3
Effect of Temperature on growth of C. violaceum in Brown Sugar
Growth of C. violaceum in brown sugar (BS) medium was observed at 25°C
and 30°C. However, pigment was only produced at 25°C as can be seen in Figure
4.5. At 37°C, no growth and pigment production was observed.
a) 25⁰C
b) 30⁰C
c) 37⁰C
Figure 4.5: Growth of C. violaceum and production of violet pigment on different
temperatures in BS
43
It can be concluded that production of pigment in BS medium is highly
temperature dependent and the optimum growth temperature for C. violaceum and
production of violacein reported as 25°C.
Production of pigment is also studied at different concentrations of BS
medium. Violacein absorbance of extracted pigments from different concentration of
BS medium suggested the highest amount of pigment production belonged to sample
number 17 (Figure 4.6).
Figure 4.6: Absorbance of violacein samples extracted from different concentration
of BS media.
44
4.1.4
Effect of Time on Growth of C. violaceum in NB and BS
Figure 4.7 shows growth profile of C. violaceum in NB and BS. To get the
required data for plotting the graph, samples were shaken for 24 hours at 25⁰C.
Log phase
Stationary phase
Lag phase
Figure 4.7: Growth profile of C. violaceum in NB and BS
Growth of bacteria in different growth media shows a similar growth phases
(lag, log and stationary phase). Death phase was not observed for BS growth medium
within 24 hours. It can be seen that growth of C. violacein gives more yield in NB
medium due to highest O.D 600 which is around two.
Production of pigment observed after 6-7 hours of inoculation (beginning of
stationary phase) in both medium (Figure 4.8). It has been mentioned earlier in
chapter 3 that production of pigment at or near the stationary phase is due to pigment
production via secondary metabolite.
45
Figure 4.8: Spectrophotometric cubes showing BS media before and after
production of pigment
4.1.5
Bacterial Preservation
Long term maintenance of C. violaceum was successfully obtained using
paraffin oil. Paraffin oil was reported to increase the lag phase of growth therefore
binary fission will be slowed down so it will take longer for the bacteria to reach to
the dead phase which occurs because of either loss of limiting nutrients or build-up
of toxins they release during log-phase growth (Hartsell, 1952).
Prepared slant was useable as long as there were some colonies remaining on
the surface of the slant. Therefore, the larger the surface area, the longer time the
slant can be used. The viability of the culture at different times is plated on NA as
shown in Figure 4.9.
46
Figure 4.9: Vitality test of preservation method for C. violaceum
There are some advantages of using paraffin oil as preservative factor such
as: it greatly reduces the frequency of contamination, especially with molds, thus
permitting cultures to be maintained with greater success in surroundings which
are not conducive to precise bacteriological work. No preliminary treatment of the
cultures and no seals, such as rubber caps, waxes is necessary (Harstel, 1952).
47
4.2
Characterization of Violet Pigment
4.2.1
Characterization of Violet Pigment from SPW
The UV-VIS spectrum of the violet pigment obtained from section 3.2.1.2
(Figure 4.10) is shown in Figure 4.11.
Figure 4.10: Pigment extracted from SPW
Figure 4.11: UV-VIS spectrum of violacein obtained from SPW at 25 and 30°C.
48
The obtained λmax was 571-773 nm that is indicating the presence of
chromophoric groups, hence indicated existing of violacein in the sample. A similar
λmax was obtained by Renee and Kendal (2001).
4.2.2
Characterization of Violet Pigment from BS
As explained in 3.2.3.2, extraction of purple pigment from BS was carried out
using two different solvents which were methanol and ethyl acetate. TLC test
showed presence of impurities in pigments extracted from BS with methanol, where
sample collected using ethyl acetate seemed to be pure (Figure 4.12). This fact can
be explained by assuming that present impurities in the sample extracted from BS
using methanol are highly polar; it is also known that ethyl acetate is a semi polar
solvent. Therefore, by using ethyl acetate in extraction stage just poorly-water
soluble violacein will migrate to the organic phase of solvent and impurities will
remain inside the supernatant.
1
2
Figure 4.12: TLC result of violacein extracted with 1) methanol and 2) ethyl acetate
49
4.2.2.1
Column Chromatography
Present of impurities in samples collected from BS extracted with methanol
was the reason of running the column. The first impurity separated from the sample
(Figure 4.13) was the brownish material, probably brown pigment of BS.
Melanoidin
Figure 4.13: Brownish fraction of melanoidin, pigment from BS.
As mentioned in 3.2.6, different fractions consisting different colors were
collected from the silica column. Unfortunately, TLC result of collected fractions
did not show any purification since there was more than one single spot observed on
the TLC paper. Although each vial seemed to be different from the other but the
same set of spots appeared on TLC sheet after each and every test (Figure 4.14). The
test was repeated again and the same results were observed. From the results, it can
be concluded that pure violacein was not obtained through column chromatography.
Figure 4.14: TLC results of violacein fractions from column chromatography.
50
4.2.2.2 UV-VIS Spectrophotometer Analysis of violacein
Results of the UV-VIS analysis by other researchers (Table 4.1) show
absorbance in the range of 550-580 nm of full range of UV light indicated from large
conjugation C=C existing in violacein.
Table 4.1: λmax of violacein using UV-VIS spectrophotometer
No
UV-VIS absorption
Reference
1
565 nm
(Inniss and Mayfield, 1979)
2
585 nm
(Renee and Kendall, 2000)
3
558 nm
(Bromberg and Duran, 2001)
4
558 nm
(Natalia and Nelson, 2001)
5
553 nm
(Gregor and Wolfgang, 2001)
6
577 nm
(Regina and Tania, 2004)
7
580 nm
(Lara et al, 2005)
8
576 nm
(Yuan et al, 2008)
Spectrum resulting from running UV-VIS of the sample collected from BS
(Figure 4.15) gave a similar peak at 567.50 nm that shows presence of violacein in
the solution. High absorption in the spectrum refers to successful production of
pigment in new medium.
Figure 4.15: UV-VIS Absorption spectrum of violacein extracted from C. violaceum
grown in BS.
51
4.2.2.3 FTIR Spectroscopic Analysis
The IR spectrum (Figure 4.16) of violacein from BS displayed broad
absorption band at 3430.1 cm-1 corresponding to O-H stretching. The N-H stretch
3350 cm-1 might be overlapped with band of O-H. A band appeared at 1640.6 cm-1
was assigned for carbonyl of amide groups. The presence of C=C of an intermediate
alkene was observed at 1615.9 cm-1. The out of plane band for =C-H was observed at
757.25-727 cm-1. As for pigment extracted from NB, the IR spectrum (APPENDIX
4) proved the existence of O-H (3445.45 cm-1), a carbonyl of an amide (1662.8 cm-1),
C=C of an olefinic band (1615.1 cm-1) and out of plane =C-H (760.4 cm-1). The
carbonyl of the lactam ring has high absorption value because the one pair on N is
delocalized away from the C=O, hence it behave as ketone C=O (1723.54 cm-1). The
above data were almost similar with the IR data of violacein spectrum reported by
other researchers (Table 4.2).
Table 4.2: Data of FTIR (Gregor and Wolfgang, 2001; Lara et al, 2005).
Reference peak
BS bands
NB bands
Peak Assignments
3430
3430.1
3445.45
3500
3330.1
3250.7
1665
1640.61
1662.85
Amide (C=O)
1613
1615.92
1615.11
C=C
1723
1723.54
1689.25
C=O
765
757.25 , 727.11
760.47
Alcohols O-H
N-H overlapping with
O-H stretching
Phenyl ring substitution
bands (=C-H)
52
Figure 4.16: FTIR spectrum obtained from KBr pellet of violacein from BS
4.2.2.4 NMR analysis of violacein
13
C-NMR was employed to identify 20 carbons present in structure of
violacein. The first sample analyzed by 13C-NMR was the one obtained from 3.2.3.2.
Result showed presence of noises due to the impurities in the sample which covered
the peaks assigned to carbon atoms.
The spectrum resulting from the sample which was filtered drying stage 20
carbons of violacein (Figure 4.17)
53
Figure 4.17: 13C NMR spectrum of preliminary purified violacein running by
DMSO and adequate amount of CDCl3
The 13C chemical shifts of all the present carbons in violacein structure have
been studied and summarized in Table 4.3. Labeled carbons shows carbon number
16 and 11 are shifted downfield because of the influence of the electronegative
oxygen atom where carbon number 13 is shielded to up field.
Table 4.3 also shows the similarities between the sample obtained from BS
and the available references. It is notable that violacein has eight CH groups, nine
quaternary carbons plus two C=O group with one C-OH bond.
54
Table 4.3: 13C-NMR peaks of violacein and its related carbons (Gregor and Will,
2001; Ruhul and Tsutomu, 1999).
No
Reference peaks
Sample peaks
Peak Assignment
1
96.6
97.5
C
2
104.2
105.1
C
3
105.4
106.3
C
4
109.7
109.3
CH
5
112.7
113.4
CH
6
113.7
113.6
CH
7
118.4
119.2
CH
8
120.5
121.1
C
9
122.0
122.9
CH
10
125.2
126.0
C
11
126.0
126.8
C
12
129.1
129.6
CH
13
129.3
129.9
CH
14
131.2
132.0
C
15
136.6
137.5
C
16
141.5
142.2
C
17
147.2
148.0
C
18
152.6
153.3
C
19
169.9
170.7
C=O
20
171.3
172.1
C=O
55
Simultaneously, 1H-NMR spectrum was recorded in DMSO-d6. The sample
showed similar peaks as reported in references (Figure 4.18).
Figure 4.18: 1H-NMR spectrum of preliminary purified violacein from BS.
Assigned peaks and available references also peaks obtained from violacein
sample is collected in Table 4.5 and Figure.
56
Table 4.4: Collected data from 1H-NMR in comparison with reference (Yoshitoshi et
al, 2002; Hartmut and Ronald, 1984)
Peak
Reference peak
Sample peaks
Multiplicity
11.89
11.88
s
H1
8.07
8.05
s
H2
7.24
7.23
s
H5
9.35
9.31
d
H6
6.78
6.78
d
H7
7.35
7.34
s
H8
10.74
10.70
s
H10
7.55
7.54
s
H13
10.64
10.59
d
H15
8.93
8.93
t
H19
6.95
6.95
t
H20
7.20
7.21
d
H21
6.82
6.82
0
H22
assignment
Overall, the characterization shows similar results as it has been reported in
the literatures and it can be concluded that purple pigment extracted using ethyl
acetate is pure violacein.
57
4.3
pH Test
Violacein pigments were tested for their stability in acidic and basic pH.
From the study, it was found that the pigment was not stable toward varying pH
range where in extreme acidic condition the solution turned to blue color and in
extreme alkaline condition, the solution was green in color (Figure 4.19). Initial pH
of solution was 5.83 which had a dark violet color (Figure 4.20).
Figure 4.20: Violacein at pH 5.85
Figure 4.21: Colors resulting from pH test on violacein in extreme acidic and
alkaline pH
58
Changes of the color due to varying the pH can be explained by changing of
the structure in extreme acidic and alkaline pH. For instance, in alkaline condition
excess OH- in the solution deprotonates N-H groups which cause formation of anion.
Localization of negative charge of the anion to the ring system due to the long
conjugation effect causes violacein to be absorbed at higher wavelength (694.7 nm).
Therefore it can be concluded that changes of the structure is changing the
absorbance of the light hence reflected color from the sample deferrers.
CHAPTER 5
CONCLUSION
5.1
CONCLUSION
In this project, the growth of Chromobacterium violaceum on agricultural
wastes such as SPW and BS was studied. To achieve that target different growth
factors (temperature, pH, concentration of media) were examined.
From the growth profile of C. violaceum, the purple pigment found to be the
secondary metabolite products because the pigments were produced after 6 hours of
the active stage of growth.
Effect of different temperatures (25°C, 30°C, and 37°C) has been studied on
different growth cultures to show the optimum temperature suitable for growth of
bacteria and production of pigment. All three media include Solid Pineapple Waste,
Brown Sugar and Nutrient Broth were capable of intensive growth and pigment
production at 25°C.
The extraction of pigment from C. violacein was carried out using methanol
and liquid-liquid extraction method using ethyl acetate. Liquid-liquid extraction was
the better method in terms of extracting the pigment in a pure form.
60
The pigment has been tested for stability towards pH. Appearance of different
colors after adjusting the pH at extreme acidic and extreme alkaline showed nonstability of the pigment in different range of pH value.
Characterization of pigment obtained from NB, SPW, and BS carried out
with employing UV-VIS, FTIR and finally
13
C-NMR and 1H-NMR. IR results
provided similar peaks assigned to functional groups present in violacein’s structure.
Meanwhile,
13
C-NMR and 1H-NMR spectra showed 20 carbons of violacein and
their respective hydrogen.
Maintenance of the bacteria through this study was carried out using paraffin
oil preservation method.
5.2
Future Study
It was mentioned earlier on in the literature review that the most crucial
amino acid required for formation of violacein structure is L-tryptophan. In this
project it was aimed to produce violacein pigment without the use of chemicals in
order to reduce manufacturing costs.
Some researchers reported that addition of extra amino acid into the growth
media helps to increase the rate of pigment production. To avoid using commercial
amino acid, natural source of tryptophan can be used in order to save cost.
61
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APPENDIX 1: UV-VIS spectrum of samples from growth media with different concentrations (1-10)
66
APPENDIX 2: UV-VIS spectrum of samples from growth media with different concentrations (11-20)
67
APPENDIX 3: FTIR spectrum obtained from KBr pellet of violacein from NB
68