Screening of Natural Dyes from Selected Fungal SpeciesExtraction

and Characterization of Natural Dyes from Fungi towards

Industrial Applications

Tasbiah Naz1, Shomaila Sikandar1, Fatima Sajjad², Alim-un-Nisa³*, Imran Afzal1,

Anum Fatima1, Nayyar Rubab1, Rabia Sattar1
1
Department of Biology, Lahore Garrison University, Lahore, Pakistan
2
Department of Life Sciences, University of Central Punjab, Lahore, Pakistan
3
Food and Biotechnology Research Centre, PCSIR laboratories Complex, Lahore, Pakistan

*Corresponding author’s email: alimunnisa64@gmail.com

Abstract: Some Fungi are described as effective pigments generating microbes that have the significance of
producing natural pigments and it has increased globally the decree for eco-friendly natural pigments. The pigment-
producing fungi were optimized and assessed towards the application in dyeing on cotton fabric. For this purpose,
five fungal strains were selected namely; Aspergillus terreus S10, Talaromyces atroroseus WW5A3, Penicillium
oxalicum WW3A4 (DG), WW5C2 and WW31DG w. These strains ere incubated for 21 days under static and non-
static conditions using MSM and PDB media. The production of pigments by fungi was optimized by changing
temperature (25-35℃) and pH (4.5-6.5) parameters under liquid state fermentation conditions . . The produced
colors for all the fungal strains were; T. atroroseus WW5A3 showed pinkish color, A. terrreus S10
displayedshowed yellow color, P.oxalicum WW3A4 (DG) presented showed yellow-greenish, WW5C2
exhibitedshowed light yellow color and, while WW31DG demonstrated showed greenish color., respectively
liquid state fermentation under optimized conditions. The results showed the maximum percentage absorbance of T.
atroroteus WW5A3 showed 90.36% at 600 nm, A. terrreus S10 showed 88% at 500 nm, P. oxalicum
WW35A4 showed 46.04% at 550nm, WW5C2 showed 59.60% at 550 nm, and WW31DG showed 81.9% at 550nm.
The natural fungal pigments were tested against bacterial pathogens to check the antibacterial activity. The results
indicated that S. aureus and E. coli were susceptible to the extracted fungal pigments and they exhibited antibacterial
activity in terms of maximum zone of inhibition. In conclusion, out of five pigments producing fungi, Aspergillus
terreus S10 and Talaromyces atroroseus produced maximum pigment and highest percentage absorbance under
liquid state fermentation conditions. Therefore, the present study found potential application in the textile and
leather industry.

Keywords: Cotton fabric, ecofriendly, natural pigments, characterization, antibacterial activity, dyeing

1. INTRODUCTION

Modern times heighten the demand for colourantss in the field of foodstuff,
prompt industrialization that has compelled medicine leather, and other industrial areas
the immediate formulation to use artificial nevertheless of their cancer-causing,
 Dyes Extraction from Fungi

immune-oppressive, and hazardous Naturally, fungi produce colored pigments,

environmental effects. To attain these secondary metabolites such as flavonoid and
objectives researchers are discovering tannin are familiar as pigments with
natural pigments from microbial resources extraordinary pharmaceutical importance.
as a substitute to artificial dyes [1]. Many filamentous fungi including
Researchers revealed that environment- Aspergillus sp, Penicillium sp,
adapted microbial colorants from microbes Paecilomyces sp, and Monascus purpureus
are better thangood to artificial dyes because can be utilized in the extraction of colored
of their fast growth, cool handling, and pigments. Thermomyces are utilized to
important roles in transcriptional and extract red colourcolor and found huge
intracellular signaling. Furthermore, their industrial applications [11]. Pigments
applications in food and cosmetic industry produced by fungal species showed
areis due to their assembly and easiness for improved dyeing ability at acidic pH (5) [12,
big scales production [2, 3]. Microbes such 13]. Shade created from Talaromyces under
as fungi and bacteria deliver the a voluntary 4.5 - 5 pH to direct antacid settings (pH 4.5
availability of naturally derived pigments and 6.0) [14, 15]. Colors from fungal
[4].. Effluents from industries that utilize species, utilized coloring tests become more
Synthetic dyes lead to the production of steady at extreme temperatures [10, 15]. A.
industrial effluents thatdifferent colors are Flavus shade appeared 93% to 96%
considered as toxinss and that steadiness on cotton and silk fabric at 26°C
exhibitedmerge numerous natural issues [14, 16]. Microorganisms can provide
ands well as therapeutic problems [5]. natural pigments that can be utilized in
Phycocyanins extracted from thermophilic industry rather than artificial dyes [17]. The
blue-green bacteria and fungi were colors extracted by Talaromyces sp. had
verbalized to use in makeup mostly an eye- plentiful benefits in the food, textile, and
shadows and lipstick shades were cosmetic industry [178]. Filamentous Fungi
manufactured in the industry by using both create a wide assortment of bio-colorants
fungi and bacteria this fungi[ [6]. Many and have secondary metabolites like
filamentous fungi are used in the production melanins, phenazines, flavins, carotenoids,
of natural pigments as they are eco-friendly quinones, violacein, indigo, and monascins
to the environment and less hazardous. [189, 1920]. Fungi species are curiously
Toxic effluents from industries utilizing good for the environment and give attractive
different colors posed threat to the natural colors, few fungal species are good
environment and caused serious health to produce consistent colorants and they
issues [7]. Natural colors in food have carotenoids present in them as
manufacturers are associated with several secondary compounds [201, 212]. These
advantages and endless therapeutic diseases filamentous fungi have been distinguished as
Numerous related benefits and uncountable they have anti-bacterial activity against
restorative chattels are interrelated to custom Gram-positive and Gram-negative microbes.
for natural dyes in food factories [8]. Consequently, the extractions of pigments
from these filamentous fungi are being used production of fungal pigments. It was
in many applications [23]. observed that in production of pigment from
M. ruber repressed color production when
For the better growth of fungal plates are covered and cannot coordinate
pigmentspigments, the optimal carbon with light [278-29]30]. Consequently, the
source, moderate pH, temperature, and extractions of pigments from these
availability of light source is very necessary. filamentous fungi are being used in many
It has been detailed that fungal species can applications [22]. The main objective of this
develop and create colors in the submerged research was to replace the artificial dyes
state containing glucose, fructose, and with natural ones.
dextrose [19, 234]. Hydrogen molecules
exceptionally play important role in the
production of eco-friendly pigments.
The development of color under 5.5 and
6.5pH were maintained for two strains of 2. MATERIALS AND METHODS
Pycnoporus [24]. The calculations [25] 2.1. Isolation and Collection of Fungi
showed the most noteworthy value of yellow
color was gotten with 6.5 pH while the Fungal strains were collected from the
greatest biomass concentration was Biology Lab of Lahore Garrison University .
measured at pH values of 8 for Talaromyces The cultures were preserved at -80°C, the
sp. [26]. fungal strains were refreshed by using PDA
media. For isolation, textile and
For illustration, [25] detailed that the ideal lignocellulosic waste were collected and
pH values for development of color stored in sterilized plastic bags. By using the
generation under 5.5 and 6.5 were serial dilution method samples were
maintained for two strains of Pycnoporus, collected and 1mL was is dissolved in 10
[26] calculate that for the most noteworthy mL of water in a test tube [31]. After that (0-
value of yellow color was gotten with pH 1 mL) will spread onto the PDA plates with
values of 6.5 whereas the greatest biomass the sterile spreader. All PDA plates were
concentration was measured at pH values of kept in an incubator at 28 °C for 3-4 days.
8 for Talaromyces sp. [27]. Temperature is Diverse fungal colonies showed up on PDA
the main element that influences colors and plates from which cultures of the fungi were
other metabolites generation by organisms. acquired by transferring them onto new
Researches showed the impacts of PDA plates and after that kept once more in
temperature on cell development, color an incubator at 28 °C for 3-4 days [32, 33].
generation by a different number of
organisms demonstrates that the ideal 2.2. Qualitative Analysis of Fungal Dyes
temperature ranges from 25 to 32°C. Fungi
2.2.1. Preparation of Media
also respond to light during development for
metabolites generation. The impact of light
on color generation is very important for the
 Dyes Extraction from Fungi

PDA is a solid medium comprising the [2.4.] Quantitative Characterization of

extract of 300 g skinned potato, 2.5 g Natural Pigements
glucose, 15 g agar. All PDA plates were
incubatedthen set aside in an incubator at 28 The soluble compounds from the fungal
°C for 3-4 days. Fungal colonies extract and culture filtrates of different
grewseemed on PDA plates and was placed fungal isolates were subjected to UV–
from which pure cultures of the fungi were Visible spectrophotometer. Compounds
found onto fresh PDA plates and then which were present in water extracts
preserved again in an incubator at 28 °C for were subjected to a wavelength scan
3-4 days [3, 34]. from 400 nm to 600 nm for the
determination of absorption spectra.
2.2.2. Cultivation of Fungi on appropriate The absorption spectra of compounds
Medium for Optimum Color Production present in water extracts were
For the cultivation of fungi, two media were determined by subjecting the samples to
used i.e. Mineral Salt Media (MSM) and a wavelength scan from 400 nm to 600
Potato Dextrose Broth (PDB). MSM was nm. The optical density of pigment was
prepared using following salts i.e., dextrose, measured by spectrophotometry for each
K2HPO4-5.0g, KH2PO4-5.0g, CaCl2-0.1g, process to know which one is better to
MgSO4.7H2O-0.5g, FeSO4.7H2O-0.01g,
get more concentrated pigment.
ZnSO4.7H2O-0.01g, MnSO4.H2O-0.03g. All
these salts were dissolved in 1000 mL of
distilled water (Fig 4). Two sets of flasks Spectrophotometry was used for the
were placed in an incubator, in dark, under measurement of optical density of
static conditions for 4-6 weeks until the bulk pigment. This was done for the
of pigments appeared [20]. determination of better option for
obtaining more concentrated pigment.
The optical density (OD) was
2.3. Extraction of Pigments from Fungi measured at 400, 450, 500, 550, and
The isolation of fungal supernatant was 600 nm (a wavelength which
caused by utilizing a sterile muslin cloth represents the absorption maximum
after the incubation period. To evacuate any for yellow, red, pink, and greenish and
remains display in it and observe for colour yellow-greenish pigments
illustrating extracellular shade production. respectively), thus yielding the so-
The residue was grinded ound in a called yellow, pink, reddish-brown,
pulverizer by mortar and pestle. Later, 2ml and red pigment production [37].
a small amount of water was added and
incubated at 130rpm for 60 minutes at 28°C. 2.4.[2.5.] Analysis of Color on Cotton
[36]. Fabric

To analyze the fabric properties, rate

retention of the dyed cotton cloth,
percentage absorption was calculated on UV
spectrophotometer at 400 nm to 600 nm and of new bacterial cultures were immunized
the percentage absorption was calculated by separately onto the agar plates by the spread
measuring OD of concentrated pigment plate method [4, 5 &, 9]..
before dipping the cotton cloth and again
OD was calculated after absorption of
pigment by cotton cloth. The final
percentage absorption was calculated by the
given formula [41]: 3. RESULTS

Percentage absorption %= 3.1. Optimization of Fungal Pigments

OD before dyeing - OD after dyeing x 100 Two different media PDB and MSM media
were used for the production of pigments. In
OD before dyeing
the case of submerged fermentation where
2.5.[2.6.] Antibacterial Activity of Fungal the flasks were incubated at rotary
Pigments conditions, the MSM showed color
production for all the five fungi (Fig 1).
The bacterial suspension was made of E. However, A. terreus S10, T. atroroseus
coli and S. aureus which was arranged by WW5A3 and WW5C2 secreted pinkish, dark,
taking ordinary saline sterilized test tube. and light yellowish color, respectively, in
Bacterial colonies were exchanged with the PDB. Moreover, the impact of incubation
assistance of a wire loop circle into the test conditions (rotating and/or static) were
tube. The circle utilized was sterilized by noted. Moreover, T the impact of incubation
warming. The test tube was swirled well conditions (rotating and/or static) were
with the assistance of a syringe. For the noted. Distinctive results were obtained with
making of media, the L-Agar and L-broth regard to all the five fungi after an
were utilizedarranged. These media were incubation period of 21- 28 days. On the
included in distilled water containing jar and other hand, in static, and dark conditions all
shake well with the assistance of a stirrer, five fungi showed color in a liquid state
the volume raise to 500 ml at that point under MSM media and in the case of PDB
closed the carafe with the assistance of a media, except WW35A4 all fungal strains
cotton plug and secured with aluminum produced color.
thwart and autoclaved for 15 minutess at
temperature 121 °C and weight 15 lb/inch2 T . atroroseus WW5A3 appeared pinkish
[10]. color and A. terreus S10 shown yellow
color. Greenish-yellow color
Antibacterial activity of the Bio- colors was advertised by WW31DG. Beneath 25
°C to 35 °C (Fig 3) WW5C2 created light
A loop fulfull of E. coli and S. aureus
yellow
bacteria were inoculated into a nutrient broth
color, whereas WW35A4 displayed light
and incubated on a shaker at 28 °C for a
pale yellow color in solid-state
period of 16–18 h. After incubation, 100 µL
 Dyes Extraction from Fungi

and fluid state mat 7

uration (Fig 4) in 6
4.5 to 6.5 pH as
5
described in (Fig
2). 4
3
a
b 2

p.H
1 PH

DG
ali s
)

W 2
W um
A. 5A3
ox u

W 5C
P. erre

31
c
W
W
t
(W
us
se
ro
c

tro
d

T.a
Fungal Strains

Fig. 2. Optimization of T. atroroseus, A. terreus, P.

oxalicum, WW5C2 and WW31DGdifferent fungal
strains on different pH ranges

40
e
35
30
Fig. 1. Results 25
of Solid Sate 20
fermentation (a) T.
atroroseus WW5A3 (b) A. terreus S10 (c) P. 15
Temperature ℃

oxalicum WW35A4 d) WW5C2 (e) WW31DG 10

5
Temp
0
A. terreus
T.atroroseus (WW5A3)

WW5C2
P.oxalicum

WW31DG

1 2 3 4 5
Fungal Strains
Fig. 3. Optimization of different fungal strains on
different temperatures ranges

Fig. 4. Production of fungal pigments under liquid state fermentation (a) T. atroroseus WW5A3 (b) A. terreus S10,
(c) P. oxalicum WW5A4 (d) WW5C2 and (e) WW31DG ( left to right)

2.5

2 Before dyeing O.D

After dyeing O.D
1.5
% Absorbance
O.D

0.5

0
Fig. 5. Results of 400nm 450nm 500nm 550nm 600nm T.
atroroseus Wavelenth nm WW5A3
before and after OD at a
different wavelength
 Dyes Extraction from Fungi

2.5

Before dyeing O.D

1.5
After dyeing O.D
O.D

% Absorbance
1

0.5

0
400nm 450nm 500nm 550nm 600nm
Wavelength nm

Fig. 6. Results of Aspergillus terreus S10 before and after OD at a different wavelength

3.2. Analysis of fungal pigments studies showed that fungi from the same
species produced diverse colors and thus
The different fungal isolates were subjected have diverse behavior toward the production
to UV–Visible spectrophotometer [28,38] of colored compounds [10, 38].
The T. atroroseus WW5A3 showed pinkish
color with a strong absorbance in the near 3.3. Application of Cotton Dyeing
UV region with a distinctive peak at about using Fungal Pigments
600 nm as shown in (Fig 5). However, in the
The extracted pigments were further used
case of A. terreus (Fig 6) showed yellow
for dyeing the cotton cloth. For dyeing
color, P. oxalicum showed Pale yellow,
ferrous sulphate was used as a mordant.
WW31DG showed yellow-greenish and
The capacity of extracted colors was
WW5C2 showed light yellow bio-colorants,
evaluated on cotton cloth which weighed
a strong peak at ~ 500 and ~ 550 nm,
around 10g. For detecting any change in
respectively, was observed. However, they
color on the cotton, a control was also held.
may have secondary metabolites like
The extracted colors were tested for their
carotenoids, melanins, azaphilones, and
ability to dye cotton cloth weighing
polyketide that showed that specific color
approximately 10 g (Fig 7). Control was
and also possess resistance to natural
maintained to determine the change in color
variables (drying up, high temperatures,
of the cotton cloth. It was observed that the
irradiations, and photo-oxidation). Similar
pinkish, green, and yellow colors, exhibited
an undeviating shade on cotton [41]. The % 3.4. Study of Antibacterial Activity of
OD was taken before dyeing and after Fungal Pigments Against Gram-
dyeing to check the absorbance of color in Positive Bacteria by Agar Well
cotton fabric and to check colorfastness on Diffusion Method
fabric [5]. [3.5.]
The antibacterial activity of fungal
pigments against two bacterial strains
was assessed by the Agar Well
Diffusion method. The extracts
exhibited varying degree of
antibacterial activity at 100 mg/mL
against gram-positive and gram-
negative bacteria. The result described
that S. aureus and E. coli are
Fig. 7. Dyed cotton cloth by fungal pigments susceptible for fungal pigments as
showing dyeing of cotton cloth from fungal described in (Fig 9) and Table 2. The
pigments anti-bacterial activity against the E. coli
and S. aureus are shown in Fig 10 and
Fig11.

Fig. 8. Showing the overall process of

pigment production from fungi
 Dyes Extraction from Fungi

12

10

0
T.atroroseuS A. terreus WW31DG WW5C2 P. oxalicum Control

E. coli S. aureus

Fig. 9. Result of Zone of Inhibition for Fungal extracted pigments against strain E. coli and S. aureus

Table 1. Different fungal stains were grown on two different media that is MSM and PDA: They were grown on
different temperatures and pH: on solid-state and liquid state fermentation the fungal strains showed pigments under
static and non-static state.

Color Media
Pigments Production
both in
Sr. Temperatu
Fungi pH solid and
No re Static Non-static
liquid PDB MSM
condition condition\Dark
condition
T.atrorose
usT.
1. 25°C 5.5 Pinkish No Yes Yes Yes
atroroseus
WW5A3
Aspergillu
2. s terreus 30°C 6.5 Yellow Yes Yes Yes Yes
S10
P.oxalicu Greenish-
3. 25°C 6.5 No Yes No Yes
m yellow
Light
4. WW5C2 35°C 4.5 Yes Yes Yes Yes
yellow

5. WW31DG 30°C 5.5 Greenish No Yes No Yes

 P.
T. A.
Bacteri oxalicu
atrorose terre WW5C WW31D Contr
al m
us us 2 G ol
Strains WW3A
WW5A3 S10
4 (DG)

Gram
Negativ 10 9 8 10 9 2
e E. coli

Gram
Positive
9 9 7 10 10 2
S.
aureus
 Dyes Extraction from Fungi

Table 2: Showed results of Zone inhibition of fungal extracted pigments against E. coli and S. aureu

Fig.10 Results of Antibacterial Activity against E. coli by Agar Well Diffusion Method

Fig.11 Results of Antibacterial Activity against S.aureus by Agar Well Diffusion Method

4. DISCUSSION yellow color under MSM media. All

In the present research, an effort was these pigments presented a double
complete to improve an eco-friendly advantage by giving bulk of natural
process for big-scale formation of pigments that minimize the use of
pigments was attained by fungi and its artificial dyes in several industries.
utilization in cotton dyeing. It was Various parameters, such as the pH of
observed that T. atroroteus and A. the culture medium and temperature,
terreus showed a bulk of production of promote the growth of fungal strains,
pinkish and yellow color pigments under resulting in good synthesis of natural
MSM media while WW31DG showed colors from fungus. The temperature
good production of greenish yellow range of 25 to 35°C and the pH range of
pigment under both media. While 4.5 to 5.5 were shown to be optimal for
WW5C2 AND WW35A4 showed pale fungus growth and pigment synthesis.
For both mycelial development and colorants, respectively. Due to the
pigment formation, the pH of the media proximity of a large conjugated
fluctuates. The growth of the fungal framework, which induces
strains is increased absorbance at longer wave lengths,
higher absorbance values were
by various factors i-e pH of the observed .observed. Shade broken
culture medium and temperature that down within the water is
give us good production for the effectively particle pulls in the
utilization of natural pigments from water particles and make cotton
fungi. From the results, it was retains water well. Shades broken
observed that the temperature 25 to up within the water is effectively
35°C and pH 4.5 to 5.5 appeared to bound and expresses its tint. The
be optimum for the growth and absorbance of A. terrreus is 88%
pigment production of fungi. The pH under 500nm of wavelength, T.
of the medium varies for both the atroroteus showed 90.36% of
mycelial growth and pigment absorbance under 600nm of
production. The optimum pH and wavelength, WW31DG showed
temperature of the fungal strain 81.9% of absorbance under 550nm
were 4.5-5.5 and 25±5 °C were of wavelength, WW5C2 showed
observed for growth and 59.60% of absorbance under
secondary metabolite production. 550nm of wavelength and
A pale yellow (537– 540 nm); WW35A4 showed 44.04% of
neutral arrangement, violet (529– absorbance under 550nm of
536 nm) and acidic arrangement, wavelength. We can see that the
ruddy (500–507 nm) [13]. The absorbance ranges from 500 nm to
samples were exposed to a 600 nm this absorbance also
wavelength scan from 400 nm to observed in [37]. The results of
600 nm to determine the maximum study advocated that the fungal
absorbance of chemicals present in extracts, were effective against
water extraction. The greenish and tested human pathogens i.e. Gram-
pinkish colour exhibited as a solid Negative Escherichia coli. The
absorber inside the UV area, with results indicated that fungal
a typical absorption crest observed pigments were more effective
around 550 and 600 nm. In against gram negative bacterial
addition, the pinkish colourcolor strain E. coli showed 10mm zone
showed a strong absorbance in the opposite to T. atroroteus ,9 mm
near UV area, with a prominent against A. terreus, 8 mm
peak at nearly 600 nm. However, adversedadverse WW31DG, 10 mm
substantial peaks at 500 and 550 WW5C2 and 9 mm differing
nm were seen in the case of yellow WW5A4. The Zone of inhibition is
greenish and light-yellow bio- respectively. Streptomycin served
 Dyes Extraction from Fungi

as control which showed 2 mm±0 of colors which can be utilized productively

zone of inhibition respectively. in industries.
Unique organisms were isolated
from the samples. These fungal 6. ACKNOWLEDGEMENT
species are safe to use due to all of The authors are sincerely grateful to the
the safety precautions taken Management, Lahore Garrison
during the generation process. University, for the constant
There are only a few ideas about encouragement and support.
how to use bio-colors from
organisms for cotton
colouringcoloring. 7. CONFLICT OF INTERESTS
No conflict of interest

5. CONCLUSION
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