ABS Report

OPTIMIZATION STUDIES
ON MYCOREMEDIATION OF
SYNTHETIC DYES BY DESIGN
OF EXPERIMENTS
A PROJECT REPORT
Submitted by
ABIVARMA.R.S (Register
ister No. 0910204002
0910204002)
BAHEERATHAN.M (Register
ister No. 0910204008
0910204008)
SHIVASHANKAR.R (Register
ister No. 0910204041
0910204041)
in partial fulfillment for the award of the degree

of
BACHELOR OF TECHNOLOGY
in
BIOTECHNOLOGY
KUMARAGURU COLLEGE OF TECHNOLOGY

(An Autonomous Institution Affiliated to Anna University, Chennai)
COIMBATORE-641 049
ANNA UNIVERSITY: CHENNAI 600 025

MAY 2013
OPTIMIZATION STUDIES ON
MYCOREMEDIATION OF
SYNTHETIC DYES BY DESIGN OF EXPERIMENTS
A PROJECT REPORT
Submitted by
ABIVARMA.R.S (Register No. 0910204002
0910204002)
BAHEERATHAN.M (Register No. 0910204008
0910204008)
SHIVASHANKAR.R (Register
ister No. 0910204041
0910204041)
in partial fulfillment for the award of the degree

of
BACHELOR OF TECHNOLOGY
in
BIOTECHNOLOGY
KUMARAGURU COLLEGE OF TECHNOLOGY

(An Autonomous Institution Affiliated to Anna University, Chennai)
COIMBATORE-641 049
ANNA UNIVERSITY:
UNIVERSITY CHENNAI 600 025
MAY 2013
i
ANNA UNIVERSITY : CHENNAI 600 025
BONAFIDE CERTIFICATE
Certified that this project report “OPTIMIZATION STUDIES ON

MYCOREMEDIATION OF SYNTHETIC DYES BY DESIGN OF
EXPERIMENTS” is the bonafide work of “ABIVARMA.R.S
(Register No. 0910204002), BAHEERATHAN.M (Register No.
0910204008) and SHIVASHANKAR.R (Register No. 0910204041)”
who carried out the project work under my supervision.
SIGNATURE SIGNATURE
Dr. A. Manickam Mr.D.R.Manimaran

HEAD OF THE DEPARTMENT SUPERVISOR
Assistant Professor (SrG)
Department of Biotechnology Department of Biotechnology
Kumaraguru College of Technology Kumaraguru College of Technology
Chinnavedampatti Chinnavedampatti
Coimbatore – 641 049 Coimbatore – 641 049
____________________ _____________________
Internal Examiner External Examiner
ii
ACKNOWLEDGEMENT
Firstly, we express our most sincere thanks to The Lord Almighty, for
his blessings.
We express our thankfulness to our SUPERVISOR,

Mr.D.R.Manimaran, Assistant Professor(SrG), Department of
Biotechnology, Kumaraguru College of Technology for his constructive
criticism and unrivalled support, without which this project would not
have come to fruition.
We express our deepest gratitude to Dr.S.Ramachandran, Principal,

Kumaraguru College of Technology, for granting us permission to carry
out our project work.
We sincerely thank Dr.A.Manickam, Professor and Head, Department

of Biotechnology, for his valuable insight during the course of our
project work.
Also we express our heartfelt gratitude to our project review members,
Mr.S.Sivamani, Assistant Professor (SrG), Dr.P.Ramalingam,
Associate Professor and Mr.M.ShamugaPrakash, Assistant Professor
(SrG), Department of Biotechnology, for their valuable feedback and
continual support.
Finally, we express our most heartfelt thanks to all our friends and
family members for their encouragement and support.
Abivarma.R.S Shivashankar.R Baheerathan.M
iii
ABSTRACT
Mycoremediation is a feasible method for degrading dyehouse effluent.

Degradation effects of 11 fungal species Mucor racemosus MTCC
7382, Rhizopus stolonifer var stolonifer MTCC 7370, Fusarium
moniliforme MTCC 2015, Fusarium oxysporum MTCC 284,
Trichoderma viride MTCC 2417, Aspergillus terreus var terreus MTCC
3006, Aspergillus ochraceus MTCC 1810 and Penicillium citrinum
MTCC 8009, Saccharomyces cerevisiae MTCC 2376, Kluyveromyces
maxianus MTCC 4059 and Trichoderma reesei MTCC 3193 against 5
textile dyes were screened. Out of 10 organisms showed potential dye
degradation, 6 species were selected for further parameter screening by
Plackett-Burman design (PBD). The effect of each process variable is
calculated from PBD and significant variables are further optimised
using Box-Behnken design (BBD). In this study, P.citrinum showed
maximum acid green removal of 96.78% at initial dye concentration of
0.2 mg/mL, contact time of 2 days and inoculum size of 5.38% (w/v).
Similarly R.stolonifer showed 90.78% acid green removal at initial dye
concentration of 0.14 mg/mL, temperature of 36.98°C and inoculum size
of 5.47% (w/v). F.moniliforme revealed 60.38% of removal with
optimal conditions of dye concentration 0.20 mg/mL , pH of 4.00 and
agitation speed 0 rpm. The experimental and statistical results showed
that the optimum conditions could be an ideal solution for acid green
degradation by Penicillium citrinum, Rhizopus stolonifer and Fusarium
moniliforme.
iv
ஆ சார
கழி கைள சிைத ப யற க ெபற ைவ

ய சிய , ைசகைள பய ப வ ஒ சிற த ைற.
ஆசி , ேபசி வேயாெல , ேபசி மெஜ டா, ெம ேநவ ,
ேம என ஐ வைகயான ெநச சா த சாய கள ம
மி க ெரசிேமாச , ைரேசா பச ெடாேலான ஃெப , ஃ ேச ய
ஆ ஸி ேபார , ஃ ேச ய ெமான லிஃெபா ெம, ைர ேகாெட மா
வ , அ ெப ஜி ல ெட ர , அ ெப ஜி ல ஒ ராசிய ,
ெபன சிலிய சி ன , சா கேராைம க ெச வ சிேய,
ெவேராைம க மஃஸியான , ைர ேகாெட மா சி ேபா ற
பதிேனா ைச வைககள தா க ப ேசாதி க ப ட . இ த
ேசாதைனய வ ப வைககள கண சமான சிைத
ப யற க க டறிய ப ட . அதி அதிக தா க இ த 2
சாய க டனான 6 ைச வைககள ேச ேசாதைனக ,
ப ளா க -ப ம ைற ல உக தா க
ேத ெத க ப டன. ஏ கிய அள க ஆ
உ ப த ப அவ றி தா க ப ளா க -ப ம ைற ல
கண கிட ப டன. அவ கிய வ வா த 3 அள க
அ தக ட ைறயான பா -ெப க ைற உக தா க
உ ப த ப டன. இ த ஆ வ 96.78% ஆசி சாய
ெப.சி ன தா 90.78% ைர. ெடாேலான ஃெபரா 60.37%
ஃ .ெமான லிஃெபா ெமவா அக ற ப ட க டறிய ப ட . ெப.
சி ன தி உக த நிைலகளாக 0.2 மிகி/மிலி சாய ெசறி , 2 நா க
ெதாட ேநர ம 5.38% (எ/அ) ய ல வ
வள கிய . ைர. ெடாேலான ஃெப 0.14 மிகி/மிலி சாய ெசறி
36.98°சி ெவ பநிைல ம 5.47% (எ/அ) ய ல வ
வள கிய . ஃ . ெமான லிஃெபா ெம 0.20 மிகி/மிலி சாய ெசறி ,
அமில-கார சமநிைல 4, ழ சி ேவக 0 ஆ .ப .எ மாக அறிய ப ட .
இ த ஆ வ க ல , ஆசி சாய ைத ெபன சிலிய
சி ன , ஃ ேச ய ெமான லிஃெபா ெம ம ைரேசா பச
ெடாேலான ஃெப ல திற மி க வைகய ப யற க
ேதைவயான உக த நிைலக ச யாக வ க ப டன.
TABLE OF CONTENTS
CHAPTER TITLE PAGE

1. INTRODUCTION 1
1.1 General 1
1.2 Motivation 8
1.3 Objectives 9
2. REVIEW OF LITERATURE 10
3. MATERIALS AND METHODS 28
3.1 Culture collection 28
3.2 Procedure for preservation of pure cultures 28
3.2.1 Revival of cultures 28
3.2.2 Immersion in distilled water 29
3.2.3 Preservation by Oil Overlay 29
3.3 Growth curve studies 29
3.4 General procedure for dye degradation 30
3.5 Screening studies 30
Optimization studies on mycoremediation of
3.6 31
synthetic dyes by design of experiments
3.6.1 Plackett Burman Design 31
3.6.2 Box Behnken Design 33
4. RESULTS AND DISCUSSION 34
4.1 Standard curve 34
4.2 Screening experiments 35
4.3 Growth Curves 36
4.4 Plackett Burman Design 39
Box Behnken Design for Penicillium
4.5 43
citrinum vs Acid green
v
Box Benkhen Design for Rhizopus stolonifer
4.6 49
vs Acid green
Box Benkhen Design for Fusarium
4.7 54
moniliforme vs Acid Green
5 CONCLUSION 61
APPENDICES 62
REFERENCES 64
vi
LIST OF TABLES
TABLE PAGE
TITLE
No. No.
2.1 Consolidated literature results of dye degradation using Fungi 26
2.2 Consolidated literature results of dye degradation using Fungi 27
3.6.1 Actual levels of variables tested with Plackett-Burman design 32
3.6.2 Plackett Burman Matrix for seven factors and eight experiments 33
4.1 Screening experimental results for dye degradation 35
4.2 Plackett Burman experimental Design 40
4.5 Process variables and level used for Box Behnken Design 43
4.6 Box Behnken experimental design 44
Model coefficients estimated by multiple linear regression and
4.7 45
analysis of variance
Regression coefficients and significance of response surface
4.8 46
quadratic model
4.11 51
4.12 51
quadratic model
4.15 56
4.16 57
quadratic model
A1.1 Composition of Potato sucrose Agar 62
A1.2 Composition of Potato Dextrose Agar 62
A1.3 Composition of Malt extract agar 62
A1.4 Composition of Czapek Yeast extract Agar –CYA 63
A1.5 Composition of Czapek Concentrate 63
A1.6 Composition of Yeast Extract Peptone Dextrose 63
vii
LIST OF FIGURES
FIGURE PAGE
TITLE
No. No.
4.1 Standard curve for Acid green 34
4.2 Standard curve for Basic violet 35
4.3 Growth curve of Fusarium oxysporum 37
4.4 Growth curve of Penicillium citrinum 37
4.5 Growth curve of Rhizopus stolonifer 38
4.6 Growth curve of Trichoderma viride 38
4.7 Growth curve for Fusarium moniliforme 39
4.8 Growth curve for Aspergillus terreus 39
4.9 Effect Vs Variables 42
Response Surface and contour plot for time vs. dye concentration for
4.1 47
at constant inoculum size on % dye removal
Response Surface and contour plot for dye concentration vs.
4.11 48
inoculum size at constant time on % dye removal
Response Surface and contour plot for time vs. inoculum size for at
4.12 48
constant dye concentration on % dye removal
Response Surface and contour plot of for temperature vs. dye
4.13 52
concentration for at constant inoculum size on % dye removal
Response S
4.14 urface and contour plot for dye concentration vs. inoculum size at 53
constant temperature on % dye removal
Response Surface and contour plot for temperature vs. inoculum size
4.15 54
for at constant dye concentration on % dye removal
Response Surface and contour plot of for agitation speed vs. dye
4.16 58
concentration for at constant pH on % dye removal
Response Surface and contour plot for dye concentration vs. pH at
4.17 59
constant agitation speed on % dye removal
Response Surface and contour plot for pH vs. agitation speed for at
4.18 59
constant dye concentration on % dye removal
viii
INTRODUCTION
1.1 Overview
Fungi are microscopic, eukaryotic organisms exhibiting growth on

various substrates and arecapable of continuing their function almost
indefinitely. These organisms, including the molds, yeasts, and filamentous
fungi are a diverse group of organisms which are ubiquitous in nature,
having their own specific adaptations to survive in their particular
ecosystem. They have contributed to human welfare since time
immemorial, from wine to dairy. Their contribution ranges from natural to
industrial use. They contribute to maintaining the ecosystem through their
complex mycelial network which aids in exchanging nutrients. The
mycelial networks sometimes cover hectares of the forest floor. The
numerous fungi found in nature co-ordinate with other fungi and microbes
alike to regulate nutrient flow and keep the ecosystem intact.
1.1.1 DYES
Dyes are colored substances, which are used along with a mordant to
give color to fibers.There are many classes of dyes.
1
 Azo Dyes
Azo dyes constitute the most versatile and largest class of synthetic
dyes employed commercially in the textile and food industries. More than
2000 different azo dyes are used to dye various materials, such as textile,
leather, plastic, cosmetics, and food. These are characterized by the
presence of one or more azo bonds (-N<=>N-) in association with one or
more aromatic systems that may also carry sulfonic acid groups. These are
also the most common class of dyes released into aquatic and terrestrial
environments through the effluents, resulting in the contamination of rivers
and ground water. Cationic dyes are more toxic, followed by anionic acid
and direct dyes.
 Phthalocyanine Dyes
Phthalocyanine dyes are widely used in the textile and dyestuff

industries, but little is known about the decolorization and biodegradation
of these compounds by microorganisms. Significant amounts of LiP or
MnP activity were not detected during the decolorization of CuPc by P.
chrysosporiumPC671 (Conneely et al., 1999). This suggests little or no
relation between the production of ligninolytic enzymes and decolorization
of this dye.
 Anthraquinone Dyes
Both tropho- and idiophasic cultures of TrametestrogiiBAFC 463

produce high levels of laccase activity under all conditions and may be
2
related to the decolorization of Anthraquinone Blue (Levin et al., 2001).
T.versicolor degrades Acid Green 27 faster by extracellular than by
intracellular enzymes (Wang et al, 1998).
 Heterocyclic Dyes
Little is known about color removal of heterocyclic dyes. This may be

due to the fact that these dyes are not used extensively in the textile
industries.
 Triphenylmethane Dyes
Triphenylmethane dyes are employed extensively in various industries

due to their versatility. Numerous applications of triphenylmethane dyes
are well known in the textile industries, such as dying cotton, nylon, silk,
and wool. These dyes are also used in the paper and leather industries, and
many are used as biological stains and in veterinary medicine. They are
also used in coloring plastics, fats, oils, and waxes and have applications in
the cosmetics and food industries. Some triphenylmethane dyes are used
extensively in medicine as dermatological agents.
1.1.2 Dye degradation methods
In recent years, worldwide efforts have been employed to develop

more effective colour removal processes. However, no universal method is
known for the treatment of dye wastewaters because of the complex and
varied chemical structure of these compounds. Research in the past two
3
decades has focused on the complexity of decolourization techniques of
dye effluents.
(a)Physicochemical Methods
Physical, chemical, and electrochemical methods produce a large

volume of sludge. These methods also exhibit noticeable differences in
color removal, volume capability, duration of operation, and capital cost.
Color in large volumes of wastewaters can be removed rapidly by
membrane technology and ozone treatment, but the capital cost is high.
Phthalocyanine dyes are quite stable in advanced oxidation processes, such
as TiO2/UV, Fenton, and photo- Fenton reagents. Dyes can be removed
more effectively by magnesium chloride than by alum and. Poly aluminium
chloride. Obviously, both physical and chemical methods have
shortcomings and are expensive.
(b) Biological Methods
 Degradation by Bacteria
Presently, most of the textile industries are developing wastewater

remediation technologies on-site in order to discharge below the criteria
limits. A variety of bacterial bioreactors have been proposed to obtain an
effective continuous anaerobic/aerobic treatment of dyes. These include (1)
aerobic activated-sludge or rotating biofilm reactors (Shaul et al., 1991;
Jiang and Bishop, 1994); (2) anaerobic fixed-film fluidized-bed reactors
followed by aerobic suspended-bed activated sludge reactors (Seshadri et
4
al., 1994; Fitzgerald and Bishop, 1995); (3) semicontinuous anaerobic
reactors (Mass and Chaudhari, 2005); (4) anaerobic upflow fixed-bed
columns with aerobic agitated tanks (An et al., 1996; Rajaguru et al.,
2000); (5) aerobic–anaerobic sequential batch or continuous-flow reactors
(Oxspring et al., 1986; Loyd et al., 1992; Ganesh et al., 1994); (6)
combination of anaerobic and aerobic rotating drum reactors (Harmer and
Bishop, 1992; Sosath and Libra, 1997); (7) anoxic–aerobic sequential
bioreactors (Khehra et al., 2006); and(8) fed-batch column reactors
(Kapdan and Oztekin, 2003).
 Degradation by Actinomycetes
A soil actinomycete, Streptomyces chromofuscus, exhibits less

decolorization than the white-rot fungus Phanerochaete chrysosporium
(Paszczynski et al., 1992). Several actinomycete strains decolorize the
reactive dyes, including azo, anthraquinone, and phthalocyanine, through
adsorption (Zhou and Zimmermann, 1993). Pseudomonas luteola
completely degrades the reactive azo dyes
 Degradation by Algae and Higher Plants
Azo dyes are degraded by an induced form of azoreductase by algae.

Several species of Chlorella and Oscillatoria degrade azo dyes to aromatic
amines and eventually, biotransform to simpler organic compounds or
CO2. Some species exhibit the ability to utilize azo dyes as the sole source
of carbon and nitrogen. These algae can be added to stabilization ponds for
the elimination of aromatic amines.
5
(c)Fungal decolorization and degradation of dyes
During the past decade, fungi have been demonstrated to degrade

various classes of dyes. It is a good alternative to replace or supplement
present treatment processes. Fungal treatment of dyes is an economical and
feasible method for decolorizing textile wastewaters.
White-rot fungal decolorization and degradation of dyes
The role of the white-rot fungus, Phanerochaete chrysosporiumin

the degradation of polymeric dyes was fi rst established by Glenn and Gold
(1983). Since then, several papers have appeared on the use of P.
chrysosporium in the decolorization and degradation of dyes. In addition,
other white-rot fungi (e.g., Trametes versicolor, Pleurotus ostreatus, and
Bjerkandera sp.) have also been found to be efficient in degrading dyes.
Several reports indicate the superiority of species of Trametes and
Bjerkandera over P. chrysosporiumin the rate and extent of decolorization
of different dyes. Factors such as media composition, pH, carbon and
nitrogen sources, TOC/N ratio, incubation time, ionic strength, and initial
dyestuff concentrations have a profound effect on the rate of color removal
and the process of biodegradation.
1.1.3 Optimization methods
Medium optimization by the classical method bychanging one

independent variable (nutrients, antifoam, pH, temperature, etc.) while
fixing all the others at certain level can be extremely time consuming and
6
expensive for a large number of variables. To make full factorial search
which would examine each possible combination of independent variable
at appropriate levels could require a large number of experiments, where x
is the number of levels and n is the number variables. This may be quite
appropriate for threenutrients at two concentrations (23 trials) but not forsix
nutrients at three concentrations. In this instance 36(729) trials would be
needed. Industrially the aim is to perform the minimum number of
experiments to determine optimal conditions. Other alternative strategies
must therefore be considered which allow more than one variable to be
changed at a time.
When more than five independent variables are to be investigated,

the Plackett-Burman design may be used to find the most important
variables in a system, which is then optimized in further studies (Plackett
and Burman, 1946). These authors give a series of designs for up to one
hundred experiments using an experimental rationale known as balanced
incomplete blocks. This technique allows for the evaluation of X-I
variables by X experiments. X must be a multiple of 4, e.g. 8, 12, 16, 20,
24, etc. Normally one determines how many experimental variables need to
be included in an investigation and then selects the Plackett-Burman design
which meets that requirement most closely in multiples of 4. Any factors
not assigned to a variable can be designated as a dummy variable.
Alternatively, factors known to not have any effect may be included and
designated as dummy variables.
The next stage in medium optimization would be to determine the

optimum level of each key independent variable which has been identified
7
by the Plackett-Burman design. This may be done using response surface
optimization techniques which were introduced by and Wilson (1951).
Hendrix (1980) has given a readable account of this technique and the way
in which it may be applied. Response surfaces are similar to contour plots
or topographical maps. Whilst graphical maps show lines of constant
elevation, contour plots show lines of constant value. Thus, the contours of
a response surface optimization plot show lines of identical response. In
this context, response means the result of an experiment carried out at
particular values of the variables being investigated. The axes of the
contour plot are the experimental variables and the area within the axes is
termed the response surface. To construct a contour plot, the results
(responses) of a series of experiments employing different combinations of
the variables are inserted on the surface of the plot at the points delineated
by the experimental conditions. Points giving the same results (equal
responses) are then joined together to make a contour line. In its simplest
form two variables are examined and the plot is two dimensional.
1.2 Motivation
It has been two years since the Madras High Court delivered a
landmark judgment ordering the closure of dyeing and bleaching units in
the Tirupur knitwear cluster for polluting the river Noyyal for decades. The
order was pronounced solely because the dyeing fraternity did not adhere
to the zero liquid discharge (ZLD) norms despite the directions from the
Supreme Court and High Court. According to the Tamil Nadu Pollution
Control Board, over 200 illegal units were identified in Tirupur as well as
the nearby districts of Namakkal, Erode and Salem .This closure of dyeing
8
units though directed towards the welfare of the environment and public
safety resulted in job losses. Since the demand for textiles is always on the
high, a suitable solution would be to develop a feasible method for
degrading the various dyes used in these industries .Thus, we were
motivated towards finding a biological alternative to the various
physiochemical dye degradation processes and fungi with all their
diversity, proved to be the perfect comrade for our mission.
1.3 Objectives
 To screen suitable fungi for remediation of synthetic dyes.

 To screen and identify the variables for mycoremediation of dyes using
Plackett-Burman design.
 To optimize the dye remediation process by Box-Behnken design from
the selected variables.
9
CHAPTER 2
REVIEW OF LITERATURE
Aspergillus foetidus
Aspergillus foetidus is a mold which is found to be allergenic for

some individuals like those with suppressed immune systems. However,
to this day, there are no severe health issues related to this type of mold.
Since there is limited information concerning its impact on human
health, people should be wary when dealing with this type of mold as
there is still no evidence available about its effects. Aspergillus foetidus
is not commonly seen in indoor environments. In fact, this type of
Aspergillus mold has several uses in various industrial processes. For
instance, it is used to produce koji for shochu which means a distilled
Japanese alcoholic beverage and it is also utilized for the production of
many useful enzymes that serve differing purposes. Aspergillus foetidus
is widely used to degrade Drimarene Red (Sumathi and Manju, 2000),
Drimarene Blue (Sumathi and Manju, 2000), Drimarene Black (Sumathi
and Manju, 2000). Aspergillus foetidus decolorized 65 % of Drimarene
Red,72 % of Drimarene Blue and 70 % of Drimarene Black .Recently,
the study of decolourization of liquid media containing bagasse-based
pulp and paper mill wastes through bioadsorption by the isolated fungus
Aspergillus foetidus was reported.(Sumathi and Manju, 2000).
10
Bjerkandera adusta
Bjerkandera adusta is a species of fungus in the Meruliaceae

family. It is a plant pathogen that causes white rot. It was first described
scientifically as Boletus adustus by Carl Ludwig Willdenow in 1787.
Bjerkandera adusta and Phlebia tremellosa cultures were obtained from
the Canadian Culture Collection, Alberta, Canada. Fungi were
maintained on PDA plates and stored at 4°C and sub cultured every
month. In recent years many studies have demonstrated that the white
rot fungi, Bjerkandera adusta, is able to degrade a broad spectrum of
structurally diverse dyes. Bjerkandera adusta decolorized 85 %
Cibracon Yellow CR (Robinson et al, 2001).
Candida zeylanoidis
The genus Candida includes around 154 species. Among these,

six are most frequently isolated in human infections. While Candida
albicans is the most abundant and significant species, Candida
tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, and
Candida lusitaniae are also isolated as causative agents of Candida
infections. Candida is an yeast and the most common cause of
opportunistic mycoses worldwide. It is also a frequent colonizer of
human skin and mucous membranes. Candida is a member of normal
flora of skin, mouth, vagina, and stool. As well as being a pathogen and
a colonizer, it is found in the environment, particularly on leaves,
flowers, water, and soil. While most of the Candida spp. are mitosporic,
some have known teleomorphic state and produce sexual spores.
Infections caused by Candida spp. are in general referred to as
candidiasis. The clinical spectrum of candidiasis is extremely diverse.
11
Almost any organ or system in the body can be affected. Candidiasis
may be superficial and local or deep-seated and disseminated.
Disseminated infections arise from hematogenous spread from the
primarily infected locus. Candida zeylanoidis is used to decolorize 85 %
of Orange II (Yesilada et al, 2003) and 90 % of Methyl orange (
Yesilada et al, 2003).
Coriolus versicolor
Coriolus versicolor also known as Trametes versicolor, turkey

tail mushroom and Polyporus versicolor — is a common polypore
mushroom found throughout the world. The protein-bound
polysaccharides or polysaccharopeptides produced by Coriolus
versicolor. These polymers are structurally distinct; they are not
distinguishable in terms of their physiological activity. In addition to its
medical applications, C. versicolor is widely used to degrade recalcitrant
organic pollutants such as pentachlorophenol (PCP). The visible form of
C. versicolor is a fan-shaped mushroom with wavy margin and colored
concentric zones. C. versicolor is an obligate aerobe that is commonly
found year-round on dead logs, stumps, tree trunks, and branches. The
fungus occurs throughout the wooded temperate zones of Asia, Europe,
and North America and may be the most common shelf fungus in the
Northern Hemisphere. The mushroom belongs to the family
Basidiomycotina. Coriolus versicolor is widely used to degrade
Remazol Briliant Blue R (Christian et al 2005), Malachite green (Levin
et al., 2004), Azure B (Levin et al., 2004), Remazol Brilliant Violet (
Sanghi et al., 2006), Crystal violet (Yesilada 1995), Poly R 478 ( Levin
et al., 2004), Xylidine(Levin et al., 2004), Carmine Indigo, Remazol
12
Red RR (Yi Chin Toh et al., 2003), Remazol Blue RR (Yi Chin Toh et
al., 2003), Everzol Turquoise Blue G (Kapdan and Kargi, 2002),
Astrazone Black FDL (Yesilada et al., 2003) and Astrazone Red FBL
(Yesilada et al., 2003). (Kapdan and Kargi, 2002; Yesilada et al., 2003;
Levin et al., 2004; Sanghi et al., 2006; Yesilada 1995; Yi Chin Toh et
al., 2003; Christian et al 2005 ). 82% of Everzol Turquoise Blue G, 89%
of Astrazone Black FDL, 98% of Astrazone Red FBL, 43 % of Remazol
Briliant Blue R, and 88% of Malachite green dye has been degraded.
Cunninghamella elegans
Studies on non ligninolytic fungi metabolizing dyes are minimal,

and only recently, has there been a report that Cunninghamella elegans
ATCC 36112 was able to metabolize 85% of the triphenylmethane dye
malachite green after 24 h of incubation. The mechanism of this
degradation process by C. elegans is yet to be elucidated; however, this
fungus is capable of metabolizing a wide range of compounds,
particularly by demethylation and oxidation. The main objective of the
present study was to examine the decolorization of three reactive azo
dyes and their mixture by mycelium of C. elegans in presence or
absence of carbon and/or nitrogen sources, as well as the determination
of the toxicity of dyes after the action of the fungus by reduction of
Escherichia coli respiration. Studies on non-ligninolytic fungi
metabolizing dyes are minimal, hence the mechanism of this
degradation process by C. elegans is yet to be elucidated; however, this
fungus is capable of metabolizing a wide range of compounds,
particularly by demethylation and oxidation. Cunninghamella elegans
employs to decolorize Reactive Black 5 (Ambrosio and Takaki ,2004),
13
Orange II (Ambrosio and Takaki ,2004), Reactive Red 198 (Ambrosio
and Takaki ,2004), Malachite green (Chang-Jun Cha et al, 2001) .
Cunninghamella elegans decolorized 40 % of Reactive Black 5, 88 %
of Orange II,75% of Reactive Red 198 ,85 % of Malachite green.
(Chang-Jun Cha et al, 2001; Ambrosio and Takaki ,2004).
Debaromyces polymorphus
The genus Debaryomyces comprises 15 species. Many

representatives can be isolated from natural habitats such as air, soil,
pollen, tree exudates, plants, fruits, insects, and faeces and gut of
vertebrates. The presence of Debaryomyces species in foods usually has
no detrimental effects and in some cases is beneficial to the food. Some
Debaryomyces species are important in the ripening of fermented foods
like cheese and meat products. In cheeses they metabolise lactic acid,
thus raising the pH to allow the growth of proteolytic bacteria, and
exhibit lipolytic activity that contributes to the development of cheese
aromas. Proteolytic and lipolytic activities of Debaryomyces species
have been described in curing of ham and ripening of sausages, and their
presence in salami influences the red coloration and improves the
quality of the product. To a lesser extent, Debaryomyces species
contribute to the ripening of pickles, where they oxidize the acids
produced by lactic acid bacteria during fermentation. Excessive growth
of Debaryomyces species may nevertheless cause undesirable sensory
changes due to the formation of off-odours and off-flavours. A high salt
concentration favours the growth of Debaryomyces species in aged
cheeses, dry salami and meat products. These species have also been
found as frequent contaminants in spoiled yoghurts, ice creams, fish,
14
shellfish, etc. Debaryomyces polymorphus decolorized 98.9 % of
Reactive Black 5 dye.( Yang et al, 2005)
Funalia trogii
White rot fungi, Funalia trogii can degrade a wide variety of

structurally diverse pollutants . In recent years many studies have
demonstrated that fungi were able to decolorize and remove (bio
sorption) textile dyes. Most research is on dyes involving two fungi,
Phanerochaete chrysosporium and Coriolus versicolor . There is not
much information on decolorization activity of Funalia trogii . Also,
there is limited study on dye decolourization ability of the pellet of
white rot Fungi. The effect of various culture conditions on the dye
decolourization activity of F. trogii ATCC 200800 pellets and its ability
to decolorize Astrazone Blue dye upon repeated addition were also
determined. Moreover, the effect of glucose concentration and cheese
whey on longevity of dye decolourization activity of F. trogii pellets
was tested. Funalia trogii decolorized Astrazone Black FDL (Yesilada
et al, 2003), Astrazone Blue FGRL (Yesilada et al, 2003), Astrazone
Red FBL (Yesilada et al,2003) and Reactive Black 5 (Ali Mazmanci and
Ali Unyayar,2005) . Funalia trogii decolorized 94 % of Astrazone
Black FDL ,92 % of Astrazone Blue FGRL ,97% of Astrazone Red FBL
and 99 % of Reactive Black 5 . (Ali Mazmanci and Ali Unyayar, 2005;
Yesilada et al, 2003).
15
Geotrichum sp
Geotrichum species are common yeast-like fungi whose primary

mode of reproduction is the formation of arthrospores. The genus
Geotrichum should not be identified by microscopic morphology alone
because many related and unrelated fungi form arthrospores.
Biochemical analysis is necessary for identification. Geotrichum
candidum is considered part of the normal microbiota of humans. Health
effects include reports of endocarditis, encephalitis, and osteomyelitis in
immunosuppressed hosts. Pulmonary infections have also been
described. Many of these reports lack proper documentation and may be
based on unreliable identifications. No information is available
regarding toxicity. Allergenicity has not been well studied. Tape lifts
and tease mounts from bulk samples may reveal the presence of an
arthrospore-forming yeast; isolation on culturable (Andersen) air
samples is possible but infrequent. This genus is cosmopolitan, isolated
from soil, plants, and many food products, most especially from milk
and milk products. Geotrichum sp. was isolated from Brewery spent
grain using standard microbiological procedures. It was identified by
Microbial ID, Inc. (Newark, USA). This strain was selected from among
several fungi tested because of its ability to decolourize synthetic dyes
incorporated in the agar growth medium. In SDA medium (containing
65 g/l of Sabouraud dextrose agar (BBL) plus 2 g/l agar) it produced
rapidly growing white powdery colonies with hyaline septate hyphae
and rectangular unicellular spores (arthroconidia). Colourless conidia
are formed in chains and later released by fragmentation of the
vegetative hyphae. Geotrichum species decolorized 80 % of Reactive
Black 5 ( Maximo et al,2003), 90 % of Reactive Red 158 ( Maximo et
al,2003) and 95 % of Reactive Yellow 27 ( Maximo et al,2003).
16
Irpex lacteus
Irpex lacteus is commoly called as the “Milk-white Toothed

Polypore.” It belongs to the phylum Basidiomycota, class
Hymenomycetes, order Aphyllorphorales, family Polyporaceae and
genus Irpex. Fruiting body appears to be similar in colour to milk, but
may range from white, to off-white or cream coloured. Pores break up
into short teeth (less than 3 mm long) when mature. Fruiting body is dry
and stiff. Irpex lacteus, a cosmopolitan white rot fungus, has been
studied in connection with proteinase and cellulase production, lignin
degradation and degradation of PAHs in shallow stationary cultures .
Good capacity for decolourization of azo, anthraquinone,
phthalocyanine and triphenylmethane dyes by I. lacteus in stationary
liquid cultures has been demonstrated . The fungus was also able to
degrade an anthraquinone dye in contaminated soil and decolorized 100
% of Remazol Briliant Blue R (Kasinath et al, 2003).
Laetiporus sulphurous
Laetiporus sulphureus is a species of bracket fungus (fungus that

grows on trees) found in Europe and North America. Its common names
are sulphur polypore, sulphur shelf, and chicken of the woods. Its fruit
bodies grow as striking golden-yellow shelf-like structures on tree
trunks and branches. Like other bracket fungi, they may last many years
and fade to pale grey or brown. The under surface of the fruit body is
made up of tube like pores rather than gills. Laetiporus sulphureus is a
saprophyte and causes brown cubical rot in the heartwood of trees on
which it grows. Unlike many bracket fungi, it is edible when young. The
17
cap is small and knob-shaped, overlapping in an irregular pattern.
Wide, shaped like a fan and attached direct to the trunk of a tree, it has a
shelf-like appearance and is sulphur-yellow to bright orange in colour
and has a suedelike texture. When it is old the cap fades to tan or white.
The shelves often grow in overlapping clumps, and each one may be
anywhere from 5 to 60 cm (2 to 24 in) in diameter and 4 cm (1.4 in)
thick. The fertile surface is sulphur-yellow with small pores or tubes and
has a white spore print. Some people have had gastrointestinal upset
after eating this mushroom. Studies have shown severe adverse
reactions, including vomiting and fever, in about 10% of the population.
The mushroom produces the Laetiporus sulphureus lectin (LSL) which
has haemolytic and haemagglutination activities. Haemolytic lectins are
sugar-binding proteins that lyse and agglutinate cells. The
haemagglutination and haemolytic activity are started by binding
carbohydrates. Laetiporus sulphureus bleach out 86 % of crystal violet.(
Yesilada ,1995).
Phanerochaete chrysosporium
Phanerochaete chrysosporium is a resupinate basidiomycetes

belonging to the family Corticiaceae typically form effused, very flat
fruiting bodies that appear in nature as no more than a crust on the
underside of a log. P. chrysosporium displays both homothallic and
heterothallic-bipolar sexuality. Microscopic examination of 2-week
mycelial mats typically reveals simple septate hyphae ranging from 3-9
µm in diameter with sparse to moderate branching as well as the
presence of thick-walled terminal or intercalary chlamydospores 50-60
µm in diameter. The blastoconidia are round to ellipsoid in appearance,
18
6-9 µm in diameter and are borne by poorly differentiated branched
conidiophores.
The white-rot fungus Phanerochaete chrysosporium secretes,

during its secondary metabolism, several lignin-degrading enzymes
including lignin peroxidise (LiP), manganese-dependent peroxidase
(MnP) and laccase. The secondary metabolism in this fungus is
triggered by nitrogen, carbon or sulphur deprivation. In addition to
lignin, the above-mentioned enzymes are also able to degrade a wide
range of hazardous environmental pollutants. Their application to
industrial processes (biobleaching, biopulping, decolourisation, etc.) on
a large scale requires the production of high amount of enzyme at low
cost. Thus, the design of a system permitting the continuous production
of ligninolytic enzymes efficiently is required [Domıńguez Alberto et
al, 2001 ]. Extensive studies on the effectiveness of dye decolourisation
by the WRF, Phanerochaete chrysosporium have been reported [Toh
Yi-Chin et al, 2003]. Although P.chrysosporium has the ability to
degrade various dyes under aerobic conditions, there are two major
constraints on its application to industrial wastewater treatment.
Phanerochaete chrysosporium, the most extensively studied white-rot
fungus, produces several extracellular glycosylated haem-proteins [1–3]
and it is a model organism for lignin and xenobiotic biodegradation
studies. This organism is able to transform a wide range of organic
compounds due to their extracellular, nonspecific ligninolytic enzymes
such as lignin peroxides, LiP (EC 1.11.1.14), manganese peroxidase,
MnP (EC 1.11.1.13) and laccases (EC 1.10.3.2) as well as hydrogen-
producing oxidases [Pazarlioglu et al, 2004]. P. chrysosporium degrades
not only natural and industrial lignin derivatives but also a broad range
of man-made aromatic compounds, including diverse environmental
19
pollutants [Pazarlioglu et al, 2004]. The white rot fungus P.
chrysosporium MTCC 787 was obtained from the Culture Collection of
Institute of Microbial Technology, Chandigarh, India and the stock
cultures were maintained by periodic subculture on malt agar medium at
48C[Radha et al, 2005]. 95 % of Astrazone Black FDL (Yesilada et al,
2003), 97 % of Astrazone Blue FGRL (Yesilada et al, 2003), 99 % of
Astrazone Red FBL(Yesilada et al, 2003), Reactive Black 5 (Ali
Mazmanci and Ali Unyayar,2005), 98.5 % of amaranth (red) (Chagas
and Durrant ,2001), 95 % of new coccine (red) (Chagas and Durrant
,2001), 96.8 % of orange G (orange) (Chagas and Durrant ,2001) ,60 %
of tartrazine (yellow), (Chagas and Durrant ,2001), 95 % of Remazol
Blue RR (Toh Yi-Chin et al,2003) , 97 % of Remazol Red RR (Toh Yi-
Chin et al,2003) ,50 % of Remazol Yellow RR (Toh Yi-Chin et
al,2003), 98 % of Acid orange (Radha et al ,2005), 89 % of Acid red
114 (Radha et al ,2005), 98 % of Methyl violet (Radha et al ,2005), 75
% of Acid green (Radha et al ,2005), 86 % of Methylene blue (Radha et
al ,2005) , 91 % of Vat magenta (Radha et al ,2005), 54 % of Congo
red(Radha et al ,2005),75 % of Indigo Blue (Balan and Monteiro,
2001), 90 % of crystal violet ( Bumpus and Brock, 1988),73 % of
Reactofix Gold Yellow ( Capalash and Sharma, 1992),54 % of congo
Red (Ollikka et al ,1993),99 %of Orange II (Cripps et al ,1990).
Pleurotus eryngii and Pleurotus florida
Pleurotus eryngii degraded 10 % of reactive black 5 (Mazmanci

and Ünyaya, 2005) and Pleurotus florida degraded 75% of aztrazone
black FDL (Yesilada et al, 2003) and aztrazone blue FGRL (Yesilada et
al, 2003). 97% of aztrazone red FBL (Yesilada et al, 2003) and 40 % of
20
Reactive black 5 was also removed by Pleurotus florida (Mazmanci
Ünyaya, 2005)
Pleurotus ostreatus
Pleurotus ostreatus, the oyster mushroom, is a common edible

mushroom. It is related to the similarly cultivated "king oyster
mushroom". Oyster mushrooms can also be used industrially for
mycoremediation purposes. The oyster mushroom may be considered a
medicinal mushroom, since it contains statins such as lovastatin which
work to reduce cholesterol.
P. ostreatus strain 32 was obtained from Dalian institute of

mushroom study and the Canadian Culture Collection, Alberta, Canada.
Fungal cultures were maintained on PDA plates and stored at 4°C and
subcultured every month. P. ostreatus strain 32 was chosen from 15
edible basidiomycete fungi secreting laccases due to its high laccase
activity. [Hou Hongman and Zhou Jiti, 2003] The white rot fungus
(WRF) Pleurotus ostreatus also produced manganese peroxidase (MnP)
and manganese-independent peroxidase (MIP). These enzymes play a
major role in the lignolytic activity of the fungi and also the dye
degradation properties. [Shrivastava, R et al,2004].
Pleurotus sajor-caju
Pleurotus sajor-caju known as the Dhingri Oyster or Grey

Albalone Oyster is the most popular cultivated strain of Oyster
mushrooms grown mainly in India and the tropics, suggesting a better
tolerance to higher growing temps. P. sajorcaju totally decolorized
21
amaranth, new coccine and orange G, but only grew on the solid
medium containing tartrazine that was not visibly decolorized. This
mushroom is cultivated on a wide range of plant wastes (cereal straw,
sawdust, bagasse, waste cotton) often enclosed by plastic bags.
Mushroom production is light dependent. Some growers operate a 12
hour light cycle using fluorescent lamps. Pleurotus mushrooms are the
second most important mushrooms in production in the world, 25% of
total world production of cultivated mushroom. Pleurotus sajor-caju
decolorized 94 % of Indigo Blue, 80 % of Astrazone Black FDL, 97 %
of Astrazone Blue FGRL and 99 % of Astrazone Red FBL. ( Balan et
al., 2001 ; Yesilada et al., 2003).
Pleurotus sapidus and Pycnoporus sanguineus
Pycnoporus sanguineus was used to degrade 99 % of

bromophenol blue (Pointing and Vrijimoed, 2000), 97.2 % of orange G
(Pointing and Vrijimoed, 2000), 91% of crystal violet (Pointing and
Vrijimoed, 2000) and 97 % of amaranth (Pointing and Vrijimoed, 2000).
Pleurotus sapidus degraded 4% of reactive black 5 (Mazmanci and
Ünyaya, 2005).
Pleurotus pulmonarius
Pleurotus pulmonarius, commonly known as the Indian Oyster,

Phoenix Mushroom, or the Lung Oyster, is a mushroom very similar to
Pleurotus ostreatus, the pearl oyster, but with a few noticeable
differences. In North America, P. pulmonarius also closely resembles
22
Pleurotus populinus, which is restricted to growing on aspen and
cottonwood (genus Populus)
P. pulmonarius CCB-19 was obtained from the São Paulo Botany

Institute Culture Collection. It was cultured on potato dextrose agar
slants for 2 weeks at 28 ◦C. When the slant was fully covered with the
mycelia, mycelial plugs measuring 10 mm in diameter were made and
used as inoculum for solid-state cultures.[ Tychanowicz et al, 2003]
Phlebia tramallosa
Phlebia tremellosa is a saprobic; resupinate fungus normally

present in overlapping clusters, mostly on decaying deciduous wood.
This funky mushroom is widespread in North America, and can be
found on the dead wood of hardwoods or, occasionally, conifers. Its
typical form is a classic example of what mycologists call an "effused-
reflexed" fruiting body; it spreads its spore-bearing surface over the
wood and musters up just enough cap-making umph to fold over its
upper edge into a slight extension. Other distinguishing features include
the translucent, orangish to pinkish spore-bearing surface, which
develops deep folds and pockets
Phlebia tremellosa cultures were obtained from the Canadian

Culture Collection, Alberta, Canada. Fungi were maintained on PDA
plates and stored at 4°C and subcultured every month. P. tremellosa
successfully degraded 79% of the artificial effluent after 9 days. There
were sharp increases in LiP and MnP activities between days 7 and 9,
corresponding to a decline in the dye concentrations left in the effluent
[Robinson et al, 2001].
23
Saccharomyces cerevisiae
Saccharomyces cerevisiae is capable of utilizing a variety of

carbon and nitrogen sources, molasses was chosen in the study due to its
high sucrose and other nutrient contents, low cost and ready availability
and ease of storage. Saccharomyces cerevisiae is in the fungi kingdom.
Saccharomyces when translated means “sugar fungus”. That is what this
yeast uses for food. They are found in the wild growing on the skins of
grapes and other fruits. The reasons for this classification are because it
has a cell wall made of chitin, it has no peptidoglycan in its cell walls,
and its lipids are ester linked. It also uses DNA template for protein
synthesis and it has larger ribosomes. It is then consider yeast because it
is a unicellular organism so it cannot form a fruiting body; like other
fungi. Saccharomyces cerevisiae has adapted in several important ways.
One is the fact that they are able break down their food through both
aerobic respiration and anaerobic fermentation. They can survive in an
oxygen deficient environment for a period. Another adaptation they
have is their ability to have both sexual and asexual reproduction. Very
few other Ascomycota can do both processes. And very few organisms
can do all four of these processes. This allows this species to live in
many different environments. Saccharomyces cerevisiae employ to
degrade Remazol Blue, Remazol Black B and Remazol Red RB dyes.
The specific dye uptake increased with increasing dye concentration up
to 410.0 mg for Remazol Black B,380.1 mg for Remazol Blue and 219.1
mg for Remazol Red RB. The percentages of color removal at all
concentrations studied were higher than 62% for Remazol Black B dye.
(Aksu 2003).
24
Thelephora sp.
Thelephora is a genus of fungi within the Thelephoraceae family.

The genus has a widespread distribution and contains about 50 species.
Fruit bodies of species are leathery, usually brownish at maturity, and
range in shape from coral-like tufts to having distinct caps. All species
in the genus are thought to be inedible. This strange fungus is well
camouflaged on the floor of needle-strewn conifer plantations. It is
nearly always found on dry sandy soil, where it forms mycorrhizae with
pines and with spruce trees, but it also occurs in mossy coastal dune
slacks, even where there are no obvious large plant associates.
Thelephora terrestris is most commonly found in association with
conifers, but it has also been shown to form ectomycorrhizal
associations with certain kinds of Eucalyptus. The saprophytic capacity
of this fairly common fungus is evident from the fact that a resupinate
(crust-forming) variety is sometimes found lightly attached to rotting
conifer wood. Thelephora terrestris degraded 99 % of amido black
(Selvam et al., 2003).
25
Table 2.1: Consolidated literature results of dye degradation using
Fungi
Pycnoporus sanguineus
Laetiporus sulphureus
Candida zeylanoidis
Bjerkandera adusta
Pleurotus ostreatus
Coriolus versicolor
Tramates cingulata
Phlebia tramallosa
Pleurotus eryngii
Pleurotus florida
Microorganism
Phanerochaete
Thelephora sp.
chrysosporium
Geotrichum sp
Funalia trogii
Irpex lacteus
Remazol
Briliant Blue - - - 43 - - 100 - 75 - - - - - - - - -
R -
Indigo Blue - - - - - - - 94 75 - - 94 - - - - - - -
Congo Red - - - - - - - - - 54 - - - - - - 97 - -
Reactive
- - - - - - - - - 76 - - - - - - - - -
Blue 15 -
Orange G - - - - - - - - - 97 - - - - - - 33 - -
Reactive
- - - - 40 - - - - - - - - - - - - -
Black 5 -
Orange II - - # - 88 - - - - 99 - - - - - - - - -
Reactive Red
- - - - 75 - - - - - - - - - - - - -
198 -
Malachite
- - - 88 85 - - - - - - - - - - - - -
green -
Azure B - - - 78 - - - - - - - - - - - - - - - -
Remazol
Brilliant - - - 60 - - - - - - - - - - - - - - -
Violet -
Acid Orange - - - - - - - - - 98 - - - - - - - - - -
Acid Red
- - - - - - - - - 89 - - - - - - - - -
114 -
Vat magenta - - - - - - - - - 91 - - - - - - - - - -
Acid green - - - - - - - - - 75 - - - - - - - - - -
Methylene
- - - - - - - - - 86 - - - - - - - - -
Blue -
Methyl
- - - - - - - - - 98 - - - - - - - - -
Violet -
Crystal violet - - - 92 - - - - - 90 - - - - - - - - - -
Poly R 478 - - - 30 - - - - - 46 - - - - - - - - - -
Poly T 128 - - - - - - - - - 48 - - - - - - - - - -
Reactofix
- - - - - - - - - 73 - - - - - - - - -
Gold Yellow -
Bromophenol
- - - - - - - - - 93 - - - - - - - 99 -
blue -
Amido Black - - - - - - - - - - 99 - - - - - - - - -
xylidine - - - 28 - - - - - - - - - - - - - - - -
Carmine
- - - - - - - - - - - - - - - - - - -
Indigo -
Methyl
- - # - - - - - - - - - - - - - - - -
orange -
Reactive
- - - - - - - - - - - - - - - - - -
Black 5 -
Remazol Red
- - - 96 - - - - - 97 - - - - - - - - -
RR -
Remazol
- - - 96 - - - - - 95 - - - - - - - - -
Blue RR -
Reactive blue
- - - 99 - - - - - - - - - - - - - - -
19 -
Reactive blue
- - - 99 - - - - - - - - - - - - - - -
49 -
SN4R - - - - - - - - - - - - - - - - - - - -
26
Table 2.2: Consolidated literature results of dye degradation using
Fungi
Pycnoporus sanguineus
Laetiporus sulphureus
Candida zeylanoidis
Bjerkandera adusta
Pleurotus ostreatus
Coriolus versicolor
Tramates cingulata
Phlebia tramallosa
Pleurotus eryngii
Pleurotus florida
Microorganism
Phanerochaete
Thelephora sp.
chrysosporium
Geotrichum sp
Funalia trogii
Irpex lacteus
SN4R - - - - - - - - - - - - - - - - - - - -
Reactive black
- - - 98 - - - - - - - - - - - - - - -
5 -
Remazol
Brilliant Blue - - - 95 - - - - - - - - - - - - - - -
R -
Acid Black - - - - - - - - - - - - - - - - - - - -
PolyR478 - - - - - - - - - 81 - - - - 55 - - - - -
Remazol Blue - - - - - - - - - - - - - - - - 89 - - -
Remazol Black - - - - - - - - - - - - - - - - 62 - - -
Remazol Red - - - - - - - - - - - - - - - - 77 - - -
Congo Red - - - - - - - - - - - - - - 93 - - - - -
Trypan Blue - - - - - - - - - - - - - - 95 - - - - -
Amido Black - - - - - - - - - - - - - - 89 - - - - -
Methylene
- - - - - - - - - - - - - - 57 - - - -
Blue -
Ethyl violet - - - - - - - - - - - - - - 88 - - - - -
Methyl violet - - - - - - - - - - - - - - 93 - - - - -
Drimarene Red 65 - - - - - - - - - - - - - - - - - - -
Drimarene
72 - - - - - - - - - - - - - - - - - - -
Navy Blue
Drimarene
70 - - - - - - - - - - - - - - - - - - -
Black
Methyl green - - - - - - - - - - - - - - 95 - - - - -
Brilliant Cresyl
- - - - - - - - - - - - - - 90 - - - -
Blue -
Direct Blue 15 - - - - - - - - - 75 - - - - - - - - - -
Drimarine dyes - - - - - - - - - - - - - - - - - - 99 -
Cibracon
- 85 - - - - - - - - - - - - - 79 - - -
Yellow CR -
Reactive Red
- - - - - - 90 - - - - - - - - - - - -
158 -
Reactive
- - - - - - 95 - - - - - - - - - - - -
Yellow 27 -
Acid orange - - - - - - - - - - - - - - - - - - - -
Acid green - - - - - - - - - 75 - - - - - - - - - -
orange G - - - - - - - - - - - - - 97.2 - - - - - -
crystal violet - - - - - - - - 86 - - - - 91.2 - - - - - -
amaranth - - - - - - - - - - - - - 97.2 - - - - - -
Everzol
Turquoise Blue - - - 82 - - - - - - - - - - - - - - -
G -
Astrazone
- - - 89 - 94 - - - 95 - 75 80 - - - - - - 84
Black FDL
Astrazone Blue
- - - - - 92 - - - 97 - 75 97 - - - - - - 89
FGRL
Astrazone Red
- - - 98 - 97 - - - 99 - 97 99 - - - - - - 97
FBL
Reactive Blue
- - - - - - - - - 83 - - - - - - - - -
19 (RBBR) -
Reactive Black
- - - - - 99 80 - - 21 10 40 - - - - - - -
5 -
27
28
CHAPTER 3
MATERIALS AND METHODS
3.1 CULTURE COLLECTION:

Eight fungal species, Mucor racemosus MTCC 7382, Rhizopus
stolonifer var stolonifer MTCC 7370, Fusarium moniliforme MTCC
2015, Fusarium oxysporum MTCC 284, Trichoderma viride MTCC
2417, Aspergillus terreus var terreus MTCC 3006, Aspergillus
ochraceus MTCC 1810 and Penicillium citrinum MTCC 8009 were
obtained from microbial type culture collection (MTCC), Institute of
microbial technology (IMTECH), Chandigarh, India. These cultures
were preserved under specified growth media as listed in Annexure 1.
3.2 PROCEDURE FOR PRESERVATION OF PURE CULTURES:

3.2.1 Revival of cultures:
1. The required growth medium for the fungal species to be preserved
was prepared.
2. Lyophilised culture vials were broken carefully under sterile
conditions and 400 l of sterile water was added to it. This
suspension was allowed to stand for 20 min before transferring into
solid media.
3. Few drops of this suspension were streaked on the specified solid
media.
4. The inoculated media was incubated at optimum temperature
(prescribed by MTCC).
28
3.2.2 Immersion in distilled water:
1. Using sterile pipette tips, discs or blocks were cut down from the
growing culture.
2. These discs were transferred in 50 mL injection bottles with 25 mL
distilled water.
3. The bottles were then sealed with rubber corks and paraffin wrapped
and preserved at 4C.
3.2.3 Preservation by Oil Overlay:

1. Liquid paraffin was sterilized at 121C for 15 min by autoclaving.
2. Fungal cultures grown on agar slants were covered with about 10 mm
of paraffin.
3. This was done in such a way that the entire agar surface and fungal
culture was submerged completely in paraffin.
4. These tubes were preserved in upright positions at 4C. (Karen, K.N,
et al, 2004)
3.3 GROWTH CURVE STUDIES:

1. Growth medium for the fungal species was prepared and sterilized at
121°C for 15 minutes.
2. 2 mL of sterile media was taken to serve as blank.
3. 2% w/v (on wet basis) of mycelia was inoculated into 100 mL of the
medium and the flask was swirled for even suspension of mycelia in
the medium.
4. An initial absorbance of this broth (0th h) was measured at 600 nm
using visible spectrophotometer (Systronics 106, India).
5. The culture was incubated at room temperature and 100 rpm.
6. Every 3 hours, 2 mL of this culture was aseptically transferred to a
cuvette and absorbance was noted.
29
7. The growth was spectrophotometrically recorded and the growth
curve was plotted till the fungal growth reached its stationary phase.
3.4 GENERAL PROCEDURE FOR DYE DEGRADATION:

1. A growth medium suitable for the fungal species of our own interest
was prepared with suitable level of nutrients.
2. The medium was sterilized at 121°C for 15 minutes.
3. The medium was inoculated with a loop full of fungal culture under
sterile conditions.
4. The inoculated medium was incubated at optimal temperature with
continuous shaking for 3-5 days, depends on fungal culture.
5. Dye solution was prepared by adding known quantity of dye in
known volume of water and the optimum pH was adjusted.
6. The dye solution was inoculated with 5 to 10% (w/v) of mid log
phase culture under sterile conditions and incubated at suitable
growth conditions of temperature and agitation speed.
7. The sample was collected at regular intervals of time for the
determination of biomass growth and dye concentration.
8. The collected sample was centrifuged at 10,000 rpm for 5 min.
9. The cell free supernatant was characterised for dye concentration
using water as blank at suitable absorption maxima of the dye
spectrophotometrically.
3.5 SCREENING STUDIES:

The effect of 5 dyes such as acid green, basic violet, basic magenta,
drimarene turquoise and drimarene navy on 11 fungal species, Mucor
racemosus MTCC 7382, Rhizopus stolonifer var stolonifer MTCC 7370,
Fusarium moniliforme MTCC 2015, Fusarium oxysporum MTCC 284,
Trichoderma viride MTCC 2417, Aspergillus terreus var terreus MTCC
30
3006, Aspergillus ochraceus MTCC 1810 and Penicillium citrinum
MTCC 8009, Saccharomyces cerevisiae MTCC 2376, Kluyveromyces
maxianus MTCC 4059 and Trichoderma reesei MTCC 3193 were
screened for dye degradation.
3.6 OPTIMIZATION STUDIES ON MYCOREMEDIATION OF

SYNTHETIC DYES BY DESIGN OF EXPERIMENTS:
Optimization of process parameters by classical method of changing one
variable while fixing the others at constant level is laborious and time
consuming, especially when the number of variables is more than 6. An
alternative and more efficient approach to this method is statistical
experimental design. There are two types of experimental design:
screening and response surface designs. Screening designs are used to
investigate which factors are significant. Response surface designs are
used to determine the optimal settings of the significant factors.
3.6.1 Plackett Burman Design

For mycoremediation of dyes, the factors influenced were dye
concentration, contact time, incubation temperature, pH of solution,
inoculum size and agitation speed of shaker. When more than five
independent variables are to be investigated, the Plackett-Burman design
may be used to find the most significant variables in a system which is
then optimized in further studies (Plackett and Burman, 1946). This
method allows for the evaluation of X-1 variables by X experiments
where X must be a multiple of 4 starting from 8. PBD was selected to
screen the six variables for dye degradation. But PBD requires a
minimum of 8 experiments and 7 variables. So the above 6 factors were
made 7 by splitting dye concentration factor into 2: dye quantity and
volume of water.
31
Eight run PBD with 7 factors were tested for their effects on dye
degradation. Low and high levels were assigned for each variable (table
3.1). The experimental design for mycoremediation of dyes is shown in
table 3.2. The percentage (%) dye removal was used as the response in
this design. The significance of variables was determined by calculating
their effects on dye removal. The dye removal percentage for each
experiment was calculated using the formula,
( )
Percentage dye removal =
where Ci and Co are the initial and final dye concentrations.
The effect of each variable on the dye removal percentage were

calculated using the formula,
Effect of each variable = (∑H − ∑L)/(N/2)
where N is the number of experiments.
The variables were ranked based on the effect values i.e., variable
with highest effect secure first rank and so on. The most significant
variables influencing the experiment were identified by the rank.
Table 3.6.1: Actual levels of variables tested with Plackett-Burman

design
VARIABLES L H
A Dye Quantity (mg) 3 6
B Volume of water(mL) 25 30
C Time(days) 2 4
D Temperature(°C) 27 37
E pH 4 9
F Inoculum size (%) 5 15
G Agitation speed(rpm) 0 100
32
Table 3.6.2: Plackett Burman Matrix for seven factors and eight
experiments
Variables
Experiments
A B C D E F G
1 H H L H L L H
2 H H H L H L L
3 L H H H L H L
4 L L H H H L H
5 H L L H H H L
6 L H L L H H H
7 H L H L L H H
8 L L L L L L L
3.6.2 Box Behnken Design:

The significant variables screened by PBD were optimised by Box-
Behnken Design (BBD). The number of experiments for k factors in
BBD based on 3 level is given by 2k(k-1)+c where c is number of centre
points. According to BBD table, 15 experiments were performed for 3
factors with 3 central points.A coefficient of the quadratic model was
calculated using the following equation,
= +∑ +∑ +∑ ∑
where Y is the predicted response and i,j are linear, quadratic

coefficients respectively, b and k are regression coefficients and the
number of factors studied in the experiment respectively.
The significance of each coefficient was determined and the
results were analysed by trial version of design expert. Three
dimensional surface plots were obtained to study the interaction effect
between variables. The optimum values were obtained based on the
humps in 3 dimensional plots.
33
CHAPTER 4
RESULTS AND DISCUSSION
4.1 Standard curve:

Beer-Lambert’s law states that when a beam of light is passed
through a solution containing a light absorbing analyte, the relationship
between the analyte concentration and the absorbance is described by
A = acl
Where A is the absorbance, a is the mass absorptivity, l is the path
length and c is the concentration of the analyte solution.
When acid green concentration increased from 0.05 to 0.25

mg/mL absorbance increased linearly; this obeys Beer-Lambert’s law.
The graph showed R2 value of 0.99 and this line was used as a standard
for the rest of the experiment. Similarly a standard graph was obtained
for basic violet with R2 value of 0.98.
0.35
y = 1.2371x
0.3 R² = 0.9949
0.25
0.2
A610
0.15
0.1
0.05
0
0 0.05 0.1 0.15 0.2 0.25 0.3
Concentration (mg/mL)
Fig 4.1 Standard curve for acid green
34
0.25
0.2 y = 0.7887x
R² = 0.9893
0.15
A590
0.1
0.05
0
0 0.05 0.1 0.15 0.2 0.25 0.3
Concentration (mg/mL)
Fig 4.2 Standard curve for basic violet
4.2 Screening experiments:

Table 4.1 Screening experimental results for dye degradation [+:
Degradation and -: No Decolourisation]
Drimarene navy (DN)

Basic magenta
Basic violet
Drimarene
Acid green
turquoise
(BM)
DYE 
(AG)
(BV)
(DT)
FUNGI
Penicillium citrinum + + + + -
Aspergillus terreus + + - - -
Aspergillus ochraceus + + - - -
Fusarium oxysporum + + + + -
Saccharomyces cerevisciae + + + + -
Trichoderma reesei - - - - -
Trichoderma viride + + - + -
Kluyveromyces marxianus + + - - -
Rhizopus stolonifer + - - + -
Mucor racemosus + - - + -
Fusarium moniliforme + + - - -
35
Eleven fungal species were selected and their degradation effect on 1
acidic, 2 basic and 2 reactive dyes were screened (Table 4.1). Twenty
five combinations showed decolourisation. Twelve combinations which
showed maximal dye decolourisation were selected for optimization
using Plackett- Burman design. The selected combinations were as
follows:
 Penicillium citrinum vs acid green
 Penicillium citrinum vs basic violet
 Fusarium oxysporum vs acid green
 Fusarium oxysporum vs basic violet
 Aspergillus terreus vs acid green
 Aspergillus terreus vs basic violet
 Fusarium moniliforme vs acid green
 Fusarium moniliforme vs basic violet
 Rhizopus stolonifer vs acid green
 Rhizopus stolonifer vs basic violet
 Trichoderma viride vs acid green
 Trichoderma viride vs basic violet
4.3 Growth Curves:

Growth curve studies for Fusarium oxysporum revealed that its
log phase started at 17th hour and ended at 32nd hour (Fig 4.3). Thus mid
log phase was found to be at 24th hour approximately. Similarly for
Penicillium citrinum was at log phase during 9th to 31st hour (Fig 4.4)
and mid-log phase was at 20th hour. From these observations mid log
phase cultures were used for dye degradation experiments.
36
0.8
0.7
0.6
0.5
0.4
A600
0.3
0.2
0.1
0
0 4 8 12 16 20 24 28 32 36 40 44 48
-0.1
Time (h)
Fig 4.3 Growth curve of Fusarium oxysporum
1.8
1.6
1.4
1.2
1.0
A600
0.8
0.6
0.4
0.2
0.0
0 4 8 12 16 20 24 28 32 36 40 44 48
-0.2
Time (h)
Fig 4.4 Growth curve of Penicillium citrinum
Growth curve studies of Rhizopus stolonifer(fig4.5) revealed its

log phase immediately after inoculation .Thus mid log phase was found
to be somewhere around 12th hour after the initial inoculation. The
stationary phase is obtained after approximately 24thhour. Similarly,
37
Trichoderma viride (fig 4.6) was at log phase at 1st hour following its
inoculation and showed maximum growth at approximately 24 hours
after the initial inoculation and the mid log phase is between 10th to 13th
hour. From these observations mid log phase cultures were used for dye
degradation experiments.
0.6
0.5
0.4
A 600
0.3
0.2
0.1
0
0 5 10 15 20 25 30
Time(h)
Fig 4.5 Growth curve of Rhizopus stolonifer
0.8
0.7
0.6
0.5
A 600
0.4
0.3
0.2
0.1
0
0 10 20 30 40 50 60
Time(h)
Fig 4.6 Growth curve of Trichoderma viride

Growth curve studies for Fusarium moniliforme showed that its
log phase started at 12th hour and ended at 19th hour (Fig 4.7). Thus mid
log phase was found to be at 16th hour approximately. Similarly
Aspergillus terreus was at log phase during 13th to 19th hour (Fig 4.8)
38
and mid-log phase was at 16th hour. From these observations mid log
phase cultures were used for dye degradation experiments.
1
0.8
0.6
A600
0.4
0.2
0
0 5 10 15 20 25 30
-0.2
Time (Hours)
Fig 4.7 Growth curve for Fusarium moniliforme
0.7
0.6
0.5
0.4
A600
0.3
0.2
0.1
0
0 5 10 15 20 25 30 35 40
-0.1
Time (Hours)
Fig 4.8 Growth curve for Aspergillus terreus

4.4 Plackett Burman Design:
The above mentioned combinations were subjected for parameter
screening using PBD by fixing high and low values for process
parameters as shown in Tables 4.2, 4.3 and 4.4. Experiments were
conducted and the percentage degradation for each trial was calculated
39
(Table 4.2 and 4.3). Using the percentage removal the effect of each
variable on dye decolourisation was calculated. A graph was plotted
between effect and variable as shown in Fig 4.9 for significant variables.
The best combination and 3 significant variables were selected for
further optimization using BBD.
From PBD results (Fig 4.9) it is clear that dye concentration, time
and inoculum size were the significant factors for degradation of acid
green by Penicillium citrinum. Similarly, for acid green degradation by
Fusarium oxysporum, the significant factors were dye concentration,
inoculum size and time. Dye concentration, pH and agitation speed had
maximum effects on basic violet degradation on Penicillium citrinum
and basic violet degradation by Fusarium oxysporum was mainly
impacted by dye concentration, temperature and pH.
Table 4.2: Plackett Burman experimental Design
Variables Percentage dye removal
Trial
A B C D E F G AG Vs FO AG Vs PC BV Vs FO BV Vs PC
1 H H L H L L H 95 45 0 43.8
2 H H H L H L L 55 60 0 0
3 L H H H L H L 0 50 0 0
4 L L H H H L H 8 75 45.8 0
5 H L L H H H L 70 33.3 31.5 12.5
6 L H L L H H H 0 20 0 0
7 H L H L L H H 38 50 31.2 87.5
8 L L L L L L L 50 58.3 23.3 20.8
AG- Acid green PC- Penicillium citrinum

BV- Basic violet FO- Fusarium oxysporum
From figure 4.9 It can be observed that for acid green vs Rhizopus
stolonifer dye concentration, temperature and volume of water were the
most influential factors. For the Acid green vs Trichoderma viridae
40
combination the experiment was influenced by agitation speed,dye
concentration and temperature. Inoculum size,agitation speed and pH
had the maximal effects on Basic violet vs Rhizopus stolonifer and
finally Basic violet vs Trichoderma viridae combination was influenced
by incubation time ,pH and dye concentration respectively.
Table 4.3: Plackett Burman experimental Design

Tria
l AG VS AG VS BV VS BV VS
A B C D E F G
TV RS TV RS
1 H H L H L L H 60 55 75 75
2 H H H L H L L 90 75 90 80
3 L H H H L H L 60 40 70 70
4 L L H H H L H 58.3 58.3 66.6 58.33
5 H L L H H H L 75 50 54.2 54.16
6 L H L L H H H 60 10 50 50
7 H L H L L H H 70.8 83.3 91.66 54.16
8 L L L L L L L 75 41.66 75 83.33
AG- Acid green TV- Tricoderma viridae

BV- Basic violet RS- Rhizopus stolonifer
In Fig 4.9 PBD results shows that dye concentration, agitation

speed and pH were the significant factors for degradation of acid green
by Fusarium moniliforme. Similarly, for acid green degradation by
Aspergillus terreus, the significant factors were dye concentration,
inoculum size and pH. Volume of water, pH and Temperature had most
effects on basic violet degradation on Aspergillus terreus and basic
violet degradation by Fusarium moniliforme strikes by dye
concentration, temperature and agitation speed.
41
Table 4.4:
4. Plackett Burman experimental Design
Trial AG VS AG VS BV VS BV VS
A B C D E F G
FM AT FM AT
1 H H L H L L H 47.6 69.42 72.5 0
2 H H H L H L L 47.6 27.33 0 0
3 L H H H L H L 40.5 4.58 0 0
4 L L H H H L H 87 49.44 0 0
5 H L L H H H L 75.2 40.27 0 0
6 L H L L H H H 84.4 0 0 0
7 H L H L L H H 80 52.08 0 9.44
8 L L L L L L L 60.1 78.23 0 18.74
AG- Acid green FM Fusarium moniliforme

FM-
BV- Basic violet AT- Aspergillus terreus
50
AG VS RS
45
AG VS TV
EFFECT OF VARIABLES
40
AG VS PC
35
AG VS FO
30
AG VS FM
25
AG VS AT
20
BV VS PC
15
BV VS FO
10 BV VS RS
5 BV VS TV
0 BV VS FM
A B C D E F G
BV VS AT
PROCESS VARIABLES
Fig 4.9 Effect Vs Variables
From the 12 combinations 3 best combinations which showed

significant dye removal percentages
percentage for all 8 PBD experiments were
chosen for further optimization using BBD. The 3 combinations were
42
Penicillium citrinum vs Acid green, Rhizopus stolonifer vs Acid green
and Fusarium moniliforme vs Acid green.
4.5: Box Behnken Design for Penicillium citrinum vs Acid green:

To optimize the dye degradation experiments of Penicillium
citrinum vs Acid green, Box Behnken design was chosen for response
surface optimization with a 3 level 3 factors i.e., dye concentration, time
and inoculum size . Tables 4.5 and 4.6 lists the experimental factor
settings and results on the basis of the experimental design. All the 15
designed experiments were conducted and the results were analysed by
regression. This showed 3 linear coefficients (A,B,C) and 3 quadratic
coefficients (A2, B2, C2) and 3 cross product coefficients(AB, BC,AC)
were significant.(table 4.7)
Table 4.5: Process variables and level used for Box Behnken Design
Levels
Variable Symbol Unit
-1 0 1
Dye concentration C mg/ml 0.12 0.16 0.2
Time T d 2 3 4
Inoculum Size I % (w/v) 5 10 15
43
Table 4.6:Box Behnken experimental design
Run dye concentration (mg/ml) Time (d) Inoculum size (% w/v) % DR

1 0.12 2 10 76.66
2 0.2 2 10 84.15
3 0.12 4 10 61.66
4 0.2 4 10 76
5 0.12 3 5 43.55
6 0.2 3 5 51
7 0.12 3 15 0
8 0.2 3 15 25
9 0.16 2 5 96.25
10 0.16 4 5 80
11 0.16 2 15 75.625
12 0.16 4 15 51.25
13 0.16 3 10 6.25
14 0.16 3 10 0
15 0.16 3 10 15.62
ANOVA for the response surface model is given in table 4.6. The
coefficients of this model is given in equation 1 were also evaluated. A
p value showed that all of the linear coefficients were more significant
than their quadratic and cross product terms. However to minimize the
error, all of the coefficients were considered in the design. According to
ANOVA analysis, we noted a few lack of fits. This indicates that the
model does indeed represent the actual relationships of reaction
parameters, which are well within the selected ranges (Table 4.7).
44
Table 4.7: Model coefficients estimated by multiple linear regression
and analysis of variance
Sum of Mean F p-value

Source df
Squares Square Value Prob > F
Model 14769.77 9 1641.085 26.273 0.0011
A-[Dye] 195.228 1 195.228 3.125 0.137
B-Time 508.406 1 508.406 8.139 0.035
C-Inoculum 1761.36 1 1761.359 28.198 0.003
AB 119.137 1 119.137 1.907 0.225
AC 75.082 1 75.082 1.202 0.322
BC 16.503 1 16.503 0.264 0.629
A^2 408.774 1 408.774 6.544 0.050
B^2 11774.52 1 11774.516 188.505 < 0.0001
C^2 504.198 1 504.198 8.072 0.036
Residual 312.312 5 62.462
Lack of Fit 171.281 3 57.093 0.809 0.593
Pure Error 141.031 2 70.515
Cor Total 15082.08 14
45
Table 4.8: Regression coefficients and significance of response
surface quadratic model
Coefficient Standard 95% CI 95% CI

Factor Df VIF
Estimate Error Low High
Intercept 7.625 1 4.562 -4.104 19.354

A-[Dye] 4.94 1 2.794 -2.242 12.122 1
B-Time -7.971 1 2.794 -15.154 -0.789 1
C-Inoculum -14.838 1 2.794 -22.021 -7.655 1
AB 5.457 1 3.951 -4.700 15.615 1
AC 4.332 1 3.951 -5.825 14.490 1
BC -2.031 1 3.951 -12.189 8.126 1
A^2 10.521 1 4.113 -0.050 21.094 1.011
B^2 56.470 1 4.113 45.897 67.043 1.011
C^2 11.685 1 4.113 1.112 22.258 1.011
The final estimative response model equation (based on the actual value)
DR=7.62+4.94*A-7.970*B-14.84*C+5.46*A*B+4.33*A*C-2.03*B*C
+10.52*A2+56.47*B2+11.69*C2
where DR is the dye removal, A,B,C are the values of dye concentration
(mg/mL), time(d) and inoculum size (% w/v) respectively. The model
coefficients and probability values are shown in Table 4.8. The model
proved suitable for adequate representation of the real relationship
among the selected factors.
46
Fig 4.10:Response Surface and contour plot of for time vs. dye
concentration for at constant inoculum size on % dye removal
Fig 4.10 represents effect of dye concentration and time at a

constant inoculum size on acid green degradation. At any designed dye
concentration from 0.12 to 0.2 mg/mL, the dye removal percentage
slightly decreased and then increased rapidly. Similarly the dye removal
decreased and then increased when time varied from 2 to 4 days. This
show that time is more significant than dye concentration at constant
inoculum size.
Fig 4.11 represents effects of dye concentration and inoculum size

at a constant time. It is evident here that dye removal decreases initially
and then increases with the increase in inoculum size from 5 to 15 %
(w/v). When dye concentration increases from 0.12 to 0.2 mg/mL the
dye removal also increases slightly. This shows that inoculum size is
more significant than dye concentration at constant time.
47
Fig 4.11:Response Surface and contour plot for dye concentration
vs. inoculum size at constant time on % dye removal
Fig 4.12: Response Surface and contour plot for time vs. inoculum
size for at constant dye concentration on % dye removal
Fig 4.12 represents effects of time and inoculums size at a

constant dye concentration. Here as the inoculum size increases from 5
48
to 15% dye removal gets lowered and then gets increased rapidly.
Similarly an increase in time from 2 to 4 days has the same effect on dye
removal. Comparatively inoculums size is more significant than time at
constant dye concentration.
From these figures, the optimal conditions for dye degradation
were found. The optimal values were obtained by solving the regression
equation using design expert 7 trial software.
4.6: Box Benkhen Design for Rhizopus stolonifer vs Acid green
To optimize the dye degradation experiments of Rhizopus

stolonifer vs Acid green, Box Behnken design was chosen for response
surface optimization with a 3 level 3 factors i.e., dye concentration, time
and inoculum size . Table 4.9 and 4.10 lists the experimental factor
settings and results on the basis of the experimental design. All the 15
designed experiments were conducted and the results were analyzed by
regression. This showed 3 linear coefficients (A,B,C) and 3 quadratic
coefficients (A2, B2, C2) and 3 cross product coefficients(AB, BC,AC)
were significant.(table 4.11)
Table 4.9:Process variables and level used for Box Behnken Design
Levels
-1 0 1
Temperature T °C 27 32 37
Inoculum Size I % (w/v) 5 10 15
49
Table 4.10: Box Behnken experimental design
Run Dye concentration (mg/ml) Temperature(°C) Inoculum % (w/v) % DR
1 0.12 27 10 75.94
2 0.2 27 10 83.33
3 0.12 37 10 86
4 0.2 37 10 74
5 0.12 32 5 44
6 0.2 32 5 50
7 0.12 32 15 32
8 0.2 32 15 25
9 0.16 27 5 75
10 0.16 37 5 89
11 0.16 27 15 70
12 0.16 37 15 49.75
13 0.16 32 10 5.9
14 0.16 32 10 42
15 0.16 32 10 16
ANOVA for the response surface model is given in table 4.11.

The coefficients of this model is given in equation 2 were also
evaluated. A p value showed that all of the linear coefficients were more
significant than their quadratic and cross product terms. However to
minimize the error, all of the coefficients were considered in the design.
According to ANOVA analysis, we noted a few lack of fits. This
indicates that the model does indeed represent the actual relationships of
reaction parameters, which are well within the selected ranges (Table
4.11).
50
Table 4.11: Model coefficients estimated by multiple linear
regression and analysis of variance
Sum of Mean F p-value
Source Df
Squares Square Value Prob > F
Model 9336.073 9 1037.341 7.325 0.02
A-[Dye] 3.934 1 3.934 0.027 0.874
B-Temperature 3.808 1 3.808 0.026 0.876
C-Inoculum 825.195 1 825.195 5.827 0.06
AB 93.993 1 93.993 0.663 0.452
AC 42.25 1 42.25 0.298 0.608
BC 293.265 1 293.265 2.07 0.209
A^2 592.254 1 592.254 4.182 0.096
B^2 7762.898 1 7762.898 54.82 0.0007
C^2 52.896 1 52.896 0.373 0.567
Residual 708.029 5 141.605
Lack of Fit 14.289 3 4.763 0.013 0.997
Pure Error 693.74 2 346.87
Cor Total 10044.103 14

Factor Df VIF
Intercept 21.3 1 6.87 3.639 38.96
A-[Dye] -0.701 1 4.207 -11.516 10.113 1
B-Temperature -0.69 1 4.207 -11.505 10.125 1
C-Inoculum -10.156 1 4.207 -20.971 0.658 1
AB -4.847 1 5.949 -20.142 10.447 1
AC -3.25 1 5.949 -18.544 12.044 1
BC -8.562 1 5.949 -23.857 6.7322 1
A^2 12.665 1 6.192 -3.254 28.584 1.01
B^2 45.852 1 6.192 29.933 61.771 1.01
C^2 3.785 1 6.192 -12.134 19.704 1.01
51
DR =+21.30 -0.70 * A -0.69* B -10.16 * C -4.85* A * B -3.25 * A *

C -8.56 * B * C+12.66 * A2 +45.85 * B2+3.78 * C2
Where, DR is the dye removal, A, B and C are the values of dye

concentration (mg/mL), temperature (°C) and inoculum size (% w/v)
respectively. The model coefficients and probability values are shown in
Table 4.12. The model proved suitable for adequate representation of the
real relationship among the selected factors.
Fig 4.13:Response Surface and contour plot of for temperature vs.

dye concentration for at constant inoculum size on % dye removal
Fig 4.13 represents effect of dye concentration and temperature at

a constant inoculum size on acid green degradation. At any designed
dye concentration from 0.12 to 0.2 mg/mL, the dye removal percentage
increased rapidly after a slight decline. Similarly the dye removal
52
decreased and then increased when temperature is varied from 27 to
37°C. This shows that temperature is more significant than dye
concentration at constant inoculum size.

vs. inoculum size at constant temperature on % dye removal
Fig 4.14 represents effects of dye concentration and inoculum size at a

constant time. It can be observed here that dye removal decreases the
increase in inoculum size from 5 to 15 % (w/v). When dye concentration
increases from 0.12 to 0.2 mg/mL the dye removal decreases initially
and increases steadily. This shows that dye concentration is more
significant than inoculum size at constant temperature.
53
Fig 4.15: Response Surface and contour plot for temperature vs.
inoculum size for at constant dye concentration on % dye removal
Fig 4.15 represents the effects of temperature and inoculum size

at a constant dye concentration. Here as the inoculum size increases
from 5 to 15% steady dye removal can be observed .Similarly an
increase in from 27 to 37°C the dye removal decreases and then
increases rapidly. Comparatively temperature is more significant than
inoculum size at constant dye concentration.
4.7: Box Benkhen Design for Fusarium moniliforme vs Acid Green

To optimize the dye degradation experiments of Fusarium
moniliforme vs Acid Green, Box Behnken design was chosen for
response surface optimization with a 3 level 3 factors i.e., dye
concentration, agitation speed and pH. Table 4.13 and 4.14 lists the
experimental factor settings and results on the basis of the experimental
design. All the 15 designed experiments were conducted and the results
were analysed by regression. This showed 3 linear coefficients (A,B,C)
54
and 3 quadratic coefficients (A2, B2, C2) and 3 cross product
coefficients(AB, BC,AC) were significant.(table 4.)
Table 4.13: Process variables and level used for Box Behnken
Design
Levels
-1 0 1
Agitation speed rpm rpm 0 50 100
pH 4 6.5 9
Table 4.14: Box Behnken experimental design

Agitation speed
Run dye concentration (mg/ml) % DR
(rpm) pH
1 0.12 0 6.5 57.90
2 0.2 0 6.5 61.76
3 0.12 100 6.5 57.02
4 0.2 100 6.5 59.38
5 0.12 50 4 26.94
6 0.2 50 4 29.36
7 0.12 50 9 24.62
8 0.2 50 9 20.50
9 0.16 0 4 55.64
10 0.16 100 4 57.13
11 0.16 0 9 63.74
12 0.16 100 9 69.50
13 0.16 50 6.5 25.31
14 0.16 50 6.5 27.04
15 0.16 50 6.5 28.62
ANOVA for the response surface model is given in table 4.15.

The coefficients of this model is given in equation 2 were also
55
evaluated. A p value showed that all of the linear coefficients were more
significant than their quadratic and cross product terms. However to
minimize the error, all of the coefficients were considered in the design.
According to ANOVA analysis, we noted a few lack of fits. This
indicates that the model does indeed represent the actual relationships of
reaction parameters, which are well within the selected ranges (Table
4.15).
Table 4.15: Model coefficients estimated by multiple linear

regression and analysis of variance
p-value
Sum of Mean Prob >
Source Squares df Square F Value F
Model 4430.11 9 492.23 16.289 0.003
A-DYE
CONCENTRATION 2.553 1 2.553 0.084 0.782
B-AGITATION SPEED 1.990 1 1.990 0.065 0.807
C-pH 10.788 1 10.788 0.356 0.576
AB 0.562 1 0.562 0.018 0.896
AC 10.69 1 10.692 0.353 0.577
BC 4.558 1 4.558 0.150 0.713
A^2 14.640 1 14.640 0.484 0.517
B^2 4307.628 1 4307.628 142.54 < 0.0001
C^2 0.909 1 0.909 0.030 0.869
Residual 151.093 5 30.218
Lack of Fit 146.863 3 48.954 23.14 0.041
Pure Error 4.230 2 2.11
Cor Total 4581.21 14
56
Factor df VIF
Intercept 26.85 1 3.173 18.69 35.008
A-Dye
0.565 1 1.943 -4.431 5.561 1
Concentration
B-Agitation
0.498 1 1.943 -4.497 5.494 1
Speed
C-pH 1.161 1 1.943 -3.834 6.157 1
AB -0.375 1 2.748 -7.440 6.690 1
AC -1.635 1 2.748 -8.700 5.430 1
BC 1.0675 1 2.748 -5.997 8.132 1
A^2 -1.991 1 2.860 -9.345 5.362 1.01
B^2 34.156 1 2.860 26.80 41.510 1.01
C^2 0.496 1 2.860 -6.85 7.850 1.01
DR=26.85+0.565*A+0.498*B1.161*C-0.375*A*B-
1.635*A*C+1.0675*B*C-1.991*A2+34.156*B2+0.4962*C2
Where, DR is the dye removal, A, B and C are the values of dye

concentration (mg/mL), temperature (°C) and inoculum size (% w/v)
respectively. The model coefficients and probability values are shown in
Table 4.16. The model proved suitable for adequate representation of the
real relationship among the selected factors.
57
Fig 4.16: Response Surface and contour plot of for agitation speed
vs. dye concentration for at constant pH on % dye removal
Fig 4.16 corresponds to effects of dye concentration and agitation

speed at a constant pH. At any designed dye concentration from 0.12 to
0.2 mg/mL, steady dye removal has been observed. Similarly the dye
removal decreased and increased sharply with increase in agitation
speed from 0 to 100 rpm. This shows that agitation speed is more
significant than dye concentration at constant pH.
Fig 4.17 corresponds to effects of dye concentration and pH at a
constant agitation speed. It is evident here that dye removal decreases
initially and then increases with the increase in pH from 4 to 9. When
dye concentration increases from 0.12 to 0.2 mg/mL the dye removal
also increases slightly. This shows that pH is more significant than dye
concentration at constant agitation speed
58
vs. pH at constant agitation speed on % dye removal
Fig 4.18: Response Surface and contour plot for pH vs. agitation
speed for at constant dye concentration on % dye removal.
Fig 4.18 corresponds to effects of pH and agitation speed at a

constant dye concentration. Here as the pH increases from 4 to 9, steady
59
dye removal has been observed .But increase in agitation speed from 0
to 100 rpm the dye removal decreases and then increases rapidly.
Comparatively agitation speed is more significant than pH at constant
dye concentration.
From these figures, the optimal conditions for dye degradation
were found. The optimal values were obtained by solving the regression
equation using design expert 7 trial software.
60
CHAPTER 5
CONCLUSION
Eleven fungal species were screened for their ability to degrade the
selected synthetic dye solutions. Out of 10 potential fungi degrading dyes,
6 species were selected for further parameter screening by Plackett-Burman
design (PBD). The growth curve studies were carried out to determine the
mid log phase of each species. The variables influencing the dye
degradation by fungi were identified and screened by PBD. From the PBD
results, three significant variables such as volume of water, time and
inoculum size that have the maximal influence on the degradation process
were identified and further optimized by Box-Behnken design (BBD). The
experimental results were run through the trial version of design expert
software). In this study, P.citrinum showed maximum acid green removal
of 96.78% at initial dye concentration of 0.2 mg/mL, contact time of 2 days
and inoculum size of 5.38% (w/v). Similarly R.stolonifer showed 90.78%
acid green removal at initial dye concentration of 0.14 mg/mL, temperature
of 36.98°C and inoculum size of 5.47% (w/v). F.moniliforme revealed
60.38% of removal with optimal conditions of dye concentration 0.20
mg/mL , pH of 4.00 and agitation speed 0 rpm. The experimental and
statistical results showed that the optimum conditions could be an ideal
solution for acid green degradation by Penicillium citrinum, Rhizopus
stolonifer and Fusarium moniliforme.
61
APPENDICES
Appendix 1
A1.1Growth Medium
Table A1.1:Composition of Potato Sucrose agar:
Components Quantity
Potatoes ( Scrubbed And Diced) 200 g
Sucrose 20 g
Agar 20 g
Distilled Water 1000 ml
Table A1.2:Composition of Potato Dextrose agar
Components Quantity
Potatoes ( Scrubbed And Diced) 200 g
Dextrose 20 g
Agar 20 g
Table A1.3:Composition of Malt extract agar
Components Quantity
Malt extract 20 g
Agar 20 g
Water 1000 ml
62
Table A1.4:Composition of Czapek Yeast extract Agar –CYA
Composition Quantity
Czapek concentrate 10 ml
Di potassium hydrogen phosphate 1g
Yeast Extract 5g
Sucrose 30 g
Agar 15 g
Table A1.5:Composition of Czapek Concentrate
Sodium Nitrate (NaNO3) 30g
Potassium chloride (KCl) 5g
Magnesium Sulphate (MgSO4.7H2O) 5g
Ferrous Sulphate (FeSO4.7H2O) 0.1g
Distilled water 100ml
Table A1.6: Composition of Yeast Extract Peptone Dextrose
Yeast extract 3g
Peptone 10.0 g
Dextrose 20.0 g
Distilled water 1000 mL
63
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OPTIMIZATION STUDIES

in partial fulfillment for the award of the degree

KUMARAGURU COLLEGE OF TECHNOLOGY

ANNA UNIVERSITY: CHENNAI 600 025

in partial fulfillment for the award of the degree

KUMARAGURU COLLEGE OF TECHNOLOGY

Certified that this project report “OPTIMIZATION STUDIES ON

Dr. A. Manickam Mr.D.R.Manimaran

Assistant Professor (SrG)

Department of Biotechnology Department of Biotechnology

Kumaraguru College of Technology Kumaraguru College of Technology

Coimbatore – 641 049 Coimbatore – 641 049

Internal Examiner External Examiner

We express our thankfulness to our SUPERVISOR,

We express our deepest gratitude to Dr.S.Ramachandran, Principal,

We sincerely thank Dr.A.Manickam, Professor and Head, Department

Abivarma.R.S Shivashankar.R Baheerathan.M

Mycoremediation is a feasible method for degrading dyehouse effluent.

கழி கைள சிைத ப யற க ெபற ைவ

CHAPTER TITLE PAGE

Fungi are microscopic, eukaryotic organisms exhibiting growth on

Phthalocyanine dyes are widely used in the textile and dyestuff

Both tropho- and idiophasic cultures of TrametestrogiiBAFC 463

Little is known about color removal of heterocyclic dyes. This may be

Triphenylmethane dyes are employed extensively in various industries

1.1.2 Dye degradation methods

In recent years, worldwide efforts have been employed to develop

Physical, chemical, and electrochemical methods produce a large

(b) Biological Methods

Presently, most of the textile industries are developing wastewater

A soil actinomycete, Streptomyces chromofuscus, exhibits less

 Degradation by Algae and Higher Plants

Azo dyes are degraded by an induced form of azoreductase by algae.

During the past decade, fungi have been demonstrated to degrade

White-rot fungal decolorization and degradation of dyes

The role of the white-rot fungus, Phanerochaete chrysosporiumin

1.1.3 Optimization methods

Medium optimization by the classical method bychanging one

When more than five independent variables are to be investigated,

The next stage in medium optimization would be to determine the

 To screen suitable fungi for remediation of synthetic dyes.

Aspergillus foetidus is a mold which is found to be allergenic for

Bjerkandera adusta is a species of fungus in the Meruliaceae

The genus Candida includes around 154 species. Among these,

Coriolus versicolor also known as Trametes versicolor, turkey

Studies on non ligninolytic fungi metabolizing dyes are minimal,

The genus Debaryomyces comprises 15 species. Many

White rot fungi, Funalia trogii can degrade a wide variety of

Geotrichum species are common yeast-like fungi whose primary

Irpex lacteus is commoly called as the “Milk-white Toothed

Laetiporus sulphureus is a species of bracket fungus (fungus that

Phanerochaete chrysosporium is a resupinate basidiomycetes

The white-rot fungus Phanerochaete chrysosporium secretes,

Pleurotus eryngii and Pleurotus florida

Pleurotus eryngii degraded 10 % of reactive black 5 (Mazmanci

Pleurotus ostreatus, the oyster mushroom, is a common edible

P. ostreatus strain 32 was obtained from Dalian institute of

Pleurotus sajor-caju known as the Dhingri Oyster or Grey

Pleurotus sapidus and Pycnoporus sanguineus

Pycnoporus sanguineus was used to degrade 99 % of

Pleurotus pulmonarius, commonly known as the Indian Oyster,

P. pulmonarius CCB-19 was obtained from the São Paulo Botany

Phlebia tremellosa is a saprobic; resupinate fungus normally

Phlebia tremellosa cultures were obtained from the Canadian