Journal of Biotechnology 260 (2017) 11–17

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Research paper

Optimization of conditions for decolorization of azo-based textile dyes by MARK

multiple fungal species
⁎⁎ ⁎
Wafaa M. Abd El-Rahima, , Hassan Moawada, Ahmed Z. Abdel Azeizb, Michael J. Sadowskyc,
a
Agricultural Microbiology Department, National Research Center, Cairo, Egypt
b
College of Biotechnology, Misr University for Science and Technology (MUST), Egypt
c
Department of Soil, Water and Climate, Department of Plant and Microbial Biology, and The Biotechnology Institute, University of Minnesota, St. Paul, MN, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Wastewater from textile industries contains azo dye residues that negatively affect most environmental systems.
Biodegradation The biological treatment of these wastes is the best option due to safety and cost concerns. Here we isolated and
Wastewater identified 19 azo dye-degrading fungi and optimized conditions resulting in enhanced degradation. The fungi
Azo dyes belonged to five species of Aspergillus and a single Lichtheimia sp. All fungi were evaluated for their ability to
Aspergillus niger
decolorize 20 azo dyes. While the most easily transformable azo dye was direct violet (decolorization ranged
A. terreus
from 71.1 to 93.3%), the most resistant to decolorization was fast green azo dye. The greatest degradation
A. oryzae
potential of azo dyes (direct violet and methyl red) was optimized using the most promising four fungal strains
and changing media glucose concentration, nitrogen source, and micronutrients. Biomass, lignin peroxidase, and
laccases production were also determined in the optimization studies. The decolorization of both azo dyes by the
four fungal strains was greatly enhanced by glucose supplementation. The fungal strains were not able to pro-
duce lignin peroxidases in the absence of organic nitrogen source. Both yeast extract and casamino acid sup-
plementation enhanced decolorization of direct violet and methyl red dyes and production of lignin peroxidase
by the fungal strains. In contrast, the laccases were absent in the similar medium enriched with the same organic
nitrogen sources.

1. Introduction Othmer, 1979).

Industrial wastewater released from textile plants often contains
residual azo-dyes, which are considered as environmental hazardous
materials. These dyes are difficult to treat biologically, mainly due to
their synthetic origin and complex aromatic structures. Chemical
methods remain the major wastewater treatment procedures used in
these factories, and often result in the production of other forms of
chemical pollutants. Consequently, a lack of remediation technologies
in industrial dye plants results in the discharge of wastewater into the
environment, which affects plant, animal, and human health. One
major problem in the treatment of textile industrial wastewater is due
to the variation in the types and chemistry of textile dyes. This is in part
due to the staining requirements of different fiber types:1) cellulose
(cotton, rayon, linen, ramie, and lyocell), 2) protein (wool, angora,
mohair, cashmere and silk) and 3) synthetic fibers polyester, nylon,
spandex, acetate, acrylic, ingeo and polypropylene (Ghaly et al., 2014).
The textile dyes used to treat these fibers are generally classified as
acid, basic, direct, fluorescent, reactive, sulphurous, or vat dyes (Kirk-
Several investigations have been done to examine the biodegrada-
tion of azo-dyes by either bacteria or fungi isolated from textile dye
effluent. Manikandan et al. (2012) isolated Achromobacter xylosoxidans
GRIRKNM11 from a textile dye effluent site able to decolorize turquoise
blue dye (100 mg/L) within 48 h. Similarly, Hassan et al. (2013) iso-
lated and identified indigenous bacteria from textile dye effluent and
tested them for decolorization of the azo dyes novacron, viz orange
W3R, red FNR, yellow FN2R, blue FNR and navy Joshi et al. (2015)
studied degradation of methyl red (MR) and carbolfuchsin (CF) by
Proteus spp., Pseudomonas spp. and Acinetobacter spp. bacteria and its
consortium isolated from textile effluents. The consortium showed the
highest potential in decolorizing MR and CF, up to 100% and 96%,
respectively, within 24 h. Karthikeyan et al. (2015) tested the efficacy
of Pleurotus platypus in decolorization of two textile dye effluents.
Variation in textile dye types and chemistries prevent the use of a
single microorganism to treat all wastewaters containing industrial
dyes. Moreover, since there are many types of azo dyes, only relatively
few isolated microorganism have been tested for decolorization of a

⁎
Corresponding author at: BioTechnology Institute, University of Minnesota, 1479 Gortner Ave, 140 Gortner Labs, St. Paul, MN 55108, USA.
⁎⁎
Corresponding author at: Agricultural Microbiology Department, National Research Center (NRC), 33 El Bohouth St., Dokki, P.O. 12622, Cairo, Egypt.
E-mail addresses: wafaa10m@gmail.com (W.M. Abd El-Rahim), sadowsky@umn.edu (M.J. Sadowsky).

http://dx.doi.org/10.1016/j.jbiotec.2017.08.022
Received 5 April 2017; Received in revised form 21 August 2017; Accepted 27 August 2017
Available online 30 August 2017
0168-1656/ © 2017 Elsevier B.V. All rights reserved.
W.M. Abd El-Rahim et al. Journal of Biotechnology 260 (2017) 11–17

Table 1 100 μg/ml, or the industrial wastewater, was poured into the plate and
Absorbance maxima (λ-max in nm) for the 20 tested azo dyes. the contents was mixed well. Plates were incubated at 28 °C for 5 days.
Colonies appearing after incubation were picked, purified by streaking
Dye No. Azo-dye λ-max Dye No. Azo-dye λ-max
(nm) (nm) multiple times on the same initial isolation medium, and maintained on
the same medium. Fungi were isolated in a similar manner except that
1 Reactive red 520 11 Tartrazin 460 the medium was adjusted to pH 5.0 prior to use.
2 Direct blue 584 12 Naphthol blue 626
black
3 Direct red 546 13 Trypan blue 626 2.5. Identification of the fungal isolates
4 Direct violet 626 14 Janus green 626
5 Reactive blue 626 15 Alirazin yellow 410 All of the isolated fungi were identified by sequencing the ITS1 and
6 Reactive 512 16 Evans blue 660
orange
ITS4 gene regions. The primer sequences used were: ITS1:
7 Fast green 418 17 Brilliant green 627 TCCGTAGGTGAAC CTGCGG (Korabecna, 2007) and ITS4: TCCTCCG-
8 Methyl red 480 18 Safranin 500 CTTATTGATATGC (Korabecna, 2007).The PCR conditions for gene
9 Crystal violet 600 19 Pararosaniline 550 amplification were: 95 °C, 10 min; 29 x (95 °C, 30 s; 52 °C, 2 min; 72 °C,
10 Alura red 491 20 Poneau S 567
30 s); 72 °C, 8 min; and 4 °C hold. The PCR products were separated by
electrophoresis on 1% agarose gels, and purified using the ExoSAP-It
limited number of azo dyes or textile dye effluents. Therefore, there is a reagent − ExoSAP-IT® PCR Product Cleanup (Affymetrix USA) as in-
need for obtaining multiple azo dye-degrading microorganisms to be structed by the manufacturer. DNA samples were sequenced at Uni-
used for biological treatment of wastewater produced from different versity of Minnesota Genomic Center (UMGC). The sequences obtained
textile plants. In the present study, we aimed to isolate and identify were trimmed and analyzed by using BLAST analysis (http://blast.ncbi.
multiple azo dye- degrading microorganisms and optimize the de- nlm.nih.gov).
gradation and decolorization process by altering growth conditions that
favored enzyme production. 2.6. Inoculum preparation

2. Materials and methods Spore suspensions were prepared by washing 3 day-old fungus
slants with sterilized saline solution (0.85% NaCl). The OD was mea-
2.1. Azo dyes sured at 600 nm and adjusted to A 600 nm = 1.0 prior to inoculation.

Twenty azo dyes (Table 1) were used in this study. These included
direct blue 71, reactive blue 4, reactive orange, fast green, methyl red,
crystal violet, alura red AC, tartrazine, naphthol blue black, trypan
blue, janus green B, alizarin yellow R, evans blue, brilliant green, sa-
franin, pararosaniline, and ponceau S. The dyes were obtained from
Sigma-Aldrich (St. Louis, MO, USA). Direct red 80, cibacron brilliant
Red 3B-A, and direct violet 51 were obtained from Santa Cruz Bio-
technology (Dallas, TX, USA).
2.2. Sample collection
Industrial wastewater from a textile plant was collected from the
Mardini Fabrics Co., 10th of Ramadan City, Egypt. Three soil and water
samples were collected from the Suez Canal and Kafr El Sheikh, in the
Nile Delta region of lower Egypt. One soil sample was collected from
cultivated soil at San Joaquin valley, near Modesto, California, USA.
2.3. Microbiological media
Mineral salts medium (MSM) was prepared as described by Chao
et al. (2006), with slights modifications. The medium contained (g l−1):
NH4NO3, 0.5; K2HPO4, 1.5; KH2PO4, 0.5; MgSO4·7H2O, 0.2 g; FeSO4,
0.02 g; CaCl2, 0.05; and CuSO4, 0.02 g. The medium was supplemented
with 0.5 g/l of yeast extract as a source of growth factors and the azo-
dyes, each at a final concentration of 100 μg/ml. The pH of this medium
was adjusted to 5.0 for fungi and 7.0 for isolation of bacteria. This
medium was solidified by the addition of 1.7% agar, for culture
maintenance. Liquid medium was used for testing the decolorization
activity of all fungi.
2.7. Assays for azo dye decolorization activity
A 10 ml aliquot of MSM medium supplemented with 100 μg/ml of
the tested azo dye was added to a 50-ml culture tube and separately
inoculated with 100 μl (OD600nm = 1.0) of each spore suspension.
Tubes were incubated at 150 rpm at 28 °C for 5 days. Three replicates
cultures were used for each dye/strain combination. Biomass was se-
parated by centrifugation at 5000 xg for 5 min, and absorbance of the
supernatant was measured at the λ-max of each azo dye (Table 1). The
λ-max of each azo dye was obtained from scanning a 10 μg/ml solution
using an Agilent G1120A spectrophotometer. The initial OD (control)
from non-inoculated MSM containing the azo dye was measured and
the relative decolorization percentage was calculated as follows:
Percent relative decolorization = 100 − [(OD measured/OD initial)] *
100
The separated fungal biomass was dried at 70 °C until constant
weight, usually only a few mg was produced. Since a relatively large
amount of dye was used in our assay, and only a small amount of dye
can be adsorbed by the limited amount of fungal biomass produced, we
did not use dead biomass controls. Moreover, dye decolorization oc-
curred in a fungal-specific manner.
Consequently, we attributed the majority of dye decolorization to
biotransformation by living fungal cells.
The biodegradation products of methyl red and direct violet azo
dyes by the four selected fungal strains was determined by LC/MS and
GC/MS to identify the biodegradation mechanism (unpublished data).
2.8. Enzyme assays

2.4. Isolation of azo dye-degrading microorganisms
A 10 g aliquot of each soil sample was independently suspended in
100 ml sterilized saline solution (0.85% NaCl) and shaken for 1 hat
150 rpm. A 1 ml aliquot of each soil suspension, or water sample, was
separately added into a petri dish and 25 ml of molten MSM medium,
containing either commercial direct blue or direct green azo-dyes at
Manganese peroxidases activity was determined by measuring the
purpurogallin formation rate at 420 nm, from the reaction between
pyrogallol and hydrogen peroxide catalyzed by peroxidase
(Bourbannais and Paice, 1988). This was done using the extinction
coefficient ε420 nm = 2640 M−1 Cm−1. One unit of peroxidase was
defined as the amount of enzyme that catalyzed the production of 1 mg
of purpurogallin in 20 s at 25 °C and at pH 6.0. Laccase activity was

12
W.M. Abd El-Rahim et al. Journal of Biotechnology 260 (2017) 11–17

measured by using 2,6-dimethoxyphenol (DMP) (Sigma, St Louis, MO,
USA) according to Palmieri et al. (1997). Dopachrome formation was
measured at 477 nm using the molar extinction coefficient
ε477 = 18400 M−1 cm−1. Activities are expressed as μΜ/min. One
unit of laccase activity was defined as the amount of enzyme that cat-
alyzed the oxidation of 1 μM of DMP/min. at 25 °C.
2.9. Effect of nutritional factors on the decolorization activity
The influence of glucose concentration, types of nitrogen sources,
and microelements on dye decolorization, biomass and enzymes pro-
duction was investigated. To study the effect of glucose concentration
on decolorization activity, MSM was supplemented with either 10 μg/
ml methyl red or 100 μg/ml direct violet and with glucose concentra-
tions of 0, 0.25, 0.5% or 1% (w/v).
The influence of organic nitrogen source on decolorization activity
was studied by substituting casamino acids or yeast extract (0.5 mg/ml)
for the ammonium sulfate in MSM. To study the effect of microelements
concentration on decolorization activity, MSM was supplemented with
0, 1, 2, or 3 ml/L of a filter sterilized (0.2 μm) solution of microele-
ments containing:0.3 g H3BO3, 0.2 g CoCl2 6H2O, 0.1 g of ZnSO4 ·7H2O,
30 mg of MnCl2·4H2O, 30 mg of NaMoO4·2H2O, and 20 mg of
NiCl2·6H2O, in 1.0 l water. Five ml aliquots of supplemented MSM in
50 ml culture tubes were inoculated with 0.5 ml (O.D. = 1) of each
spore suspension. The tubes were incubated at 28 °C, with shaking for
2 days. The decolorization percentage of direct violet and methyl red,
as well as the activity of laccases and peroxidases were determined after
2-days of incubation. Each test contained three replicates.
2.10. Statistical analysis
All results were analyzed for significance by using ANOVA and
Duncan-Wallis multiple range analysis at α = 0.05.
2.11. Deposition of strains
All fungal isolates used in this study have been deposited in the
Egyptian Microbial Culture Collection Network (http://www.emccn.eg.
net), Academy of Scientific Research and Technology (ASRT), Egypt,
under accession numbers 3031–3049 (Table 2).
3. Results and discussion
3.1. Fungal isolation and identification
Nineteen fungal isolates were obtained from soil and wastewater
samples on growth medium containing direct green azo dye. Since the
sole source of carbon in the MSM isolation medium is the azo dye, all
isolated fungal isolates used the azo dye as a sole carbon source. The
isolation was also based on the capacity of isolates to decolorize and
degrade the dye. The taxonomic status of each strain was determined by
sequencing of ITS genes. Results in Table 2 show that the isolated fungi
belonged to five species of Aspergillus: A. niger, A. terreus, A. oryzaem, A.
flavus, A. fumigatus, and A. alabamensis and a single L. corymbiferastrain.
Also, our results showed the presence of two pairs of similar fungi
strains 4 and 5, both similar to Aspergillus fumigatus strain FJAT-31052,
and strains 16 and 17, both 100% identical to Aspergillus terreus strain
FJAT-31011. The identification of fungi using ITS gene has been used
previously for assigning fungal taxonomic status criteria (Paolocci
et al., 1999; Abd El-Rahim et al., 2015).
3.2. Screening for azo dyes decolorization activity
All unique fungal strains were tested for decolorization of 20 azo
dyes (Table 3). The most easily transformable azo dye was direct violet,
where decolorization ranged from 71.1 to 93.3%. In contrast, the most
resistant azo dye was fast green, where the decolorization ranges from
0.2 to 8% (Table 3). Abd El-Rahim et al. (2009) and Abd El-Rahim and
El-Ardy (2011) previously reported that direct violet was an easily
degradable azo dye as compared to fast green. The four fungal strains
with greatest degradation capacity: A. niger strain A40, A. terreus strain
CSV2, A. oryzae strain QRF355, and A. fumigatus strain S45 18S, were
selected for optimization studies. These results are in agreement with
those of Wesenberg et al. (2003) who reported that A. niger, A. flavus
and Penicillium sp. are active azo dyes-degrading fungi. Gaanappriya
et al. (2012) also reported that A. niger and A. flavus showed the highest
decolorization percentages of direct red by (87% and 83%) after 48 h,
respectively. Madhuri and Lakshmi (2014) evaluated A. niger, A. fumi-
gates and A. flavus for decolorization capacity of three azo dyes (Congo
red, trypan blue and methyl red). A. fumigatus showed the highest de-
colorization percentage of Congo red after 21 days of incubation and an
Aspergillus sp. strain has been reported as degrader of Coomassie Bril-
liant Blue (Aditee et al., 2014) and Congo red azo dye (Srinivasan and
Devi, 2010).

Table 2
Fungi isolated in this study and identified by sequencing of the ITS1- ITS4 genes.

Strain No. Closest organisms (% identification to nearest relative) Accession number in EMCCNa

1 Aspergillus alabamensis strain CBS (100%) 3031

2 Aspergillus flavus isolate AfKSA13-06 (100%) 3032
3 Aspergillus flavus strain FJAT-31042 (100%) 3033
4 Aspergillus fumigatus strain FJAT-31052 (100%) 3034
5 Aspergillus fumigatus strain FJAT-31052 (100%) 3035
6 Aspergillus fumigatus strain MSEF106 (97%) 3036
7 Aspergillus fumigatus strain S45 18S (100%) 3037
8 Aspergillus niger strain 211 (100%) 3038
9 Aspergillus niger strain A40 (100%) 3039
10 Aspergillus oryzae isolate AoKSA13-04(100%) 3040
11 Aspergillus oryzae strain QRF355 (100%) 3041
12 Aspergillus terreus ATCC 1012 (95%) 3042
13 Aspergillus terreus isolate E 18S (95%) 3043
14 Aspergillus terreus strain CSV2(99%) 3044
15 Aspergillus terreus strain CSV2i (100%) 3045
16 Aspergillus terreus strain FJAT-31011 (100%) 3046
17 Aspergillus terreus strain FJAT-31011 (100%) 3047
18 Aspergillus terreus strain FJAT-31038 (100%) 3048
19 Lichtheimia corymbifera isolate 1–5-f-9(100%) 3049

a
EMCCN: Egyptian Microbial Culture Collection Network (http://www.emccn.eg.net).

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W.M. Abd El-Rahim et al. Journal of Biotechnology 260 (2017) 11–17

Table 3
Decolorization percentages of 20 azo dyes by the isolated 17 fungal strains.*

*
The shaded cells show the highest decolorization percentages of each dye.

14
W.M. Abd El-Rahim et al. Journal of Biotechnology 260 (2017) 11–17

Table 4
Effect of glucose concentration on the decolorization percentage of methyl red and direct violet azo dyes by the four fungal strains.

Fungus Glucose Concentration (%)

0 0.25 0.5 1

Methyl red Direct violet Methyl red Direct violet Methyl red Direct violet Methyl red Direct violet

A. niger 35.95 i 0.333 m 60.42 g 41.50 i 87.11 b 60.24 f 84.75 b 53.39 g

A. terreus 33.94 i 11.087 l 53.27 h 59.66 f 60.98 g 45.76 h 80.97 c 70.15 e
A. oryzae 0.333 j 26.79 k 72.76 d 83.75 b 61.43 fg 83.82 b 64.10ef 78.96 d
A. fumigatus 0.969 j 39.33 j 66.52 e 85.86 a 91.34 a 79.36 d 52.95 h 82.27 c
LSD 2.774 0.7821 2.774 0.7821 2.774 0.7821 2.774 0.7821

Table 5
Effect of glucose concentration on the lignin peroxidase (Lip) and laccase (Lac.) production (U/ml) by fungi grown in MSM supplemented with methyl red or direct violet azo dyes.

Fungus Glucose Concentration (%)

0 0.25 0.5 1

Methyl red Direct violet Methyl red Direct violet Methyl red Direct violet Methyl red Direct violet

Lip Lac Lip Lac Lip Lac Lip Lac Lip Lac Lip Lac Lip Lac Lip Lac

A. niger 0 0 0 4.90a 0 0 0 3.05e 0 0 0 3.05e 0 0 0 4.25c

A. terreus 0 0 0 4.91a 0 0 0 3.89d 0 0 0 5.13a 0 0 0 3.92d
A. oryzae 0 0 0 4.40bc 0 0 0 2.44g 0 0 0 2.79f 0 0 0 2.95ef
A. fumigatus 0 0 0 4.52b 0 0 0 3.06e 0 0 0 4.36bc 0 0 0 2.51g
LSD 0.2416 0.242 0.2416 0.2416

Table 6 3.4. Effect of glucose concentration on laccases and lignin peroxidases

Effect of nitrogen source types on azo dyes decolorization by fungi grown in MSM sup- production in MSM supplemented with methyl red and direct violet
plemented with either methyl red (MR) or direct violet (DV) azo dyes.

Fungus Nitrogen Source

Results of enzyme analyses showed that glucose supplementations
led to significant inhibition of laccases production by all tested fungi in
Ammonium sulfate Casamino acids Yeast extract direct violet containing medium. This effect may be due to the utilizing
of glucose as a carbon source instead of the azo dye which leads to a
MR DV MR DV MR DV
decrease in the laccase production. In contrast, laccase was completely
A. niger 35.95h 0.000L 2.970j 0.341k 64.93f 54.14f absent in the MSM supplemented with methyl red (Table 5). Methyl red
A. terreus 31.94i 11.87j 51.62g 49.04g 68.42e 55.13e does not appear to be a suitable substrate for laccases, as it does not
A. oryzae 0.000L 26.79i 78.79d 82.48d 90.62b 91.40b contain hydroxyphenyl group. Laccases have been extensively studied
A. fumigatus 0.480k 39.33h 93.45a 91.95a 86.08c 83.57c
for their degradation efficiency of azo dyes (Blanquez et al., 2004;
LSD 0.053 0.053 0.053 0.053 0.053 0.053
Novotný et al., 2004). Laccase production by A. niger, A. fumigatus and
A. flavus on MSM supplemented with Congo red was previously in-
3.3. Influence of glucose concentration on decolorization of direct violet and vestigated by Madhuri and Lakshmi (2014).
methyl red Lignin peroxidase was absent in the MSM supplemented with either
direct violet or methyl red azo dyes at all of the tested glucose con-
The decolorization of direct violet and methyl red by the four fungi centrations (Table 5). The peroxidase was detected previously (Prabin
was significantly enhanced by glucose supplementation. The highest et al., 2016) when these fungi were grown in MSM supplemented with
decolorization percentage (85.8%) was obtained by A. fumigatus at a 0.5 g/L yeast extract. Since yeast extract is considered as an organic
glucose concentration of 0.25%, followed by A. oryzae (83.7%) at the nitrogen source as well as source of growth factors (vitamins and es-
same sugar concentration. In contrast, the maximum methyl red deco- sential amino acids). Therefore, these fungi are not able to produce the
lorization (91.3%) was obtained by A. fumigatus at sugar concentration lignin peroxidase in absence of either organic nitrogen sources or some
0.5%, followed by A. niger (87.1%) at the same sugar concentration growth factor, or both.
(Table 4). The enhancement of fungal decolorization by glucose sup-
plementation was likely due to an increase in biomass production,
which in turn, adsorbs the azo dyes. These results are in agreement with 3.5. Effect of nitrogen source on azo dyes decolorization
Madhuri (2014) who showed that the maximum decolorization of
trypan blue by A. niger, A. fumigatus and A. flavus occurred with In this experiment, the inorganic nitrogen source (ammonium sul-
medium supplemented with 2% glucose at pH 4. fate) in MSM was replaced by either yeast extract or casamino acids.
In the MSM supplemented with methyl red, the pH of the medium When the ammonium sulfate was used as a sole source of nitrogen, the
was strongly decreased up to 2.5 units by all of the tested fungi; how- decolorization of direct violet or methyl red were the lowest by all of
ever, the methyl red color was not reconstituted after pH adjustment to the tested fungi. The yeast extract enrichment significantly enhanced
the initial value (pH 5). The pH decrease was likely due to production of direct violet decolorization by A. niger (0% to 54%). Similar trends were
organic acids from glycolysis by A. niger. These acids are not produced found with the decolorization activity by A. oryzae and A. terreus. Direct
in absence of glucose (Belén Max et al., 2010; Lei et al., 2014; Abd El- violet decolorization by A. fumigates was significantly enhanced in
Rahim et al., 2009). medium containing casamino acids. The same trend was also observed
in the methyl red supplemented MSM; where the absence of either yeast

15
W.M. Abd El-Rahim et al.
Table 7
Effect of nitrogen source types on lignin peroxidase (Lip) and laccases (Lac.) production (U/ml) by fungi grown in MSM supplemented with either methyl red or direct violet azo dyes.
Fungus Nitrogen Source
Ammonium sulfate Casamino acids
Methyl red Direct violet Methyl red
Lip Lac Lip Lac Lip
A. niger 0.0f 0 0.0d 0 0.91b
A. terreus 0.0f 0 0.0d 0 0.317d
A. oryzae 0.0f 0 0.0d 0 0.070e
A. fumigatus 0.0f 0 0.0d 0 0.297ef
LSD 0.5355 0.5355 0.5355
extract or casamino acids completely inhibited methyl red decoloriza-
tion activity by A. oryzae and A. fumigatus. The enhancement of the
fungal decolorization activities by yeast extract and casamino acids
supplementation (Table 6) is likely due to presence of the required
growth factors such as essential amino acids and vitamins in these
sources (Cruz et al., 2002; Abd-El Rahim et al., 2003).
3.6. Effect of nitrogen source on laccases and lignin peroxidase production
In presence of ammonium sulfate as sole nitrogen source the lignin
peroxidase and laccases were absent in MSM supplemented with either
methyl red or direct violet azo dye. When the MSM was enriched with
either casamino acids or yeast extract, the lignin peroxidase was sig-
nificantly enhanced by all of the tested fungi. The presence of organic
nitrogen source (casamino acids or yeast extract) in the media was
found to enhance the production of lignin peroxidase. This enhance-
ment may be due to the increase in biomass production as has been
noted by Kaal et al. (1993).
Peroxidase production in the medium was negligible in nitrogen
limited medium (Kaal et al., 1993). The fungus A. niger showed the
highest lignin peroxidase production in the direct violet or methyl red
containing MSM enriched with either casamino acids or yeast extract.
Lignin peroxidase production was also significantly enhanced by A.
terrreus in MSM supplemented with the organic nitrogen sources.
However, the production in MSM supplemented with methyl red was
significantly higher than in the presence of direct violet in MSM con-
taining the tested organic nitrogen sources (Table 7). These results in-
dicate the induction effect of the organic nitrogen source and/or the
growth factors on lignin peroxidase production by the tested fungal
strains. In contrast, the laccases were absent in the MSM enriched with
either yeast extract or casamino acids as nitrogen sources. This shows
the importance of ammonium sulfate for fungal laccase production
(Table 7). These findings are in agreement with the results obtained by
Manimozhi and Kaviyarasan (2012), where they stated that laccases
production was stimulated when both ammonium tartrate and yeast
extract were used as nitrogen sources. Laccase production was found to
be dependent on both carbon and nitrogen sources. Similar results were
previously reported by Eggert et al. 1996) who found that the nitrogen
source plays a key role in the laccase production. It is clear that the
presence of inorganic nitrogen source in the medium suppressed laccase
production, whereas the organic nitrogen sources enhanced the laccases
yields. This is in agreement with the results indicating that yeast extract
is one of the best organic nitrogen sources that increases the laccase
yield (Arora and Rampal, 2002).
4. Conclusions
The bioremediation of textile azo dye residues represents the most
efficient approach to remove these pollutants from water before
reaching the surrounding environment. This study focused on isolation
and identification azo dye of degrading fungi and on optimization of
16
Journal of Biotechnology 260 (2017) 11–17
Yeast extract
Direct violet Methyl red Direct violet
Lac Lip Lac Lip Lac Lip Lac
0 0.851b 0 0.989a 0 0.921a 0
0 0.020d 0 0.570c 0 0.170c 0
0 0.050d 0 0.330d 0 0.020d 0
0 0.040d 0 0.020ef 0 0.012d 0
0.5355 0.5355 0.5355
degradation conditions. Nineteen fungal isolates were isolated and
identified. These fungi belonged to five species of Aspergillus and a
single species of Lichtheimia. The maximum degradation of direct violet
and methyl red dyes was optimized using the most promising 4 fungal
strains on media containing glucose concentration and organic nitrogen
source. The fungal strains were not able to produce lignin peroxidase in
the absence of organic nitrogen source. Peroxidase and laccases were
absent in both azo dyes supplemented MSM medium lacking organic
nitrogen source. The supplementation of MSM medium with either
casamino acids or yeast extract markedly enhanced the lignin perox-
idase by all tested fungi.
Acknowledgments
This study was funded, in part, by a grant from the US-Egypt Joint
fund, project No 4462, and by a grant from the University of Minnesota
Agricultural Experiment Station (to MJS). The funding sources had no
involvement or role in the study design; in the collection, analysis and
interpretation of data; in the writing of the report; and in the decision to
submit the article for publication.
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