AASCIT Journal of Biology 2015; 1(2): 15-24 Published online April 20, 2015 (http://www.aascit.org/journal/biology) Keywords Waste Water Treatments, Bioremediation of Direct Green Dye, Biosorption, RFLP and Sequencing of 18S, ITS rRNA, Fungi Identification Received: March 29, 2015 Revised: April 11, 2015 Accepted: April 12, 2015 Biodiversity Among Dominant Fungi Involved in Water Production from Non-Traditional Water Resources Wafaa M. Abd El-Rahim 1, * , Abdelaal Shamseldin 2 , Fatma H. Abd El Zaher 1 , Hassan Moawad 1 , Eman Refaat 3 1 Department of Agriculture Microbiology, National Research Centre, Dokki, Cairo, Egypt 2 Environmental Biotechnology Dept, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technology Applications, Alex 3 Department of Microbiology, Faculty of Science, Al-Azhar University, Girls' Branco, Egypt Email address [email protected] (W. M. A. El-Rahim) Citation Wafaa M. Abd El-Rahim, Abdelaal Shamseldin, Fatma H. Abd El Zaher, Hassan Moawad, Eman Refaat. Biodiversity Among Dominant Fungi Involved in Water Production from Non-Traditional Water Resources. AASCIT Journal of Biology. Vol. 1, No. 2, 2015, pp. 15-24. Abstract Now a day it is very important to remove industrial pollutants with minimum cost. The present work focused on the assessment of extracellular laccases produced by different fugal strains and its potential for Direct Green (DG) bio-removal from textile effluent water, which can be used as a resource of non-traditional water. Ten fungal strains from polluted sites were screened to test their capability of DG bioremoval to clean up waste water containing-dye. Fungal dye removal capacity was assessed by measuring the residual of DG dye in supernatant using spectrophotometer, over production of fungal biomass and reduction of COD values after 28h of incubation. Results showed that the fungal isolates could remove the simulated effluent containing-DG dye and they reduced the COD values indicating on bio-removal of dye from the growth media. Strain FRD 11 was the most effective strain which removed about 85% of (300 mgl -1 ) dye in 28h. Molecular characterization of fungal isolates was done by using the restriction fragment length polymorphisms (RFLP) and sequencing of ITS rRNA region as a reproducible molecular method for fungal species identification and differentiation. RFLP of 16S rRNA with CfoI proved the suitability of this technique for strain typing, species identification and could divide them into three genetic clusters. Cluster 1 included strains (FRD1, 2, 3 and 5), cluster 2 (FRD 4, 7, 8, 10 and 11) and cluster 3 (FRD 9). Phylogenetic tree of ITS rRNA of five representative strains showed that strains FRD 7 and 11 were identified as Aspergillus tubigenesis, strain FRD 9 was identified as Aspergillus niger and strains FRD1 and 3 were belong to Aspergillus terreus. These results indicated that these fascinating new fungal strains can play significant role in bioremediation and detoxification of effluents polluted with DG dye due to their ability to biosorb or produce laccase enzyme-remediating dye. 1. Introduction Industrial pollutants are the major environmental threats. Untreated industrial effluents discharged into ecosystems cause serious problems to the surrounding ecosystems. One of the greatest pollutants generated from industrial activities is the textile organic pollutants. Textile effluents contain carcinogenic aromatic amines, dyes, organic and inorganic materials (Ramachandran et al., 2013). Removing of textile dye from effluent gained tremendous concern by environmentalists dye to the negative impact on the
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AASCIT Journal of Biology 2015; 1(2): 15-24
Published online April 20, 2015 (http://www.aascit.org/journal/biology)
Keywords Waste Water Treatments,
Bioremediation of Direct Green
Dye,
Biosorption,
RFLP and Sequencing of 18S,
ITS rRNA,
Fungi Identification
Received: March 29, 2015
Revised: April 11, 2015
Accepted: April 12, 2015
Biodiversity Among Dominant Fungi Involved in Water Production from Non-Traditional Water Resources
Wafaa M. Abd El-Rahim1, *
, Abdelaal Shamseldin2,
Fatma H. Abd El Zaher1, Hassan Moawad
1, Eman Refaat
3
1Department of Agriculture Microbiology, National Research Centre, Dokki, Cairo, Egypt 2Environmental Biotechnology Dept, Genetic Engineering and Biotechnology Research Institute,
City of Scientific Research and Technology Applications, Alex 3Department of Microbiology, Faculty of Science, Al-Azhar University, Girls' Branco, Egypt
Aspergillus was reported by Muthezhilan et al.(2008). The
biodegradation of three azo dyes (Congo red, Orange II and
Tropaeolin O) by the fungus Phaenerocheate
chrysosporium was reported by Cripps et al. (1990).
Revankar and Lele (2007) found that 73% of RB19 dye
(100 mgl-1
) could be removed in 8 hours by Ganoderma sp,
while in our study fungal isolates could remove (300 mgl-1
)
in 28h.
Changes in COD values by different isolates can be used
as a good evidence for progress of bioremediation process
(Ademorotti et al., 1992; Patheet al., 1995), consequently. In
this study the COD values measured at the end of experiment
(28h of inoculation) of the simulated effluent containing dye
and treated by ten fungal isolates are presented in Fig3. The
results showed that the fungal growth reduced the organic
load of the synthetic effluents by 56 to 81%. Fungal isolates
FRD 4, 8, 9 and 10 were the highest efficient strains in
removing (81% removal) of the organic dye contents of the
synthetic effluent. The rest of the isolates were capable of
removing up to 55% of the growth media dye contents. This
clearly showed that the fungal isolates included in this study
are good candidates for bioremediation of green textile dye.
These results are in agreement with previous studies stating
that fungi can play important role in detoxification of waste
water from hazardous organic pollutants (Muthezhilan et al.,
2008; Cripps et al. (1990) and Revankar and Lele (2007).
3.3. Activity of Lac Enzyme in
Biodegradation Process
Lac enzymes were reported to be found in eukaryotes, e.g.,
fungi, plants, and insects. Lac was used for the treatment of
different industrial effluents containing chlorolignin or
phenolic compounds (Bollag and Leonowicz, 1984; Brenna
and Bianchi, 1994; Jonssonet al., 1998; Ullahet al., 2000;
Bohmeret al., 1998; Call and IMucke, 1996). The lac
activities of the ten fungal strains used in this study are
presented in Fig 4. The fungal isolates FRD 1 and 6 had the
same pattern of lac enzyme activity. However, the
bioremoval of green dye by fungal biomass differed
significantly (Fig 4) being 34% with isolate FRD 6 compared
to (72%) removal by isolate FRD 1 after 28h incubation.
Four strains FRD2, FRD3, FRD4 and FRD6 were actively
produced lac enzyme throughout the incubation period.
Based on molecular identification which will presented later
in this study, two of these strains; FRD2 and FRD6 belonged
to Aspergillus terreus, whereas the other two strains; FRD3
and FRD4 were Aspergillus tubigenesis. These four strains
expressed steady increase in lac activity starting with early
incubation till the end of incubation 28h. The strains of A.
terreus FRD3 and FRD4 were distinguished, among all other
strains, with the highest lac activity. The results showed that
the lac activity of these selected strains is strain specific and
not species specific. Strain FRD1 that belonged to A. terreus
did not show lac activity as strain FRD2 and FRD3 which
belonged to the same species. Similar results were found with
strains A. tubigenesis FRD8 and FRD10. None of the strains
belonging to A. niger was distinguished as a strong lac
producer. The results of lac activity did not follow the same
trends obtained in dye bioremoval experiment presented in
(Fig 4). The strains which showed high dye removal capacity
namely, FRD8, FRD9, FRD10 and FRD11 were very poor in
lac production. This clearly showed that the dye bioremoval
takes place by more than one mechanism. It is likely that the
high bioremoval without high production of lac could be
mainly due to the high biosorption of the dye on fungal
biomass. The high biosorption of dyes by fungal growth was
reported before (Wafaa and El-ardy 2011, Wafaa et al., 2009,
Wafaa and Moawad 2010, Wafaa, 2006). The over production
of lac by other strains namely FRD2, FRD3, FRD4 and
FRD6 indicated that these strains contributed to remove of
AASCIT Journal of Biology 2015; 1(2): 15-24 19
the dye residues not only by biosorption but also by
biodegradation through lac production. It has been reported
that lac induce degradation of azo dyes and phonemic
compounds, which exist in commercial azo dyes (Nyanhongo
et al., 2002). Rodriguez et al. (1999) also reported that the
de-colorization of dye by Trametes hispida strain is mainly
ascribed to extracellular enzyme activity of lac that plays a
major role in de-colorization. The bioremediation of textile
dye effluents by fungi is a complex process that include
direct dye removal by fungal biomass (Heinflinget al. 1997;
Asher et al. 2008: Singh and Singh 2010: Tan et al., 2013)
and/or possible dye bioremediation by certain enzymes
existing in fungal cells such as Lac enzyme (Brenna and
Bianchi 1994; Nyanhongo et al., 2002).The reduction of lac
activity during the incubation time was associated with FRD
8, 10 and 11. However, these isolates had high efficiency in
dye bioremoval (85%). This may be attributed to the decrease
in dye concentration as a result of dye degradation which can
cause less induction of the enzyme due to decrease in
substrate. The presence of very low concentration of dye
within 28h could be the reason for decrease in the enzyme
activity. Similar results were reported by Kalme et al. (2007)
for LiP, lac and tyrosinase. On the other hand, Wafaa et al.
(2003b) reported that the rapid biosorption of dyes on the
intact fungal biomass caused a decrease of free dye
concentration in the medium.
3.4. Molecular Identification of Fungal
Isolates
Standard biochemical tests, and morphological
characteristics have conventionally been used for the
identification of fungal species, but these methods of
identification are costly, time-consuming, and require
special skills. In addition, these tests fail to identify near
fungal species due to lack of precise test for species
identification. An amplified fragment of 600 bp of
ITSrRNA was generated by PCR amplification (data not
shown).We used the RFLP technique using two restriction
enzymes (CfoI and MspI) to digest the targeted fragment of
ITS rRNA to differentiate among fungal isolates. The
digestion of the PCR product by enzyme CfoI could classify
the ten fungal isolates into three genetic profiles (Fig5).
Genetic profile I included (strains FRD 1, 2, 3 and 5);
genetic profile II contained (strains FRD 4, 7, 8, 10 and 11)
and genetic profile III (strain FRD 9). Strain FRD 6 had the
same genetic profile of group I (data not shown). The RFLP
of PCR product with enzyme Msp1 confirmed the results
obtained from digestion with CfoI (Data not show). The
obtained RFLP results confirmed the possibility of using
this part of ribosomal rRNA to differentiate among fungal
strains as reported previously by Colin et al. (1999) who
used the same part of ITS to identify Dermatophyte Fungi
strains. The use of RFLP analysis of ITS interagenic regions
of the rRNA repeat is a valuable technique both for
molecular strain differentiation of T. rubrum and for species
identification of common dermatophyte fungi. This study
showed that internal transcribed spacer region analysis
using polymerase chain reaction based on RFLP using CfoI
and MspI enzymes is useful for rapid differentiation among
the fungal isolates capable of removing textile dye.
Paolocci et al. (1997) successfully used the same technique
to identify commercially important black truffle fungi
(Tuber melanosporum). Results of phylogenetic tree (Fig 6)
confirmed the results of RFLP analysis. Strains FRD1 and 3
shared the genetic clade with reference strain of Aspergillus
terreus KALM 104, while strains FRD 7 and 11 similar to
Aspergillus tubigenesis and strain FRD 9 was similar to
Aspergillus niger. However there is no clear difference
between these last three strains in the phylogenetic tree,
although, strain FRD 9 had a unique RFLP pattern. This
may be due to mutation in into restrictions sites that made
strain FRD 9 differed than other strains or due to the high
similarity between species of Aspergillus niger and
Aspergillus tubigenesis. There are many papers on the
bioremediation of Azo-dyes but for our knowledge this is
the first report about the bioremediation of this specific dye
(green direct dye). Our results reflected the importance of
such fugal isolates to be use as a safe bio resource fungal
product to reduce the environmental pollution caused by the
industrial effluents. This study also identified three different
fungal species equipped with enzymatic machinery that can
play important role in textile dye bioremediation.
20 Wafaa M. Abd El-Rahim et al.: Biodiversity Among Dominant Fungi Involved in Water Production from
Non-Traditional Water Resources
Figure 1. Bioremoval of direct green dye by the biomass of fungal isolates throughout 28 hours of inoculation.
AASCIT Journal of Biology 2015; 1(2): 15-24 21
Figure 2. Fungi biomass accumulation after 28 hours in the medium amended with direct green dye.
Figure 3. Efficiency of DG dye bioremediation as indicated by COD reduction after 28 hours of inoculation.
22 Wafaa M. Abd El-Rahim et al.: Biodiversity Among Dominant Fungi Involved in Water Production from
Non-Traditional Water Resources
Figure 4. Activity of Laccase enzyme as expressed in OD associated with growth of fungal isolates.
AASCIT Journal of Biology 2015; 1(2): 15-24 23
Figure 5. RFLP of ITS rRNA fragments for fungal isolates using Cfo1
enzyme. From left to right, lanes: 1, 100 pb ladder, then FRD 1, FRD2,
FRD3, FRD4, FRD5, FRD7, FRD8, FRD9, FRD10, FRD11, and 100 bp
ladder.
Figure 6. Neighbor-Joining phylogenetic analysis of five representative
strains using 600 bp of ITS rRNA, Bootstrap probabilities of 1000 pseudo
replicates are indicated.
4. Conclusions
This study demonstrated that several fungal strains belong
to three different species of Aspergillus were highly active in
removing direct green textile dye from aquatic polluted
effluents through the two pathways of biosorption and
biodegradation. Strains FRD 1 and FRD7 were proven to be
fast bio-removing of the DG dye residue most likely by
biosorption mechanism. A. terrous strains FRD 3 and FRD4
were actively bio degrading dye due to their ability for lac
production. The consortium of these strains is recommended
as bioremediation agent for fast and save removal of the DG
dye residue.
Acknowledgement
Authors wish to thank Egypt/US joint funded for
sponsoring the project on “Developing of Enzymatic
Bioremediation Technology For Textile Dye Bioremoval”
which supported this research.
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