Comparison of Alkali-Tolerant Fungus Myrothecium Sp. IMER1 and White-Rot Fungi for Decolorization of Textile Dyes and Dye Effluents

2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from Sigma Chemical Company (St. Louis, MO, USA). RBBR (C.I. 61200) was purchased from Sigma (St. Louis, MO, USA), Congo Red (C.I. 22120) and Indigo Carmine (C.I. 73015) from Tianjin Damao Chemical Reagent Factory (Tianjin, China). All other chemicals used were analytically pure. Highly colored effluents (dark red) were collected from a textile dye-producing plant situated in Foshan, Guanzhou, China. The dye effluents contained both acid and direct dyes. The main components were azo dyes. Apart from azo phenyl and cyclohexyl substituted anthraquinonic dyes were coloring the effluent. It was an alkaline wastewater with a pH value of 10-10.5. Prior to laboratory decolorization treatment, the effluent was centrifuged at 10,000×g for 15 min to remove large suspended particles and then was sterilized. Sample absorbances were measured using a Varian CARY50 UV-Vis (St. California, USA) spectrophotometer; pH values were measured with a pHS-3A pH-meter (Hangzhou Wanda Instrument Factory, China).
 

Fungal strains used in this study were: SDK (Polyporus sp. SDK), AX3 (Leutinus sp. AX3), DS1 (Schizophyllum sp. DS1), CD3 (Irpex sp. CD3), BP2 (Pleurotus sp. BP2), IMER1 (Myrothecium sp. IMER1). The strain Myrothecium sp. IMER1 was isolated from soil from the suburb of Wuhan, P.R.China. On the basis of the comparison of sequences of the ITS regions and 5.8SrRNA gene with those found in databanks, its morphology, and microscopy observations, it was identified as Myrothecium sp. (GenBank accession no. EF458487). These strains were isolated and identified in our laboratory. They were grown in potato dextrose broth medium (PDB), or on potato dextrose agar medium (PDA). Every strain was maintained on PDA medium slant. The slant was inoculated and incubated at 28°C for 5-6 days, and then stored at 4°C and periodically sub-cultured.
 

Dye agar plates were prepared using PDA containing 60mg L^-1 of each individual dye. Plates were inoculated with a 1 cm² piece of the fungus isolated from a six day PDA plate growth, cut out from the actively growing fungal culture. Dye agar plates that were not inoculated served as controls. All plates were incubated at 28°C. After six days, the size of the decolorization halo was measured.
 

For the decolorization experiments, five agar plugs (6 mm diameter) of active mycelium from PDA plate was transferred aseptically into 500 ml Erlenmeyer flasks containing 100 ml of autoclaved PDB medium and RBBR (60mg L^-1) or the dye effluent at different concentration of 30% (v/v). The pH of PDB medium was adjusted using 1.0 M NaOH or 1.0 M HCl. The cultures were incubated at 28°C in rotary shakers running at 150 rpm. Biotic (sterilized medium, without dye addition) and abiotic (sterilized medium containing the effluent but not inoculated with the fungus) control experimental sets were also prepared and maintained in parallel with the decolorization experiments. The cultivations were carried out in triplicate. Culture samples were withdrawn at defined intervals, and after centrifugation at 10,000×g for 20 min at 10°C, the supernatants were used as samples for decolorization assay. Biomass production was evaluated by determining the dry mass of mycelia. Mycelia were harvested from the cultivation liquid by filtration using a piece of filter paper, dried at 105°C for 24 h and weighed.
 

Bilirubin Oxidase (BOX) or laccase activity was quantified using an assay based on the oxidation of ABTS. The assay reaction mixture consisted of 1.5 mM ABTS, sodium acetate buffer (0.2 M, pH 4.0) and a suitable amount of enzyme to create a total reaction volume of 3.0 ml that was incubated at 25°C. The unit activity was expressed as 1μM of product formed per min under the assay conditions [23,24].
 

Decolorization of RBBR was analyzed in culture filtrates and monitored spectrophotometrically at the maximum wavelength of absorbance (595 nm). The intensity of effluent color was measured at its maximum absorbance wavelength 510 nm. Color removal rate (P) was calculated according to the following formula: P=(A₁-A₂)/A₁×100%. Where A₁ represented the absorbance of the control, A₂ represented the absorbance of the corresponding untreated sample, and P was the dye/effluent color removal rate.
 

All decolorization experiments were performed in three sets. The results reported are an average of the three data points with standard deviations calculated. The data from phytotoxicity studies were analyzed by a one-way analysis of variance (ANOVA) using a Tukey-Kramer multiple comparison test.
 

The selected dyes (Indigo Carmine, RBBR, Congo red, Malachite green and Bromophenol Blue) have never been tested before with Myrothecium sp. IMER1 on solid medium (Figure S1). First, Indigo Carmine was completely decolorized, as revealed by the >78 mm diameter of the decolorized zone after six days. Decolorization of RBBR was less extensive with a decolorized zone diameter of between 63 to 70 mm. Second, partial decolorization was obtained for Congo red and Malachite green. Finally, little decolorization was observed for Bromophenol Blue. Myrothecium sp. IMER1 showed different potentials to decolorize five different dyes which belong to four classifications. The class of the dye which defines its structure is influential in deciding the extent to which dye is decolorized. Wong and Yu observed that anthraquinone dye was Trametes versicolor laccase substrate while azo and indigo dyes were not the substrates of laccase [25].

Thus it is seen that that dye decolorization by fungus was dependent on dye structures. Spadaro et al reported that aromatic rings with substituents such as hydroxyl, amino, acetamido, or nitro functions were mineralized to a greater extent than unsubstituted rings in dye decolorization by Phanerochaete chrysosporium[26]. Knapp et al reported that relatively small structural differences could markedly affect decolorization and this might be due to electron distribution and charge density as well as steric factors [27].
 

RBBR is a typical anthraquinone dye used in the textile industry, represents an important class of toxic and recalcitrant organopollutants [28]. It was reported that the dye can be decolorized by many white-rot fungi and Myrothecium sp. IMER1 [20,28]. Therefore we compared decolorization capability of Myrothecium sp. IMER1 for Remazol Brilliant Blue R (RBBR) with five different white-rot fungi over a pH range of 5.0 to 10.0. The decolorization rate of RBBR was measured in culture filtrates after 6 d of incubation. As shown in Figure 1a, more than 65% of decolorization was observed with these white-rot fungi in the acidic pH range of 5.0-6.0, whereas color removal rate was less than 30% in the basic pH range of 8.0-10.0. By contrast, 60-70% color removal was obtained with Myrothecium sp. IMER1 in the acidic pH range of 5.0-6.0, and more than 65% of decolorization was observed in the basic pH range of 8.0-10.0. These results showed that Myrothecium sp. IMER1 exhibited excellent decolorizing performance at all the pH value tested (5.0 to 10.0). Kapdan et al. reported the optimum growth pH of Coriolus versicolor as 4.5 [29]. Decolourization of Solar golden yellow R by Schizophyllum commune indicated that maximum decolourization efficiency (73%) was observed at pH 4.5 after 6 days [30]. It has been widely reported that for majority of the fungi the optimum pH for dye decolorization is in the acidic range. Unfortunately, such a low pH is not suitable for the wastewater treatment. Therefore, fungal strain showed an appreciable decolorization of dye over a wide range of pH, which is desirable for industrial applications since it can be used for wastewater treatment without a previous pH adjustment stage [31].

Decolorization of dyes by most fungi could be due to adsorption to microbial cells or to biodegradation [32,33]. If the dye removal were attributed to biodegradation, either the major UV-VIS light absorbance peak would completely disappear or a new peak would appear. In adsorption, cells of fungus become deeply colored because of adsorbing dyes [34,35]. The absorbance spectra of the dye were scanned before and after decolorization and changes in its absorption spectrum (350-800 nm) were recorded. The absorption peaks of RBBR in the visible region disappeared, suggesting that removal of dye by Myrothecium sp. IMER1 should be partly attributed to biodegradation (Figure 1b). In addition, in the decolorization of RBBR by Myrothecium sp. IMER1, the cells were stained deeply colored at pHs (5.0-10.0), indicating that the dye was adsorbed on the cell surface, which was partly due to biosorption (Figure 1c). It suggested that decolorization by Myrothecium sp. IMER1 involved biosorption and biodegradation. This result was in agreement with our previous studies [20,21].

To investigate the effect of pH on growth of Myrothecium sp. IMER1, biomass production was evaluated by determining the dry mass of mycelia. No decrease or increase in biomass production was observed throughout the study in the pH range of 5.0-8.0 (Figure 2a). Interestingly, cells of Myrothecium sp. IMER1 grew almost as well as the control at pH 9.0 and 10.0 (Figure 2b). Although biomass production of Myrothecium sp. IMER1 decreased slightly with increase pH value (9.0-10.0), data statistically analyzed showed that there were no significantly difference in biomass production between control group and experimental group. In comparison with Myrothecium sp. IMER1, growth of white-rot fungi tested such as Pleurotus sp. BP2 was strongly inhibited and low biomass production was observed in the basic pH range of 9.0-10.0 (Figure 2c). The results indicated that biomass production of Myrothecium sp. IMER1 might not be affected by changes in the pH range of 7-10, which also confirmed that Myrothecium sp. IMER1 is alkali-tolerant fungus. Previously, Sulistyaningdyah et al. had reported that Myrothecium verrucaria 24G-4 is an alkali-tolerant fungus and can grow at pH10.0 [36].
 

Although a large number of lab-scale studies have been conducted on decolorization of single synthetic dye solutions and simulated dye wastewaters by fungal biosorption/biodegradation, there is a need to generate relative performance data on real dye effluents [37,38]. Decolorization of industrial effluent from dyeing industry is shown in Figure 3. At pH 7.0 and 9.0, decolorization by Myrothecium sp. IMER1 at the effluent concentrations of 30% (v/v) was 73 and 70%, respectively, whereas in the case of white-rot fungi tested less than 25% of decolorization was observed. The result showed that Myrothecium sp. IMER1 had higher color removal efficiency than these white-rot fungi tested under neutral and alkaline conditions. On the one hand, when UV-VIS spectra of dye effluent-containing culture fluid for defined intervals were scanned from 350-800 nm, a marked decrease in absorbance maximum at 510 nm was observed at 6-days incubation, which was related to the breakdown of the chromophoric group in dyes of the effluent (Figure 3b and 3c). On the other hand, the mycelia became deeply colored at the beginning of decolorization, and then became faint, finally almost disappeared (Figure 3d). Spectra analyses of culture supernatants and color changes in fungal cells suggested that dyes in the effluent were removed by Myrothecium sp. IMER1 mainly due to enzymatic reaction.

It was reported that most white-rot fungi only exhibit good decolorization ability in the acidic pH range of 3-6, which due to low pH for the optimum activity of extracellular ligninolytic enzymes [39,40]. The decolorization capability of Myrothecium sp.IMER1 under alkaline conditions will be an advantage in the case of practical applications to dye effluents, because pulping, bleaching and dye-producing are mainly performed under highly alkaline conditions and their effluents generated are also alkaline.

It has previously been reported that BOX as a main extracellular oxidoreductase of Myrothecium sp. IMER1 plays a major role in the dye decolorization, while in the case of many white-rot fungi; main decolorization enzyme is laccase [20-22]. Therefore, BOX/laccase activity and the biomass production of Myrothecium sp. IMER1/white-rot fungi tested were evaluated during decolorization. Both enzyme activity and biomass production of Myrothecium sp. IMER1 were higher than that of all white-rot fungi tested, which attributed to the fact that Myrothecium sp. IMER1 is alkali-tolerant fungus and BOX activity keeps good under neutral and alkaline pHs (Figure 4).