Abstract: The aim of this study is to evaluate amaranth textile dye decolorization by white-rot fungus Pleurotus eryngi F019 isolated from tropical rain forest. The isolate was able to decolorize an azo dye in wide range (up to 200 mg), at temperature 25°C. The results revealed that the removal azo dye amaranth was rapid at the initial incubation period (15 days) and became slowly with the time (30 days). The maximum decolorization was observed after 30 days incubation. Addition of Cu2+, Mg2+, Mn2+ gave the positive results while decolorization was inhibited by addition of Fe2+. Metal ion also affected the level of enzyme production during decolorization of amaranth. Induction in the activity of laccase and lignin peroxidase was observed during decolorization of amaranth in the culture, which represented their important role in biotransformation. The biodegradation of amaranth dye was monitored by UV-Vis spectrometer and gas chromatography. These promising results suggest the application of Pleurotus eryngi F019 to treat dye containing wastewater having higher concentrations of metals.
INTRODUCTION
Azo dyes are an important and the largest classes of synthetic organic compounds are widely used as coloring agent in the production of textile, paint, ink, fibers, plastics, leather, paper, mineral oils, waxes, foodstuffs and cosmetics. Azo dyes have complex molecular structures which resist their elimination in the environment, resulting in direct and indirect exposure of human life and other organisms. These dyes are characterized by the presence of one or more azo groups (-N = N-) bonds to aromatic rings, which may also carry sulfonic acid groups. A large amount of wastewater with azo dyes is discharged from these industries into the environment (Bras et al., 2005; Zhang et al., 2011). Previous study reported that azo dyes contribute mutagenic activity of aquatic environment polluted by textile effluents. This may cause a significant impact to human health as well as other animals due to mutagenic and carcinogenic effects of majority of azo dyes and their metabolites (Pierce, 1994).
Many researchers have studied in the field of biological treatment due to the potentially low cost. Treatment of wastewater containing synthetic dyes involves chemical and physical methods such as ultrafiltration, ozone oxidation, coagulation, precipitation, adsorption and ionizing radiation. The negative aspect these treatments are not only very expensive but also commercially unattractive and still be toxic (by product), which are danger to dispose off and are difficult in application. Microbial processes provide a promising method to existing technologies because they are more environmentally friendly, less cost and transform a toxicity of dye structure (Borchert and Libra, 2001; Lopez et al., 2002; Hadibarata and Tachibana, 2009).
Several microorganisms such as fungi, bacteria, yeasts and algae, have ability to decolorize and even completely transform many azo dyes structure under certain optimum conditions. The effectiveness of decolorization depends on the dye structure and characteristic even though relatively small structural differences can affect the decolorization rate. The capability of white-rot fungi is due to extracellular non-specific and non-stereoselective enzyme production such as laccases (EC 1.10.3.2), lignin peroxidases (EC 1.11.10.14) and manganese peroxidases (EC 1.11.1.13) (Novotny et al., 2004). Most azo-dye-degrading microorganisms cleave the azo bond (s) of the respective azo dye and produce decolorized products (Liu et al., 2004). Several factors can influence the ability of fungi in biodegradation, such as the fungal growth conditions and the chemical structure of azo dye. Azo dyes with ortho-and para-substituted mono- and polyphenol bond or aromatic group in their chemical structure are easy and faster to decolorize than azo dyes with non-substituted aromatic structure (Selvam et al., 2003). Some white-rot fungi have been reported to completely degrade azo dyes without the formation of aromatic amines bonds (Chivukula and Renganathan, 1995). Dye decolorization is always performed using enzymes secreted by microorganism. These enzymes can be permanently inhibited by some heavy metal compounds. Some metal compounds had shown an enzymes inactivation effect on decolorization of anthraquinone dye Remazol Brilliant Blue R (RBBR) by white-rot fungus Polyporus S133 (Hadibarata and Kristanti, 2012). In previous study, we have isolated white-rot fungi which have potential to decolorize another azo dye (Remazol Black 5). Also we showed the acceleration of decolorization by addition of carbon and nitrogen source, agitation and aramaticion in the culture (Hadibarata et al., 2011, 2012). In the present investigation, we have investigated the heavy metals effect on decolorization ability of Pleurotus eryngi F019 during decoloriztion of amaranth and their effect on enzyme production.
MATERIALS AND METHODS
Dye and chemicals: Malt extract and polypeptone were purchased from Difco (Detroit, USA). Amaranth and other chemicals were purchased from Wako Pure Chemical Industry Co. Ltd. (Osaka, Japan) at the highest purity available and of an analytical grade. The structure of Amaranth is shown in Fig. 1.
Microorganism and culture condition: Pleurotus eryngi F019 was isolated from a tropical rain forest in Samarinda, Indonesia. It was isolated by cultivating a small pieces of the internal tissue removed from the fruiting body on a Malt Extract Agar (MEA) plate at 5°C. The MEA contained 20 g L-1 of malt extract, 20 g L-1 of glucose, 1 g L-1 of polypeptone and 15 g L-1 of agar. Pleurotus eryngi F 019 was selected for this investigation because of the extracellular enzymes activities demonstrated during the decolorization of reactive black 5 (Hadibarata et al., 2011). The pure culture was maintained on dye containing nutrient agar slants at 4°C.
Decolorization study and effect of metals: Experiments were performed by using 100 mL Erlenmeyer flasks in incubation room. Flasks were prepared in triplicates and contained 20 mL nutrient media with dyestuff.
| Fig. 1: | The chemical structure of amaranth |
An 5 mm active plug cut from the pure fungal culture grown on agar plates was used for inoculation of flasks under sterile conditions. In order to determine the effect of metal ions on the decolourization of amaranth, 10 mm of: Fe2+ (as FeCl2), Mg2+ (as MgSO4), Mn2+ (as MnCl2) and Cu2+ (as CuCl2) were used in the reaction mixture which consisted of acetate buffer 50 mm. The pH of the culture medium was 4.5. Control flasks contained only dyestuff and nutrients, but no fungi. All of the experiments were performed with Amaranth concentration of 200 mg L-1. Samples (triplicate flasks) were taken periodically, centrifuged at 8000 g for 20 min at 15°C and the clear supernatant obtained was used for determination of decolorization rate spectrophotometrically by reading UV-Vis Spectrometer with the absorbance at λmax (520 nm). The percentage of decolorization was calculated as follow:
where, C0 is initial dye concentration and C is final dye concentration.
Assay of manganese peroxidase and laccase: Enzyme activities were determined spectrophotometrically at 25°C. Laccase production was assayed by oxidation of 2,22-azinobis-(3 ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 420 nm. Manganese Peroxidase (MnP) activity was assayed by monitoring the increase in absorbance at 270 nm due to the oxidation of 50 mmol L-1 malonate buffer and dimethoxyphenol in 20 mmol L-1 MnSO4. The activities were expressed in U L-1.
Statistical data analysis: Data were analyzed by one-way analysis of Variance (ANOVA) using SPSS multiple comparison test. Mean values separation was performed using the Least Significant Difference (LSD) test (p = 0.05), where the F-value was significant. The Standard Error (SE) was used where required.
RESULTS AND DISCUSSION
Identification of fungi: A white-rot fungus, isolated from decayed wood in a tropical rain forest, Samarinda, Indonesia, was designated F 019. The strain F 019, when grown on me agar, had a whitish spore (7-10x2.5-5 μm), smooth; cylindric to long-elliptical. and has a clamp connection between hyphae. F 019 has a cap (2-14 cm), convex, becoming flat or somewhat depressed; lung to semicircular, or nearly circular if growing on the tops of logs, fairly smooth, whitish to beige or pale tan, usually without dark brown colorations. The gills are running down the stem; close or nearly distant; whitish. Based on these microscopic and macroscopic morphological characteristics, F019 was identified as Pleurotus eryngi F019 (Laessoe, 2002; Pace, 1998).
Decolorization of amaranth: The visible absorbance spectrum for amaranth after treatment with Pleurotus eryngi F 019 is shown in Fig. 2. A maximal absorbance was seen at 520 nm and this peak decreased with time, which is associated with oxidation of the dye. The rapid delocorization of amaranth was observed after 15 incubation days and then become slow with the time. Following 15 and 23 days of incubation, the percentage of decolorization was 66 and 68 %, respectively. The final overlay shows that highest decolorization occurred within 30 days (Fig. 3). This was caused by strong attractive force between the amaranth molecule and the fungi cell; fast diffusion onto the external surface was followed by fast diffusion into the fungi cell to attain rapid equilibrium (Omar, 2008). The amaranth might be easier to be delocorized and adsorbed into cellular compartment of the fungi, because of the sulphonic group was bonded to azo structure in amaranth was a strong electron-with drawing groups through resonance to cause an enhanced of amaranth removal to be easily decolorized (McMurry, 2004).
Enzyme analysis: The activity of ligninolytic enzyme especially MnP and laccase was shown in Fig. 3. The productions of ligninolytic enzymes like MnP and laccase have a key role in mineralization of amaranth. The role of ligninolytic enzymes in the decolorization of synthetic dyes may be different for each microorganism (Pointing and Vrijmoed, 2000). The enzyme production rapidly increased in the first 15 days of incubation and then becomes slow and reached a maximum of 1,474 U L-1for laccase. Thereafter, a drop in activity was observed (Fig. 3). MnP were tested for its activity from the day 1 but there was no significant quantity of MnP production till the end of experiment, after which 229 U L-1 was observed after 30 days. From these results, it was observed that laccase was the key enzyme responsible for the decolorization process.
Effect of metals on decolorization: Figure 4 shows the results of effect of metal ion on decolorization of amaranth. Metal choosen were Fe2+, Mg2+, Mn2+ and Cu2+. Addition of Cu2+, Mg2+, Mn2+ gave the positive results while decolorization was inhibited by addition of Fe2+. highest decolorization was shown in addition of Cu2+ (100%). Our results are comparable with previous study reported that decolorization was increased in the presence of heavy metals (Bakshi et al., 1999).
| Fig. 2: | Visible absorbance spectra of amaranth at 0 day (solid lines) and last day of biodegradation (dashed lines) |
| Fig. 3: | Monitoring of color removal and enzyme activity |
| Fig. 4: | Effect of metal on decolorization of amaranth |
| Fig. 5(a-b): | (a) GC chromatogram of control amaranth and (b) Degradation products |
On the other hand, copper is a component of active site of laccases. It has been observed in previous studies that the addition of Cu2+ enhanced the laccase activity. When the concentration was increased to 100 mm, laccase stability decreased in the presence of all the metals assayed, in particular against Fe2+. Whereas in the presence of Fe2+, a severe inhibition of the decolorization (less than 80% decolorization) was observed. This could be explained by the instability of the enzymes especially laccase occurred in the presence of Fe2+ at high concentration (Rodriguez et al., 2005).
Metabolites of amaranth: The absorbance peaks, corresponding to amaranth dye, diminished which indicated that the dye had been degraded. The spectrum of amaranth in visible region exhibits a main peak at 520 nm. The decrease of absorbance peak of amaranth at λmax= 520 in the Fig. 5 indicated degradation of dye. Decolorization of synthetic dyes by white-rot fungi could be due to adsorption or to biodegradation by microbial activity. In the case of adsorption, the absorbance of the dye decreases approximately in proportion to each other, whereas in biodegradation, either the major visible light absorbance peak disappears completely, or a new peak appears in chromatogram (Ambrosio and Campos-Takaki, 2004). Comparison of GC chromatogram of control dye with extracted metabolites after complete decolorization clearly indicated the biodegradation of the dye by Pleurotus eryngi F 019 (Fig. 5). GC analysis of extracted sample showed original dye at retention time 6.2 min (Fig. 5a) and after complete decolorization peaks for metabolites were at retention time 6.9 and 7.4 min (Fig. 5b).
CONCLUSION
The rapid decolorization processes of Amaranth throughout biodegradation system by fungi, Pleurotus eryngi F 019, were highly linked with enzymes production in the medium. The rapid delocorization of amaranth was observed in the first 15 days of incubation correlated with the production of laccase and MnP. In the process of decolorization, laccase was playing a key role in the biotransformation of amaranth. Pleurotus eryngi F 019 can decolorize higher concentration of amaranth dye and it can be used repeatedly for decolorization. The UV-Vis and GC analysis confirmed biodegradation of dye. These promising results suggest the application of Pleurotus eryngi F 019 to treat dye containing wastewater having higher concentrations of metals.
ACKNOWLEDGMENTS
A part of this project was financially supported by Universiti Teknologi Malaysia (RUG Vote QJ1.3000. 2522.02H65) and Ministry of High Education, Malaysia (ERGS Vote R.J130000.7822.4L053).