Biodegradation of Turquoise Blue Textile Dye by Wood Degrading Local Fungi Isolated from a Plantation Area

This research aimed to investigate the potential screening of wood degrading fungi to degrade the textile dye. Nine fungi were selected based on their ability to remove the turquoise blue textile dye on liquid potatoes dextrose médium. The fungi which have a high color removal efficiency were identified and analyzed qualitatively in terms of the manganese peroxidase and lignin peroxidase enzymes and their enzyme activity was determined. The selected fungi were investigated for their ability to degrade turquoise blue under variations in pH, dye concentration, and contact time. The results showed that three of the nine strains of wood-degrading local fungi, identified as Trametes hirsuta , Microporus xanthopus , and Ganoderma applanatum , were able to degrade textile dye. The color removal efficiency of dye treatment was monitored under the initial pH, dye concentration, and contact time variation. The study analyzed that the optimal color removal efficiency of the turquoise blue textile dye with a concentration of 30 mg/L in an aqueous solution was achieved at 78.50 and 85.84% at pH 6 for Microporus xanthopus and Ganoderma applanatum , while the color removal efficiency was 82.17% at pH 5 for Trametes hirsuta with seven days of incubation time.


INTRODUCTION
In the fabric dyeing process, large amounts of wastewater containing various toxic compounds, such as heavy metals, caustic soda, and dyes, are produced. About 10 to 15% of the total dye used is lost during the dyeing process and released into the environment as wastewater [Nam and Renganathan, 2000; Wilkolazka et al., 2002]. The discharge of these effluents into receiving water bodies can cause negative environmental effects. The presence of color in water bodies causes a reduction in environmental aesthetics. It can inhibit light penetration into the water, produce the various amines compounds, and unpleasant odor products under anaerobic conditions [Slokar and Lemarechal, 1998]. In addition, some dyes, commonly used in the fabric dyeing process are known to be toxic, carcinogenic, and mutagenic. Hence, they are harmful to aquatic life, as well as human life. Among the dyes, turquoise blue dye, a wellknown reactive dye, is often used in the dyeing process and produces a dark blue color in wastewater effluent. A study by Xu et al. in 2010 found that the azo dyes have a carcinogenic effect on the liver and bladder of mammals, and are potentially mutagenic in humans. A bladder tumor has been reported more frequently for the workers in the dye industry than in general society [Suryavathi et al., 2005]. Therefore, the pollutants in textile wastewater must be removed before discharging into water bodies.
Generally, the methods used for color removal and other contaminants in textile wastewater involve various combinations of chemical, physical and biological methods. Several physicochemical methods have been applied for textile wastewater treatment, such as electrochemical oxidation , adsorption using chitin-based adsorbent [Wang et al., 2020], and Khamir (Saccharomyces cerevisiae) as adsorbent of Remazol Red RB textile dyes [Sukarta et al., 2021], and photocatalytic method using TiO 2 /UV-irradiation [Azzaz et al., 2021] Among these methods, the biological method, mainly using wood-degrading fungi, has attracted much attention as one of the efficient techniques for removing color from dyes due to their environmentally friendly, cost-competitive, easy operation, and high color removal efficiency. Degradation of dyes by fungi is associated with the ability of fungi to produce extracellular oxidizing enzymes, such as ligninolytic extracellular enzymes, including manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase. The ligninolytic enzymes have been widely applied in food, paper, and textile [Canas and Camarero, 2010]. Fungi use extracellular enzymes to transform aromatic compounds, such as lignin, dyes, and polycyclic aromatic hydrocarbons into less toxic products [Yesilada et al., 2003]. Several strains of fungi have been reported to degrade the color of textile wastewater, including laccase enzymesproducing soil fungi of Aspergillus niger and Phanerochaete chrysosporium [Rani et al., 2014 [Machado et al., 2005].
The main objective of the present study was to investigate and select the wood degrading local fungi that grow on wood decayed pieces located in the Buleleng-Bali plantations area for color removal of textile dye. The selected wood degrading fungi were further investigated for their ability to remove the color of textile dyes under various conditions of initial pH, dye concentration, and incubation time.

Dye
The turquoise blue textile dye used in this study was obtained from Sigma Chemical Company. This dye has the molecular formula C 40 H 25 CuN 9 O 14 S 5 with a molecular structure, as shown in Figure 1.

Sampling and screening of wood degrading local fungi
In this study, nine local fungi growing on rotting wood pieces of tropical plants in the Buleleng-Bali area were investigated for their ability to degrade textile dye. Each fungus culture was placed in a plastic bag and then kept in a freezer at 0 o C. The nine wood degrading local fungi were selected by growing them in Potatoes Dextrose broth containing 10 mg/L of turquoise textile dye and then incubated for 10 days at room temperature while shaking at 150 rpm using an automatic shaker. The color removal of dye was measured by a double beam Shimadzu-1700 UV-Visible spectrophotometer at 624.5 nm.

Identification of selected wood degrading local fungi
Three of the nine wood degrading fungi with high potential to degrade textile dye were identified based on their macroscopic and microscopic characteristics. The three selected fungi have distinctive characteristics, such as pores on the lower cap, and the presence of pores indicates the fungi class of Basidiomycetes. The macroscopic and microscopic characteristics of three wood degrading local fungi were presented in Table 1.

Qualitative analysis of ligninolytic enzymes
Manganese peroxidase (MnP) and lignin peroxidase (LiP) produced by wood degrading fungi were qualitatively analyzed; 2.00 mL of the culture filtrate was placed in the test tube and added with 1 mL of 1 mM MnSO 4 , 0.3 mL of 1 mM phenol red, 1 mL phosphate buffer (pH 5), and 1 mL of 50 mM H 2 O 2 to investigate the manganese peroxidase enzyme production, The formation of a brownish orange color indicates a positive presence of the manganese peroxidase enzyme. During the experiment, 2.00 mL of the culture filtrate was placed in the test tube and then added with reagents comprised of 1 mL of 100 mM n-propanol, 1 mL of 250 mM tartaric acid, and 1 mL of 50 mM H 2 O 2 to assess the lignin peroxidase enzyme production, the formation of a yellowish white color is positive for the lignin peroxidase enzyme.

Ligninolytic enzymes activity assay
The manganese peroxidase (MnP) and lignin peroxidase (LiP) activities were assayed using a double beam Shimadzu-1700 UV-Visible spectrophotometer. The MnP activity was determined using phenol red as a substrate in the presence of H 2 O 2, according to Geethanjali and Jayashankar, 2017. The oxidized phenol red was observed by reading the absorbance at 610 nm. The LiP activity was assayed using n-propanol as the substrate in the presence of H 2 O 2 modified from Shanmugam et al. 1999. The formation of propanaldehyde was The results of observations on the macroscopic and microscopic properties of wood-degrading local fungi Name of fungi The cap is quite thick (2.5-3 cm), with a hard texture like wood, known as bracket shaped fungi, with a white surface/cap color and a clean pinky color at the bottom. At the bottom of the cap, there are medium-sized pores, as shown in Figure B.

Trametes hirsuta
Main characteristics: hard fruiting bodies like wood but thin texture like paper. These fungi resist living in a dry environment by attaching to wood stems as their hosts. On the cap, there are concentric circles/lines or radial lines surrounding the cap. The bottom cap is cream/light brown and observed using a stereomicroscope. There are pores, as shown in Figure B.

Microporus xanthopus
The main characteristics: are having a hard fruit body like wood (A), a hard hood surface, and brown (A, B) with a white bottom cap color (C). At the bottom of the cap, there are pores as a place for spores of these fungi (D) Ganoderma applanatum monitored at 310 nm in the reaction mixtures containing 10 mM n-propanol, 250 mM tartaric acid, and 1 mM H 2 O 2 . All the enzyme activity assays were run in triplicate and compared with reference blanks containing all the reagents except an enzyme. One unit of enzyme activity was defined as the amount of enzyme consumed to oxidize 1 µmol of substrate per minute (de Oliveira et al., 2009). The MnP and LiP enzyme activity (Uml -1 ) was calculated using the formula: where: EA = enzyme activity (U/mL), A = absorbance, V = total volume of reaction mixture (mL), t = incubation time (min), ϵ = extinction coefficient (M -1 cm -1 ) and v = volume of enzyme (mL). The value of ϵ for the oxidized phenol red is 22 mM -1 cm -1 , while 20 mM -1 cm -1 for monitoring the formation of propanaldehyde.

The color removal efficiency of turquoise blue textile dye by wood degrading fungi
Different strains of three selected wood-degrading fungi species: Trametes hirsuta, Micropurus xanthopus, and Ganoderma applanatum, were separately investigated for their performance in degrading turquoise blue textile dye on Czapex media. A total of 50 mL of remazol turquoise blue dye with a concentration of 100 mg/L was transferred to Erlenmeyer in 100 mL and 3 mL of fungal spore suspension was added. Then, the mixture in Erlenmeyer was covered with aluminum foil and incubated for 10 days while shaking using an automatic shaker with a rotation speed of 150 rpm. After the degradation process, the mixtures were filtered through Whatman No.1 paper filter, and the supernatant was centrifuged at 3000 rpm for ten minutes. The absorbance of remazol turquoise blue before and after treatment was measured using a double beam Shimadzu-1700 UV-Visible spectrophotometer at 624.5 nm. The effect of initial pH, dye concentration, and contact time on color removal of turquoise blue textile dye was investigated, and each treatment was replicated three times. The color removal efficiency was calculated using the formula: where: A initial and A final are the initial and final absorbance of the turquoise blue solution before and after days of growth, respectively.

Screening of wood degrading local fungi
The nine isolates of wood degrading local fungi were collected from decaying wood in the plantation area located in Buleleng -Bali. Further, each fungus was assayed for its ability to degrade turquoise-blue textile dye. The visual appearance of the nine types of fungi is presented in Figure 2, while the color removal of turquoise blue textile dye by nine fungi cultures is shown in Figure 3. Figure 3 shows that the nine wood degrading fungi obtained could degrade turquoise textile dye. However, it was found that three of the nine fungal strains produced the highest color removal efficiency, namely Trametes hirsuta (78.40%), (81.25%), and Ganoderma applanatum (84.10%). The strains of Trametes hirsuta and Ganoderma applanatum were obtained from decayed pieces of teak wood, while Microporous xanthopus came from the decay of melinjo wood.

Qualitative test of Ligninolytic enzyme from wood degrading fungi
MnP and LiP enzymes are extracellular enzymes produced by wood-degrading fungi. These two enzymes play an important role in the degradation of turquoise blue textile dye. MnP enzymes can be tested qualitatively using phenol red and H 2 O 2 reagents, while LiP enzymes are tested using n-propanol reagents in the presence of H 2 O 2 . The qualitative analysis result of the MnP and LiP enzymes from wood-degrading fungi were presented in Figure 4.
As shown in Figure 4, the three types of wood degrading local fungi, namely Trametes hirsuta, Microporous xanthopus, and Ganoderma applanatum, were able to secrete extracellular enzymes, Manganese peroxidase and lignin peroxidase. In the MnP enzyme assay, the formation of brownish yellow color was due to the oxidation of phenol red by MnP enzyme in the presence of hydrogen peroxide. The oxidation of n-propanol in the presence of hydrogen peroxide by lignin peroxidase enzyme forms the color yellowish-white of propionaldehyde. These two enzymes play a significant role in degrading the turquoise blue dye solution.

Enzymes activity from wood degrading local fungi
The manganese peroxidase and lignin peroxidase enzymes produced by wood-degrading fungi Trametes hirsuta, Microporous xanthopus, and Ganoderma applanatum were tested for enzyme activity and their role in dye degradation. The enzyme activity of manganese peroxidase was determined using phenol red as a substrate in the presence of H 2 O 2 . In contrast, the lignin peroxidase activity was assayed using n-propanol as the substrate in the presence of H 2 O 2 . The ligninolityc enzyme activity from wood degrading local fungi is shown in Table 2. The degradation of dye by fungal culture is often associated with the ligninolytic enzyme activity [Selvam et al., 2003]. The conducted study monitored the enzyme activity of manganese peroxidase and lignin peroxidase after being cultured on a liquid Czapek medium for 10 days. The maximum lignin peroxidase activity from Trametes hirsuta, Microporus xanthopus, and Ganoderma applanatum were 0.32, 0.28, and 0.24 UmL -1 , while the manganese peroxidase activity was 0.27, 0.24, and 0.35 UmL -1 , respectively.

Color removal of dye at different pH of the medium
The pH of the culture media greatly affects the growth, the ability of the ligninolytic enzymes produced, and dye degradation by wood-degrading fungi. The effects of media pH on turquoise blue textile dye removal efficiency is shown in Figure  5. The result reveals that Trametes hirsuta, Microporus xanthopus, and Ganoderma applanatum could decolorize turquoise blue dye over a pH range of 4.0-9.0. The optimum color removal efficiency was achieved at pH 5 for Trametes hirsuta. In contrast, at pH 6 for Microporus xanthopus and Ganoderma applanatum, the percent color removal at 7 days of incubation time was recorded at 85.26, 82.30, and 83.14%, respectively.
The obtained result indicates that the initial media pH is one of the important factors influencing the biodegradation process. This finding is in line with Kunjadia et al. 2020, who investigated the decolorization of azo dye from aqueous solution by ligninolytic producing fungi strain of Pleurotus spp. They found that the maximum color removal was obtained at pH 6. Syafiuddin and Fulazzaky, 2021, also examined the effect of

Color removal of dye at various concentrations of dye
The color removal of turquoise blue dye by three selected wood degrading fungi was investigated in the varying dye concentration (10,20,30,40, and 50 mg/L). The percent decolorization of dye by each fungus is presented in Figure 6.
On the basis of the graph in Figure 6, it was observed that the ability of three selected wood degrading fungi to decolorize turquoise blue dye was increased by the increasing dye concentration from 10-30 mg/L. However, it was decolorizing ability decreased sharply with higher dye concentration treatment. The maximum dye degradation was obtained at a dye concentration of 30 mg/L with a color removal efficiency of 78.82% and 88.22% at pH 6 for Microporus xanthopus and Ganoderma applanatum, respectively, while the color removal efficiency was 87.21% at pH 5 for Trametes hirsuta. Most of the previous studies also showed a decrease in the ability of fungi to degrade dyes at higher concentrations, because dyes with higher concentrations would be toxic to metabolic activity. Zhuo et al. 2011, reported that the color removal efficiency of methyl orange using white-rot fungus decreased drastically from approximately 100% to less than 60%, when the dye concentration was increased from 25 mg/L to 100 mg/L.

Color removal of dye at different incubation times
The influence of incubation time on the degradation of the turquoise blue textile dye process was studied by varying the contact time between 6 to 10 days. The other operational conditions were 30 mg/L of dye and adjusted at pH 5 for Trametes hirsute, while at pH 6 for Microporus xanthopus and Ganoderma apllanatum. The effect of incubation time of three selected wood degrading local fungi on the color removal efficiency of turquoise blue textile dye is shown in Figure 7. Figure 7 illustrates that the color removal efficiency of dye by all wood degrading fungi increased from 6 to 7 days and then tended constantly from incubation time of 7 to 10 dye. The result revealed that the dye decolorization rate ranged from 78.50 to 85.84% after 7 days, while after 10 days of incubation time it ranged between 78.51 and 85.65%.

CONCLUSIONS
On the basis of the results obtained, three local fungi, namely Trametes hirsuta, Microporous xantophus, and Ganoderma applanatum, that grew on rotten wood could degrade turquoise textile dye effectively. The dye decolorization capabilities of the fungal strains are related to the extracellular excretion ability of ligninolytic enzymes, such as manganese peroxidase and lignin peroxidase. The color removal efficiency of turquoise blue textile dye by Tramete hirsuta, Microporous xantophus, and Ganoderma applanatum reached 82.17, 78.50, 85.84%, respectively. The optimum operational parameters were obtained at a dye concentration of 30 mg/L, an initial pH of 5 for Trametes hirsuta, while at pH 6 for Microporus xanthopus and Ganoderma applanatum with seven days of incubation time.