Decolorization of Synthetic Dyes by Ligninolytic Enzymes from Trametes hirsuta D7

The ability of ligninolytic enzymes from Trametes hirsuta D7 to decolorize several synthetic dyes was investigated. A crude enzyme powder was produced by fermenting oil palm empty fruit bunch fibers for one month. The dye decolorization by the enzymes occurred at an efficient 0.25 U/mL. The enzymes degraded 100, 200, 300, 400, and 500 ppm Remazol Brilliant Blue R (RBBR) within 7 h by 95.57 ± 0.32%, 93.46 ± 3.09%, 91.84 ± 0.65%, 86.44 ± 0.97%, and 82.14 ± 0.52%, respectively. The enzyme also decolorized anthraquinone (Acid Blue 129), monoazo (Acid Orange 7), diazo (Reactive Black 5), and trimethyl methane (Methyl Violet) dyes within 7 h by 94.59 ± 7.97%, 13.99 ± 0.30%, 7.61 ± 0.01%, and 7.59 ± 0.18%, respectively. Addition of MnSO4, H2O2, and violuric acid enhanced the dye decolorization rate by up to 10-fold. This study shows the potential for application of ligninolytic enzymes from T. hirsuta D7 in the treatment of wastewater effluent of textile industries.


Introduction
Synthetic dyes are extensively used in the textile, paper, rubber, cosmetics, pharmaceuticals, and food industries. However, the presence of dyes in wastewater from these industries can be particularly problematic. The dyes have a complex chemical structure; therefore, they are resistant to oxidation by light and water. The accumulation of undegradable dyes in surface water can therefore reduce aquatic diversity by blocking the passage of sunlight, thereby affecting the ecological balance of aquatic ecosystems. In addition, many synthetic dyes are toxic, mutagenic, and carcinogenic [1,2].
Dyes can be classified based on their chemical structure (e.g., azo, anthraquinone [AQN], indigo, triphenylmethane, etc.), the method of usage (e.g., direct, reactive, chromic, metal-complexes, disperse, sulfur, vat, pigments, etc.), and their chromogens (e.g., donor, acceptor, cyanine, polyene, etc.) [3]. The major classes of synthetic dyes are azo and AQN dyes, and their complex structure makes decolorization a challenge. Currently available physical or chemical treatments, like adsorption, precipitation, filtration, and chemical transformation, are expensive, environmentally unfriendly, and inefficient. As an alternative to these physicochemical methods, biologi-cal treatment of dyes using ligninolytic enzymes provides a lower energy cost and an eco-friendly process [4].
The aim of the present study was to evaluate the ability of ligninolytic enzymes from T. hirsuta D7 to decolorize various dyes at different concentrations. The effect of enzyme mediators, such as manganese sulfate (MnSO 4 ), hydrogen peroxide (H 2 O 2 ), and violuric acid (VA), on dye decolorization was also investigated.

Methods
All experiments were carried out in the Laboratory of Biomass Conversion Technology and Bioremediation and the Laboratory of Microbiology, Research Center for Biomaterials. Oil palm empty fruit bunches (OPEFB) were used as a substrate for enzyme production fungus was T. hirsuta D7, a white rot fungus isolated from a peat swamp forest area in Riau, Indonesia. The test dye substrates were Remazol Brilliant Blue R (RBBR), Acid Blue 129 (AB129), Acid Orange 7 (AO7), Reactive Black 5 (RB5), and Methyl Violet (MV) purchased from Sigma-Aldrich. The characteristics of these substrates are shown in Table 1. Malt extract, tone, glucose, corn steep liquor, malonate buffer (pH 4.5), MnSO 4 , 2,6-dimethoxyphenol (DMP), H acid (VA), and acetate buffer (pH 4.5) were purchased from Wako Pure Chemical Industries, Ltd. (Japan).
Fungal culture. The T. hirsuta D7 strain was grown on malt extract agar and incubated at room temperature ( 25-30 C) for 7 days. The fungus culture was then i oculated in a malt extract-glucose-peptone broth co taining 20 g/L malt extract, 20 g/L glucose, and 1 g/L peptone. An inoculum was prepared by inoculating 4 plugs ( 8 mm) of the fungal colony into a 300 mL Erlenmeyer flask containing 100 mL medium broth and agitated at 150 rpm at room temperature for 7 days. A D7 strain was grown on malt extract agar and incubated at room temperature ( C) for 7 days. The fungus culture was then inpeptone broth containing 20 g/L malt extract, 20 g/L glucose, and 1 g/L peptone. An inoculum was prepared by inoculating 4 8 mm) of the fungal colony into a 300 mL Erlenmeyer flask containing 100 mL medium broth and ated at 150 rpm at room temperature for 7 days. Af-ter incubation, 1% (w/v) corn steep liquor and the culture was homogenized with a Waring blender twice for 2 sec. The homogenized mixture was used as the inoculum for enzyme production.
Substrate preparation and enzyme production enzyme was produced by solid state fermentation (SSF), as follows: 10 meshes of 150 g OPEFB, 150 mL di tilled water, and 150 mL malt extract broth were combined in a heat substrate (53% water content) was sterilized at 121 ºC for 15 min and then cooled at room temperature. A 10% (w/v) of T. hirsuta D7 inoculum was inoculated into the sterilized substrate and incubated at room temperature for 1 month [14].
Enzyme extraction. The crude enzyme was extracted from the substrate by adding pH 4.5, at a ratio of 1:3 to the weight of substrate. The mixture was homogenized using an ACE AM mogenizer (Nissei, Japan) at 10,000 rpm for 10 min on ice and then filtered. The filtrate was centrifuged at 10.380 x g and 4 C for 20 min. Ammonium sulfate was then added to the supernatant to give a 50% saturated solution. The solution was stirred with a magnetic stirrer for 1 h and then centrifuged at min. The pellet was dissolved in er, pH 4.5 and freeze-dried for 1 enzyme powder. corn steep liquor was added and the culture was homogenized with a Waring blender twice for 2 sec. The homogenized mixture was used as the inoculum for enzyme production.
preparation and enzyme production. The enzyme was produced by solid state fermentation (SSF), as follows: 10 meshes of 150 g OPEFB, 150 mL dismalt extract-glucose-peptone broth were combined in a heat-resistant plastic bag. The bstrate (53% water content) was sterilized at 121 ºC for 15 min and then cooled at room temperature. A 10% D7 inoculum was inoculated into the sterilized substrate and incubated at room temperature The crude enzyme was extracted from the substrate by adding 50 mM malonate buffer, pH 4.5, at a ratio of 1:3 to the weight of substrate. The mixture was homogenized using an ACE AM-11 homogenizer (Nissei, Japan) at 10,000 rpm for 10 min on ltered. The filtrate was centrifuged at C for 20 min. Ammonium sulfate was then added to the supernatant to give a 50% saturated solution. The solution was stirred with a magnetic stirrer for 1 h and then centrifuged at 10  March . MnP activity was determined using DMP as a substrate. The reaction mixture consisted of buffer (pH 4.5), 20 mM manganese and 20 mM DMP. The MnP activity was observed at 470 nm with a UV-Vis spectrophotometer and calculated from the molar extinction coeffi-. One unit of MnP is defined as the amount of enzyme needed to oxidize 1 mmol of (III) in 1 min. Laccase activity was determined by the laccase assay using syringaldazine as the substrate. The reaction mixture contained 10 mM ngaldazine and was mon-Vis spectrophotometer. Laccase activity was calculated from the oxidation of syringaldazine to its quinone using a molar absorptivity ]. Enzyme activity (U/L) was μL : 10 6 ) × 10 6 × μL : 10 3 ) (1)

Influence of RBBR dye concentration on
Crude enzyme at optimum activity was added to 100, 200, 300, 400, and 500 ppm RBBR dye.
. Crude enzyme at optimum activity was added to 3 mL of 100 ppm dye:

Influence of mediators on decolorization of dyes.
and 0.1 mM H 2 O 2 ) were 100 ppm dye. In a separate experiment, 2 mM VA was added to activate the laccase.
Decolorization experiment and assay mixtures were incubated at room temperature for 7 h. The decolorization process was measured at an interval of 1 h with UV-Vis spectrophotometer at the λ each dye. Each decolorization experiment was pe formed in triplicate and the mean of the decolorization percentages was reported. The percentage of decolorization was calculated using Eq. (2).

Decolorization (%) = A i -A
A i where A i and A f are the initial and final absorbances of the mixture, respectively.

Results and Discussion
The enzymatic activities of MnP and laccase were 0.038  0.001 U/mg and 0.314  0.010 U/mg, respectively, in the crude powder.
Effect of enzyme loading. Enzyme loading is one of the reaction parameters required to achieve maximum dye degradation. The decolorization of RBBR dye at various enzyme concentrations during the 7 h reaction time is shown in Figure 1a. The dye degradation was more efficient at 0.25-4.00 U/mL, with maximum decolorization of 95.57-100% after a 7 h reaction time (Fig. 1b). In general, increasing the enzyme concentr tion enhanced the decolorization activity. This result agrees with the findings of Ang et al. [6], who reported that the initial decolorization efficiency of RBBR i creased with the increase of enzyme concentration. However, an excessively high enzyme concentration would increase the cost of the treatment process [1 Therefore, an enzyme concentration of 0.25 U/mL was presumed adequate for use in further decolorization experiments in this study. Decolorization experiment and assay. All reaction mixtures were incubated at room temperature for 7 h. process was measured at an interval Vis spectrophotometer at the λ max of each dye. Each decolorization experiment was performed in triplicate and the mean of the decolorization percentages was reported. The percentage of calculated using Eq. (2).

Percentage of RBBR Decolorization at Various Enzyme Concentrations (a) During the 7 h Reaction Time, (b) at
A f x 100 (2) are the initial and final absorbances of The enzymatic activities of MnP and laccase were 0.038 0.010 U/mg, respectively, in Enzyme loading is one of the reaction parameters required to achieve maximum decolorization of RBBR dye at various enzyme concentrations during the 7 h reaction 1a. The dye degradation was 4.00 U/mL, with maximum 100% after a 7 h reaction time ral, increasing the enzyme concentration enhanced the decolorization activity. This result agrees with the findings of Ang et al. [6], who reported that the initial decolorization efficiency of RBBR increased with the increase of enzyme concentration.
ver, an excessively high enzyme concentration would increase the cost of the treatment process [16]. Therefore, an enzyme concentration of 0.25 U/mL was presumed adequate for use in further decolorization B

Percentage of RBBR Decolorization at Various Enzyme Concentrations (a) During the 7 h Reaction Time, (b) at
Effect of dye concentration. The RBBR dye concentrations at 100, 200, 300, 400, and 500 ppm were treated with a constant enzyme concentration of 0.25 U/mL for a 7 h reaction time (Figure 2a.). Maximum decolorization (95.57  0.32%) was found at a lowest dye concentration (100 ppm). The decolorization activity by the enzyme decreased with increasing dye concentration (Fig. 2b.). This is possibly related to the maximum capacity of enzymes at a certain concentration for bleaching the dyes [17]. Sabarathinam et al. [16] also reported that the maximum decolorization of 96.90% was achieved at 25 ppm of RBBR and the decolorization was reduced to 53.21% at 250 ppm of RBBR.
Decolorization of various dyes. Synthetic anthraquinone, azo, and trimethyl methane dyes are common dyes used in the textile industry. In this study, AB129, AO7, RB5, and MV were tested in the decolorization process using the crude ligninolytic enzymes extracted from T. hirsuta D7. The enzyme was most effective at decolorizing anthraquinone AB129 (94.59%) in a 7 h reaction period, followed by monoazo AO7, diazo RB5, A B
The chemical structure of dyes generally consists of a conjugated system of double bonds and aromatic rings. The major classes of dyes are composed of anthraquinoid, indigoid, and azo aromatic structures [3]. AQN is the basic unit of dye classes, as its quinonoid system acts as a chromophore. AB129 is an acidic dye or an AQN-type dye that has groups containing nitrogen and sulfur (amine and sulfonate, respectively). It has two structural units, AQN and 1,3,5-trimethylbenzylamine, which constitute the chromophore core [3]. The azo dye class is the largest group of dyes, with -N=N-as a chromophore in an aromatic system [19]. The azo groups are generally connected to benzene and naphthalene rings, but they can also be attached to aromatic heterocyclic or enolizable aliphatic groups [20]. AO7 is mono-azo dye that has three aromatic rings and one sulfonate group. RB5 is diazo dye that contains stringent aromatic molecules and bis-azo bonds and is difficult to degrade due to its complex structure [21]. MV belongs to a class of intensely colored organic compounds called triphenylmethane dyes, due to the presence of three aryl groups, each of which is bonded to a nitrogen atom that interacts with one or two methyl groups [22].
Dyes with simple structures exhibit high rates of decolorization. Lavanya et al. [19] and Lade et al. [23] showed that the color removal rate is faster for monoazo dyes than for diazo and triazo dyes. Azo compounds with hydroxyl or amino groups are more easily degraded than are those with methyl, methoxy, sulfo, or nitro groups. In this study, decolorization proceeded in the order of AB129 > AO7 > RB5 > MV. Anthraquinone dye is more susceptible to laccase enzymatic activities [24]. This was proven in this study, where AB129 and RBBR dyes were more susceptible to the crude ligninolytic enzyme activity, while the triphenyl methane dye (MV) was resistant to the enzyme activity.   [29].
In a separate experiment, the addition of 2 mM VA led to a greater decolorization of the dyes than was achieved with addition of MnSO 4 and H A previous study showed that the addition of 2 mM and 5 mM VA could enhance the decolorization of the diazo dye Acid Red (AR97) by 3-fold. VA was reported as  4). A previous study also found that industrial dyes, such as RBBR, Congo red, trypan blue, methylene blue, MV, methyl green, and brilliant cresyl blue, were decolorized when the reaction medium contained an enzyme as well as 1 mM MnSO 4 ]. Asgher et al. [26] reported that MnP requires hydrogen peroxidase as well as Mn 2+ for its activity. The addition of manganese (MnSO 4 ) at a 1 mM also can enhance the MnP activity. Mn is a specific mediator in the MnP catalytic ], while hydrogen peroxidase acts as a is also accelerates the MnP ] through a Fenton reaction, where H 2 O 2 OH) and/or superoxide arate experiment, the addition of 2 mM VA led to a greater decolorization of the dyes than was and H 2 O 2 ( Figure 4). A previous study showed that the addition of 2 mM and 5 mM VA could enhance the decolorization of the diazo fold. VA was reported as one of the most effective redox mediators for laccase oxidation when compared to the natural mediators [ In the present study, the addition of mediators such as MnSO 4 , H 2 O 2 , and VA enhanced the decolor of the dyes up to 10-fold when compared to the rate without the mediator.
In the present study, the dye decolorization was a result of a combination of MnP and laccase enzyme activity. ) and with the Addition of 2 mM VA ( ) one of the most effective redox mediators for laccase oxidation when compared to the natural mediators [30].
In the present study, the addition of mediators such as , and VA enhanced the decolorization rate fold when compared to the rate In the present study, the dye decolorization was a result of a combination of MnP and laccase enzyme activity. The MnP oxidizes Mn (II) to Mn (III), and Mn (III) then xidizes many phenolic compounds [17]. The laccase decolorizes azo dyes by catalyzing the oxidation of phenolic compounds and aromatic amines through the reoxygen to water. The enzymes attack the double bonds or aromatic rings of the dyes and change the configuration of the dye structure. Disruption of the dye structure results in a reduction in the color intensity. Changes in the absorption spectra of the dyes (AB 129, AO 7, RB 5, and MV) as a result of the wn in Figure 5.

Conclusion
The ligninolytic enzyme from T hirsuta D7, at 0.25 U/mL, can effectively decolorize 100 ppm of RBBR dye. The decolorization activity of this enzyme decreases with increasing dye concentration. Dyes with simpler structure show higher rates of decolorization than do dyes with a more complex structure. The addition of mediators, such as MnSO 4 , H 2 O 2 , and VA enhances the dye decolorization rate.