The effect of addition of bacterium Pseudomonas aeruginosa on biodegradation of methyl orange dye by brown-rot fungus Gloeophyllum trabeum

The methyl orange (MO), one of common textile dyes from azo groups, has negative impact in human life and the environment. Therefore, many attemps have been devoted to find the most effective method for MO degradation. Brown-rot fungus Gloeophyllum trabeum has identified as the biodegradable agent of MO, but its efficiency is still low, and it requires a long incubation time. In this work, the biodegradable performance of brown-rot fungus Gloeophyllum trabeum was investigated for MO degradation in the presence of bacterium Pseudomonas aeruginosa with various volumes (2-10 mL, 1 mL = 5.05 x 1012 Colony Forming Unit (CFU)). The addition of 10 mL of bacteria into G. trabeum culture showed the maximum degradation of 88.67% in potato dextrose broth (PDB) medium for the 7-day incubation. The identified metabolites were 4-((4-(dimethyliminio) cyclohexa-2.5-dien-1-ylidenehydrazinyl) phenolate (C14H15N3O, compound 1), 4-((4-iminiocyclohexa-2.5-dien-1-ylidene) hydrazinyl) benzenesulfonate (C12H10N3O3S, compound 2), 4-((hidroksi-4-iminioyclohexa-2.5-dien-1-ylidene) hydrazinyl) benzenesulfonate (C12H10N3O4S, compound 3), 4-((4-(dimethyliminio) hydroxy-cyclohexa-2.5-dien-1-ylidene) hydrazinyl) methoxy benzenesulfonate (C15H16N3O5S, compound 4), and 4-((4-(dimethyliminio) dihydroxy-cyclohexa-2.5-dien-1-ylidene) hydrazinyl) dimethoxy benzenesulfonate (C16H18N3O7S, compound 5). Based on the identification of metabolic products, the mixed cultures transformed MO via three pathways: (1) desulfonylation, (2) demethylation, and (3) hydroxylation. These results indicate that P. aeruginosa can enhance MO biodecolorization by G. trabeum.


Introduction
The development and progress of the textile industry in addition to providing many benefits to the community also has a negative impact on the environment because this industry always produces textile dye liquid waste in the production process. A large number of dyes in the textile industry cannot fully be absorbed into the colored fiber fabric so that it will be released as waste [1]. Wastewater that contains dyes is very dangerous because most dyes are difficult to biodegrade (non-biodegradable), resistant, and toxic. Thus, if dyes are released into the aquatic environment without prior treatment, it can cause serious pollution to water sources which result in damage to the ecosystem environment and threats to human health [2]. Methyl Orange (MO) is a type of azo dyes that is found in textile industry waste and is a toxic compound and harms the environment [3]. To prevent the risk posed by such pollution, efforts are needed to reduce the entry of dyes into the aquatic environment. A variety of methods have been developed for handling dyestuff liquid waste from the textile industry [4]. Bioremediation is considered a quite cheap and efficient method for handling dyes waste [5][6][7][8][9][10]. Brown-rot fungi are one of the  [11][12][13][14] by involving the Fenton reaction [15][16][17][18].
Other research has been carried out to degrade MO dye by utilizing the brown-rot fungus Gloeophyllum trabeum which can produce radical hydroxides through the Fenton reaction [13]. Purnomo et al. [13] reported that G. trabeum was able to degrade MO in liquid PDB media by 47.53% for 14 days of incubation. The yield of MO degradation by G. trabeum was still relatively low and requires a long incubation time, hence a culture modification is needed to improve its ability.
Some studies suggest that the use of mixed cultures of fungi and bacteria can increase the ability of culture degradation [11,14,[19][20][21][22][23]. One type of bacterium that can degrade some organic pollutants is Pseudomonas aeruginosa because it can produce degrading enzymes such as azoreductase [23][24][25][26][27]. P. aeruginosa had been reported to be able to enhance DDT degradation in mixed culture with some woodrot fungi [28,29]. Therefore, in this study, the effect of the addition of P. aeruginosa on the ability of biodegradation of MO by G. trabeum was investigated as an innovation in the effort to deal with the problem of textile dye waste.

Chemicals
A number of chemicals needed are as follows. Methyl Orange (MO) was purchased from SAP Chemicals, potato dextrose agar (PDA), nutrient agar (NA), and nutrient broth (NB) were purchased from Merck, Germany, while potato dextrose broth (PDB) was purchased from Himedia, India.

Culture condition 2.2.1 Regeneration of G. trabeum.
The brown-rot fungus G. trabeum NBRC 6509 species was used as the first degradation agent. The fungal mycelium was taken with a sterile ose needle and inoculated into a petri dish containing the sterile PDA medium then incubated at 30±0.5 ºC for 7 days until the mycelium covered the entire surface of the PDA medium [30].

2.2.2
Regeneration of P. aeruginosa. The bacterium P. aeruginosa NBRC 3080 was used as a second degradation agent. One bacterial colony of P. aeruginosa was taken with a sterile ose needle and inoculated into a petri dish containing the sterile NA medium then incubated at 37±0.5 °C for 24 hours [31,32].

Preparation of Liquid
Culture of G. trabeum. One petri dish containing G. trabeum in the PDA medium (2.2.1) was homogenized by using a sterile blender that contained 25 mL of sterile distilled water. One milliliter of homogenate was inoculated into 8 mL of sterile PDB medium in 100 mL of Erlenmeyer and then pre-incubated at 30±0.5 ºC for 7 days under a static condition [33].

2.2.4
Preparation of liquid culture of P. aeruginosa. One colony of P. aeruginosa (2.2.2) was inoculated into 10 mL of sterile NB medium. The culture was incubated at 37±0.5 °C for 24 hours with shaking at a speed of 180 rpm as a starter. After 24 hours, one milliliter of P. aeruginosa bacterium starter was inoculated into 500 mL of sterile NB medium and then pre-incubated at 37±0.5 °C under a shaking condition with a shaker at a speed of 180 rpm until it reached a maximum OD of the stationary phase of P. aeruginosa bacterium [34,35]. 3 the addition of MB (the final concentration was 100 mg/L) and PDB to the total volume of 20 mL. As an abiotic control, 1 mL of 2000 mg/L MO (the final concentration was 100 mg/L) was added into 19 mL of PDB medium (the final volume was 20 mL) without an addition of fungal and/or bacterial cultures. All cultures and abiotic control were incubated under a static condition for 7 days at 30±0.5 °C. After 7 days, the cultures were separated by using a centrifuge at 3000 rpm for 15 minutes. The supernatant was taken and filtered by using Whatman filter paper. The supernatant absorbance was measured with a UV-Vis spectrophotometer. The remaining supernatant was stored in a refrigerator for the characterization of metabolites. The calculation of the percentage of MO dye decolorization was according to equation 1 at a wavelength of 465 nm [14].
where Ac is absorbance abiotic control, and At is absorbance treatment.

Product metabolite identification
The identification of metabolites was performed by using the LC-TOF/MS (Impact II, Bruker). The source of ionization was the electrospray ionization (ESI) with a mass range of 50-500. The elution method used was the gradient method with a flow rate of 0.2 mL/min in the first 3 minutes and the next 7 minutes using a flow rate of 0.4 mL/min. The mobile phase was methanol and water with a ratio of 99:1 in the initial 3 minutes and 61:39 for the remaining 7 minutes. The column was the Acclaim TM RSLC 120 C18 with a size of 2.1 x 100 mm with a column temperature of 33 °C [12][13][14].

The effect of addition of P. aeruginosa on biodegradation of MO by G. trabeum
In this study, the addition of P. aeruginosa on the biodegradation of MO by brown-rot fungus G. trabeum was investigated. The MO degradation by G. trabeum fungus was also carried out as the biotic control. The absorbance profile of MO degradation is shown in figure 1. The maximum absorbance of MO for abiotic control is detected at a wavelength of 465 nm. However, in the biotic control and the mixed cultures, the absorbance of peaks decrease which indicates decolorization. Besides, the other peaks appears at the wavelength of 300-390 nm indicating the peaks of metabolites. Since the MO peak appears in the wavelength range of 400-600 nm which is the wavelength region of visible light, the metabolites might be colorless [3]. The absorbance of biotic control of G. trabeum is lower than that of the abiotic control, indicating G. trabeum decolorized MO. Besides, the biotic control peak is lower than the mixed cultures with the addition of 2, 4, and 6 mL of bacteria. This suggests that the mixed cultures still adapt to the addition of MO. The MO degradation by the mixed cultures of G. trabeum and P. aeruginosa occurs maximally in the additions of 8 and 10 mL of bacteria as evidenced by the lower peaks at 465 nm compared with the other peaks. The ability of mixed cultures of G. trabeum and P. aeruginosa to degrade MO was determined quantitatively by measuring the percent of decolorization (table 1). The percent of MO decolorization by the mixed cultures increased along with the increase in the amount of added bacteria. The more the bacteria added, the greater were the biosurfactants as well as dye degradation enzymes produced by P. aeruginosa. Thus, it caused an increase in the MO decolorization. Based on table 1, the degradation of MO by mixed culture occurs optimally in the addition of 10 mL of bacteria (1 mL = 5.05 x 10 12 CFU) approximately 88.67%. However, the percent of decolorization at the additions of 8 mL to 10 mL of bacteria is not significantly different due to the competition of bacteria for surviving that might occur rather than to decolorize MO. Some toxic metabolites might be produced during the stationary phase under an abundant population of bacteria [22].  The significant differences occur in the mixed cultures with the additions of 6 mL and 8 mL of bacteria, from 75.32% to 87.53%. It suggests that each degradation agent can adapt to each other when living in the same medium in degrading MO. The ability of G. trabeum to degrade MO is related to its ability of producing oxalic acid and H2O2 which play a role in the Fenton reaction [36]. In addition to involving the Fenton reaction, G. trabeum also produces several extracellular enzymes to degrade wood and some pollutants, such as endoglucases and xylanases that are known produced by G. trabeum in the very large quantities [37]. The ability of bacterium P. aeruginosa in degrading MO is also related to its ability to produce a degrading enzyme, i.e. enzyme azoreductase, lignin peroxidase, DCIP reductase, and tyrosinase [38,39]. Apart from producing degrading enzymes, Scheibenbogen et al. [40] reported that P. aeruginosa was able to produce biosurfactants of rhamnolipid types which could also be applied in increased degradation of hydrocarbon pollutants.

Metabolites identification
The profile of chromatogram of MO metabolites is shown in figure 2. It can be seen that the treatment MO peak is lower compared with the control MO peak, identified at the retention time of 7.29 minutes. Besides the MO peak, in the treatment chromatogram, there are 5 new peaks appear that do not yet exist in the control chromatogram. They are identified as the metabolites produced during the process of MO degradation by the mixed cultures of G. trabeum and P. aeruginosa. Identification of metabolites is determined based on the similarity between MS spectrum and time retention from database (table 2). As seen in table 2, the peak at the retention time of 1.29 minutes has m/z 241 which is identified as 4-[((4-which approximately 88.67%. However, the percent decolorization at the addition of 8 mL to 10 mL bacteria was not significantly different, due to the competition of bacteria for surviving might occur rather than to decolorize MO. Some toxic metabolites might be produced during stationary phase under abundant population of bacteria [22]. The significant differences occurred in mixed cultures with the addition of bacteria 6 mL to 8 mL, from 75.32% to 87.53%. It suggested that each degradator agent can adapt to each other when living in the same media in degrading MO. The ability of G. trabeum to degrade MO is related to its ability of producing oxalic acid and H2O2 which play a role in the Fenton reaction [36]. In addition to involving the Fenton reaction, G. trabeum also produce several extracellular enzymes to degrade wood and some pollutants, such as endogluxases and xylanases, that are known produced by G. trabeum in very large quantities [37]. The bacterium P. aeruginosa in degrading MO is related to its ability to produce a degrading enzyme, namely the enzyme azoreductase, lignin peroxidase, DCIP reductase, and tyrosinase [38,39]. Apart from producing degrading enzymes, Scheibenbogen et al. [40] reported that  There were three MO degradation pathways by the mixed cultures of G. trabeum and P. aeruginosa proposed. The first pathway: the ionized MO was transformed into compound 1 by desulfonylation. The second pathway: the ionized MO was transformed into compound 2 by demethylation, followed by hydroxylation to compound 3. Last, the third pathway: the ionized MO was transformed into compound 4 by hydroxylation and methylation, followed by hydroxylation and methylation again into compound 5. Hydroxylation occurred due to the presence of radical hydroxides that might be produced from the Fenton reaction by G. trabeum that attacked MO [20]. Previously, G. trabeum transformed MO via three pathways: (1) demethylation, followed by hydroxylation reactions; (2) hydroxylation, followed by demethylation; and (3) desulfonylation [13].    The MO degradation pathway by mixed cultures of G. trabeum and P. aeruginosa was proposed to divide into three ways. The first pathway: the ionized MO was transformed into compound 1 by desulfonylation. The second pathway: the ionized MO was transformed into compound 2 by demethylation, followed hydroxylation to compound 3. The third pathway: ionized MO was