Published at : 30 Dec 2022
Volume : IJtech Vol 13, No 8 (2022)
DOI : https://doi.org/10.14716/ijtech.v13i8.6111
|Adi Setyo Purnomo||Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya, 60111, Indonesia|
|Adelia Sabilah Prameswari||Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya, 60111, Indonesia|
|Hamdan Dwi Rizqi||Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya, 60111, Indonesia|
|Taufiq Rinda Alkas||Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya, 60111, Indonesia|
|Ratna Ediati||Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya, 60111, Indonesia|
|Yuly Kusumawati||Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya, 60111, Indonesia|
This study investigated the biotransformation of
methylene blue (MB) by mixed fungal cultures of Gloeophyllum trabeum and
Aspergillus oryzae. Equal volumes of A.
oryzae and G. trabeum cultures were applied to Erlenmeyer containing
MB and incubated at 30°C for 7 days. The change in absorbance of the MB control
solution and the solution after application, measured with a UV-Visible
spectrophotometer, was used to calculate the decolorization. The addition of A. oryzae to G. trabeum cultures showed MB biodecolorization reaching 69.34%,
greater than single cultures of G.
trabeum and A. oryzae, which were
31.50% and 36.82%, respectively. Metabolite identification from MB
biodecolorization by mixed culture using LC-QTOF-MS found the following
metabolites: C16H20N3S, C19H22N3SO4,
C31H48N3S+. The results of this
study showed that the addition of A. oryzae enhanced the percentage of
MB decolorization from G. trabeum culture.
Aspergillus oryzae; Biodecolorization; Gloeophyllum trabeum; Methylene blue; Mixed culture; Pollutants
Indonesia is one of the main textiles producing countries in Asia, which one of the world's top 10 exporters of textiles and textile products, along with countries such as China, India, Thailand, Brazil, and the United States. Indonesia's main attractions are its cheap labor force and large domestic market (AHK Indonesien, 2022). With the increasing population growth rate, textile production as a need for clothing increases rapidly.
This industrial process always produces waste, especially liquid waste. Many methods have been tried to manage textile wastewater, such as ozonation, photochemistry, adsorption, ion exchange, floatation, and electrokinetic coagulation. Removal efficiencies are in the range of 70-95%, but these processes still have drawbacks. The disadvantages of these methods include the need for large areas and the generation of a large amount of sludge, which causes problems with waste disposal (Istirokhatun et al., 2021). However, sludge from industrial by-products can also be processed and activated into biosorbents (Extracellular polymeric substances/EPS) (Kistriyani et al., 2020).
Approximately 10.000 types of dyes are used in the textile industry, and during the dyeing process, 10-15% of the textile dyes used will be removed with wastewater. One of the dye often used in the textile industry is methylene blue (MB) because it is economical, easy to obtain, and has a very strong adsorption power (Pratiwi et al., 2021). Meanwhile, although MB has some advantages, it can be toxic to humans and the environment. It can also cause human health problems such as respiratory disorders, stomach disorders, blindness, and digestion and mental disorders. Furthermore, MB also triggers nausea, diarrhea, vomiting, cyanosis, shock, gastritis, jaundice, methemoglobinemia, tissue necrosis, and increased heart rate, causing premature cell death in tissues and skin/eye irritation (Khan et al., 2022). The toxicity of MB dye was studied and reported had the no observed adverse effect level (NOAEL) value of 25?mg?kg?1 for MB in rats (Bharti et al., 2019). Besides, MB had the LD50 1180 mg kg-1 (oral acute toxicity rat), LC50 18 mg L-1 (96 h, Mystus vittatus), and EC50 2.26 mg L-1 (48 h, Daphnia magna) (LabChem, 2019). The MB molecular structure is figured out in Figure 1.
Figure 1 MB molecular structure
Many methods have been applied for MB degradation including advanced oxidation processes (AOPs), photodegradation, ozonation, oxidation with UV/H2O2, photocatalytic degradation etc. Several studies using nanocomposites have been carried out with excellent photodegradation results (88-100% removal), such as CuO/Bi2O3 nanocomposites, SnO2-bentonite, TiO2/Seashell, and ZnO-nanorods/activated carbon fibers (Khan et al., 2022). However, most of them need high costs and require elevated energy costs.
One of the effective methods for reducing dye wastewater is using microorganisms as a biological activity through biodegradation. Biodegradation, also called bioremediation, is a very broad field and the most reliable mechanism for removing organic and inorganic pollutants from the environment is by using microorganisms (Zahari et al., 2022). One of the microorganisms used for biodegradation is brown rot fungi (BRF). BRF produce hydroxyl radicals generated from the Fenton reaction to degrade cellulose, hemicellulose, and some dyes (Purnomo et al., 2022). According to Riadi et al. (2021), the initial organic compound degradation reaction is faster and more economical with Fenton's reagent than other chemical treatments, and the degradation yield can reach 70–99% (Riadi et al., 2021). In addition to producing hydroxyl radicals, these fungi produce cellulase enzymes used to degrade cellulose as a source of carbon and energy. It is an advantage of brown rot fungi compared to white-rot fungi which only use ligninolytic enzymes as degrading agents (Kim et al., 2014).
In a previous study by Purnomo et al. (2021), MB biodegradation was carried out using brown rot fungus Gloeophylum trabeum in liquid PDB media resulting in a decolorization percentage of 71.61% for 14 days of incubation. This result indicates that the biodegradation of MB using brown rot fungus G. trabeum takes a long time. Hence, it is necessary to modify the culture through mixed cultures. Aspergillus oryzae can decolorize several types of azo dyes from aqueous solutions such as Direct Red 23 and Direct Violet 51 because A. oryzae is used as a biosorption substrate by azo dyes (Corso et al., 2012). A. oryzae was reported that it can be used for the remediation of hydrocarbon polluted soils (Asemoloye et al., 2020) and degrade a mycotoxin compound (Ochratoxin A) that can contaminate agricultural products (Xiong et al., 2021). The combination of brown rot fungus G. trabeum and filamentous fungi A. oryzae is a new combination that has never been studied for MB degradation. This study aims to determine the degradation ability of this combination against methylene blue dye, predict the metabolite products, and propose the degradation pathways.
2.1. Fungi and Chemicals
The fungi used in this study include G. trabeum and A. oryzae taken from the Microorganism Chemistry laboratory collection. The chemicals were used such as methylene blue (SAP Chemicals), potato dextrose agar (PDA, Merck), potato dextrose broth (PDB, Difco), distilled water (Brataco), alcohol (70%, Brataco), and filter paper (Whatman).
2.2. Microorganisms Culture conditions
Stock cultures of G. trabeum and A. oryzae from the collection were taken ± 1 cm2 of mycelium and then inoculated on PDA sterile petri dish that had been incubated statically at 30 °C for 7 days. G. trabeum and A. oryzae mycelia (diameter 1 cm) were inoculated into 9 mL of PDB medium separately and then pre-incubated statically for 7 days at 30 °C. The regenerated G. trabeum and A. oryzae fungi were put into a sterile cup blender (Waring, J-SPEC LB10BUJ) containing 25 mL of sterile distilled water and then homogenized until evenly crushed. The homogenate (1 mL) was inoculated into Erlenmeyer containing 9 mL of PDB liquid medium using a micropipette and then pre-incubated statically for 7 days at 30 °C (Pratiwi et al., 2021).
2.4. Biotransformation of MB by Mixed Cultures
A. oryzae liquid culture (10
mL) was added to the pre-incubated G.
trabeum culture (10 mL), and then added 1 mL of 2000 mg/L MB (final culture
concentration 95.24 mg/L). The mixed cultures were incubated at 30 °C for 7
days. The cultures were separated by using a centrifuge at 3000 rpm for 5
minutes after 7 days, the supernatant was analyzed by using a UV-Vis
spectrophotometer. Abiotic control was made from 20 mL PDB liquid medium added
with 1 mL MB dye to reach a final concentration of 95.24 mg/L. In contrast, the
biotic control was made from mixed cultures of G. trabeum and A. oryzae
fungi which were turned off by heating with an autoclave before adding MB. The
percentage of MB dye decolorization was calculated using Equation 1.
2.5. Analysis of Biotransformation of MB and Its Metabolite Products
3.1. Biotransformation of MB by Single and Mixed Cultures
Figure 2 The absorbance profile graph of the MB decolorization result on the 7th day
Figure 3 The decolorization of MB by all treatments
3.2. Identification of Metabolites of MB Biotransformation by Single Culture of G. trabeum and A. oryzae
Based on the chromatogram, there was the same peak between the control and treatment at a retention time of 5.57 min (Figure 4). Based on the QTOF-MS analysis, the two peaks have m/z of 284 which was the m/z of the MB. This assumption was based on the research by Rauf and colleagues in 2010 where MB had a peak m/z of 284 (Rauf et al., 2010) and Rizqi and Purnomo (2017) also found this peak in MB decolorization by Daedalea dickinsii fungus. The MB peak intensity in treatment culture was lower than the MB peak in the abiotic control. This phenomenon showed that MB had been transformed (degraded) and the MB concentration was reduced. In the chromatogram treatment, new peaks appeared at the retention times of 1.63, 2.64, 3.04, 4.66, and 5.40 mins. The identification of metabolite was performed based on references of previous studies and databases (Table 1).
Figure 4 Profile Chromatogram of MB Biotransformation by G. trabeum. Black chromatogram: abiotic control (MB + PDB), while Red: treatment chromatogram (G. trabeum)
Table 1 Metabolites of MB decolorization by G. trabeum
The chromatogram profile of MB biotransformation by A. oryzae showed the same peak between control and treatment at a retention time of 6 min, with m/z of 284.122 identified as MB (Figure 5). The chromatogram treatment showed a new peak at the 5.17 min retention time, which was identified as C31H48N3S+ (Table 2).
Figure 5 Profile Chromatogram of MB Biotransformation by A. oryzae, Black chromatogram: abiotic control (MB + PDB), while Red: treatment chromatogram (A. oryzae)
Table 2 Metabolites of MB decolorization by A. oryzae
3.3. Identification of Metabolites of MB Biotransformation by Mixed Cultures
The LC chromatogram showed the same peak between control and treatment
at a retention time of 5.78 mins, which identified MB (m/z 284). Based on the chromatogram in Figure 6, the MB peak on
treatment showed a lower intensity than the MB peak in control. This indicates
a decolorization process in the treatment so that the MB concentration was
reduced. The treatment chromatogram showed that new peaks appeared at the
retention times of 2.79, 4.81, and 7.44 mins (Table 3).
The LC-QTOF MS profile showed a decrease in the intensity of MB in treatment compared to control, which indicated a decrease in MB concentration. The appearance of several new peaks in the treatment indicated the metabolites of MB degradation. MB biodegradation pathway was proposed, as shown in Fig. 7. G. trabeum transformed MB via 3 initial pathways were oxidation of the sulfide group be 3-((3-dimethylamino) phenyl) sulfinyl-N-N-dimethylbenzen1,4-diamine; oxidation be 5-(dimethylamino)-2-nitrobenzenesulfonic acid; and demethylation to 3,7-diaminophenothiazin-5-ium, then oxidation cleavage to 2,5- diamino benzenesulfonic acid, and to 2-aminobenzenesulfonic acid.
Besides the transformation process of MB by A. oryzae, the product metabolites were N-(8-(dimethylamino)-2-pentadecyl-3H-phenothiazin-3-ylidene)-N-methylmethanamin- ium. From the transformation process of MB by mixed cultures, the product metabolites were 3,7-bis (dimethylamino)-9a,10 dihydrophenothiazin-5-ium; 3-2-amino-3-methyl-5-(N-methylformamido) phenyl) sulfinyl) 2,6-dimethylphenyl) (methyl) carbamic acid; and N- (7-(dimethylamino)-2-pentadecyl-3H-phenothiazin-3-ylidine)-N-methylmethanamin- ium. The estimated pathway for MB degradation using mixed culture G. trabeum and A. oryzae is shown in Figure 7. This study indicated that the mixed culture could be used to transform MB.
Figure 6 Profile Chromatogram of MB Biotransformation by mixed cultures, Black chromatogram: abiotic control (MB + PDB), while Red: treatment chromatogram (mixed cultures)
Table 3 Metabolites of
MB decolorization by mixed cultures
Figure 7 MB degradation proposed pathway by single cultures and mixed cultures
The single culture of G. trabeum and A. oryzae decolorized methylene blue (MB) by 31.50% and 36.82%, respectively. In comparison, the mixed cultures of G. trabeum and A. oryzae decolorized MB by 69.34% after incubation for 7 days. Based on the LC-QTOF MS analysis, the MB metabolite from biodecolorization by A. oryzae was C31H48N3S+, while that by G. trabeum were C6H8N2SO3, C6H7NSO3, C8H10N2SO5, C16H20N3SO, and C12H10N3S. On the other hand, the MB metabolites by mixed cultures were C16H20N3S, C19H22N3SO4, and C31H48N3S+. This study indicated that mixed cultures of BRF of G. trabeum and filamentous fungus A. oryzae were effective in decolorizing MB dye.
This study was funded by the Directorate of Research, Technology, and Community Service, Ministry of Education, Culture, Research and Technology of Indonesia in accordance with the World Class Research Scheme Number: 008/E5/PG.02.00.PT/2022.
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