Textile dye decolourisation of yeast isolated from Tuak starter culture

The textile industry is one of the largest industries in the world and contributes positively to the economic growth of a country. However, the improper disposal of textile dye effluents results in discharging of high concentrations of textile dyes into the environment. Moreover, the inherent stability of textile dyes makes them hard to remove from the environment, which can harm organisms in polluted areas. Biological decolourisation was considered as a strategy to remediate textile dyes. Microbial decolourisation of textile dyes involves using various species of bacteria and fungi from different sources that can degrade or accumulate dye molecules. Although Indonesia is a well–known country with rich biodiversity, research on microbial decolourisation is still limited. Therefore, this study explored the decolourisation potential of microbes from the starter culture of Tuak, a traditional Indonesian alcoholic beverage made from fermented sugar palm or rice. Potential microbes were screened on yeast peptone dextrose solid agar (YPD) supplemented with various textile dyes. Isolates that formed a clear zone around the colony were further examined for decolourisation activity in broth culture media, and the decrease in dye concentration was measured spectrophotometrically. The results showed that two potential yeast isolates could decolourise Congo red and malachite green. Identification of the isolates by observation of colony and cell morphology and biochemical characteristics indicated that the two isolated yeasts belonged to the genera Saccharomyces and(or) Candida.


Introduction
The textile industry makes a significant financial contribution to the global economy.The textile industry generates about 1 trillion dollars, or about 7% of total world exports, and provides employment for 35 million people worldwide [1].In contrast to the economic benefits generated by the textile industry, improper disposal of industrial effluents has led to environmental pollution.Due to the inherent stability of textile dyes, it takes a very long time to remove textile dyes naturally.Moreover, the destructive properties 1255 (2023) 012054 IOP Publishing doi:10.1088/1755-1315/1255/1/012054 2 of the dye to organisms exacerbate the problem.Apart from affecting the aesthetics of water bodies, the light-absorbing property of dyes also impairs photosynthesis in water, which inhibits plant growth, disturbs the balance of the ecosystem, and affects habitability.Furthermore, the dye can accumulate in fish tissue, enter the food chain, and eventually reach humans, causing toxicity and promoting mutagenicity and cancer [2].
Various methods have been proposed for the remediation of textile dyes, ranging from physical, chemical, and biological approaches.Physical approach uses numerous strategies such as filtration, coagulation, and adsorption, while chemical approach is based on the use of oxidation-reduction reactions, for example by ozonation [3].Biological approach, on the other hand, uses the ability of microbes (fungi and bacteria), or plants to decolourise textile dyes.Microbial decolourisation is known for its relative ease of use and cost effectiveness.There are 3 main mechanisms involved in the decolourisation process: extracellular biosorption, intracellular bioaccumulation and/or intracellular and extracellular biodegradation [4].An example of biosorption is the use of water hyacinth biomass (Eichhornia crassipes) in the decolourisation of malachite green (MG) [5].Saccharomyces cerevisiae has been reported to decolourise malachite green dyes through biosorption and biodegradation [6].The extracellular biodegradation of Congo red (CR) was described using Pichia sp.[7].
Tropical countries such as Indonesia have great climatic advantages that promote biodiversity of organisms and microorganisms [8].Nevertheless, the potential use of biological agents for many problematic aspects, especially textile dye pollution, has rarely been reported.Importantly, the preferred species of usable microorganisms from fermented foods and beverages are safe to use and have no negative impact on the environment [9].Tuak is an Indonesian traditional alcoholic beverage made from the sap of palm or rice [10].The fermentants contained in the starter culture could provide non-pathogenic microorganisms suitable for the decolourisation of textile dyes.Therefore, this study explored the decolourisation potential of microbes from the starter culture of Tuak.

Materials
Agar powder and lactophenol cotton blue were obtained from HIMEDIA.Tryptic soy broth (TSB), Malachite Green (927.01 g/mol, dye content ≥ 90 %), phenol red, glucose, Tween 80, urea and sodium chloride were purchased from Merck.Yeast extract and peptone were purchased from Liofilchem.Congo Red (≥ 35% dye content), Reactive Orange (≥ 70% dye content) and Acid Violet (40% dye content) were purchased from Sigma-Aldrich.Textile dyes (Arabian Night, Pagoda Red, and Windsor Purple) were purchased from Dylon.Cornmeal was purchased from MUGO.Phosphate Buffered Saline (PBS) was purchased from Vivantis.Tuak starter culture was purchased from the local market in Jakarta.

Isolation of dye decolourising microorganisms
The Tuak starter culture was rehydrated by incubating one gram of the ground powder in a 10 mL phosphate buffer solution (PBS) at 37°C for 30 minutes.The rehydrated sample was applied to YPD agar medium enriched with 50 ppm final concentration of commercial textile dyes and grown at 30°C for two days.Isolates with decolourisation potential were selected based on the clear zones that formed around the colonies.Before assessing the decolourisation potential of the isolates, they were then purified by streak cultures.Multiple subcultures were performed until the observed colony and cellular morphology is homogenous.

Decolourisation capability analysis
Evaluation of the potential of the isolates to decolourise dyes was done by two quantitative tests: decolourisation capacity for a range of dyes and then decolourisation of the selected dye at different concentrations that had the highest decolourisation percentage.First, the isolates were incubated in YPD broth media supplemented with different dyes at a concentration of 50 ppm for two days at 30°C.The decrease in dye concentration after incubation was measured with a UV/Vis spectrophotometer in a wavelength range of 300-900 nm, using YPD broth as a blank.The percentage of decolourisation was calculated using the following equation [11]: the isolate with the highest decolourisation percentage was selected for evaluation of the ability of the isolate to survive and decolourise the dye at higher concentrations.The isolates were incubated in YPD broth at a temperature of 30°C for two days while exposed to higher dye concentrations.The percentage of decolourisation was calculated as described above.

Morphology of the isolates and biochemical identification
The morphology of the colonies was observed after incubation of the isolates on YPD agar.To identify cell morphology, each isolate grown on YPD agar was stained with lactophenol cotton blue and observed under a light microscope.Biochemical identification was done using the urease activity test, the germ tube formation test and the chlamydospore formation test.For the urease activity test, each isolate was inoculated onto Christensen Urea (CU) agar and incubated at 30°C for 4-5 days.The germ tube test was performed by inoculating each isolate onto Tryptic Soy Broth (TSB) at 37°C for 2 hours.Isolates grown on TSB were observed under the light microscope.The chlamydospores formation test was performed by inoculating each isolate onto Cornmeal Tween 80 (CT) agar at 25°C for 48 hours [12].

Isolation of dye decolourising microorganisms
Isolation of microorganisms from the Tuak starter culture was performed by spreading the rehydrated Tuak starter culture on solid growth media supplemented with seven different textile dyes.A total of 9 isolates were growing on Acid Violet, Congo Red (CR), and Reactive Orange supplemented media (Table 1).Two isolates from CR media, with identification codes SSCR1 and SSCR2, were selected for further analysis due to high CR decolourising potential indicated by clear-zone formation around the colony (Figure 1)

Isolates Decolourisation Assay
To better understand the ability of the two isolates to decolourise commercial textile dyes, a quantitative measurement of dye removal was performed by incubating the isolates in a liquid medium supplemented with seven different dyes i.e.: Arabian night, acid violet 7, CR, MG, pagoda red, reactive orange 16, or Windsor purple.Measuring the decrease in light transmission with a UV/Vis spectrophotometer after two days of incubation shows that SSCR1 was able to decolourise MG in addition to CR, while SSCR2 was only able to decolourise CR.Based on these results, a further analysis of the decolourising activity towards MG and CR was carried out.The decolourising ability of the isolates was further evaluated by determining the maximum concentration that could be removed by the two isolates.The two isolates were inoculated in a liquid medium supplemented with MG or CR at concentrations up to 800 ppm.The decrease in dye concentration was measured with a UV/Vis spectrophotometer, using the liquid medium as a blank.Both isolates still effectively decolourised CR up to 300 ppm, while decolourisation decreased at higher concentrations (Figure 2).The maximum decolourisation of CR by SSCR1 was observed at a dye concentration of 50 ppm.SSCR2, on the other hand, showed great decolourisation activity toward the dye CR with maximum activity at a dye concentration of 150 ppm.Recently, reports have accumulated on the ability of various microorganisms to decolourise CR.Some examples include the ability of Colletotrichum gloeosporioides, Penicillium oxalicum, Aspergillus tubingensis, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus to optimally decolourise CR at 100 ppm [13,14,15], Streptomyces sviceus and septate endophyte at 50 ppm [16,17].This study demonstrated that the isolates SSCR1 and SSCR2 were capable of decolourising high concentrations of CR, which is comparable to recent findings.Meanwhile, results from the decolourisation of different MG concentrations showed that SSCR1 decolourises MG maximally at a dye concentration of 50 ppm, while the subsequent addition of MG concentration reduced the activity (Figure 3).Conversely, SSCR2 showed weak decolourisation capability in the MG.The decolourisation capability of both isolates decreased significantly as the MG concentration increased to 150 ppm.The fungicidal property of MG presumably inhibits growth and reduces the viability of both isolates, resulting in retarded decolourisation [18].In comparison, some recently published findings on optimal MG decolourisation performance by fungi include Pseudopestalotiopsis theae, Astrocystis bambusae, and Fusarium oxysporum at 100 ppm and Lasiodiplodia sp. at 50 ppm [19,20,21].Thus, SSCR1 is capable of MG decolourisation, which agrees with recent publications.Interestingly, even though the isolates were capable of decolourising MG in liquid media, the initial discovery in solid media failed to detect their growth and decolourisation (Table 1).This is presumably due to the low initial cell count during spreading, leading to low viability of the isolates [22].
Preliminary determination of the decolourisation mechanisms of both isolates was performed on YPD media supplemented with 50 ppm of either CR or MG by observing the colour of the colony and analysing the decolorization activity of the cell-free extract (supernatant).The change in colony colour during incubation suggests that the decolourisation mechanism is due to either biosorption or bioaccumulation.Otherwise, a constant colony colour suggests that intracellular enzymes may be involved.In the MG decolourisation assay of SSCR1, the colour of the SSCR1 colony was consistently white throughout the incubation period, indicating possible enzymatic MG degradation by either extracellular or intracellular enzymes.In contrast, when the CR dye was decolourised after incubation, both SSCR1 and SSCR2 developed a reddish colour, suggesting that biosorption or bioaccumulation was the dominant mechanism of CR decolourisation.To determine whether extracellular enzymes contributed to dye decolourisation, the cell-free extract obtained by separating the YPD broth medium, was mixed with 50 ppm of CR and MG and incubated at 40°C for 1 hour.The decrease in dye colour indicated the contribution of extracellular enzymes to dye decolorization.Qualitative observations indicated that no colour change was observed in the mixed cell-free extract with either MG or CR dye.In conclusion, SSCR1 decolorizes MG via intracellular enzymes, whereas the decolorization of CR is due to biosorption or bioaccumulation.SSCR2 was also predicted to decolorize CR by biosorption or bioaccumulation.

Isolates Identification
Isolates SSCR1 and SSCR2 were identified using two approaches: identification based on cell and colony morphology and identification based on biochemical properties of the isolates.The colony and cell morphology showed that the two isolates were yeasts (Table 2).Under a microscope, yeasts of the genus Saccharomyces normally have cells with spherical shape.However, identification based on cell and colony morphology was insufficient to identify the isolate.Therefore, identification based on the biochemical properties of isolates is required to support morphological identification.Identification of the isolates based on a biochemical approach was performed using three different tests: urease activity, germ tube formation, and chlamydospore formation [11].To determine the genera of the isolates, further biochemical tests were performed (Table 3).The urease activity test was performed by growing the isolates on CU agar [23].The results show that both isolates do not produce urease and belong to either the Saccharomyces spp. or Candida spp. group.Germ tube and chlamydospore formation tests were then performed to exclude possible taxonomic groups of the two isolates.Under the microscope, germ tube formation after incubation of the isolates in TSB and chlamydospore formation after incubation of the isolates in CT agar were the defining characteristics of Candida albicans.The observation showed that neither isolate formed a germ tube or chlamydospore, which ruled out the possibility that either isolate was C. albicans.According to a recent report, the dominant yeasts in the Tuak starter culture usually consisted of S. cerevisiae and C. tropicalis, which may indicate the identity of the isolates [24].Saccharomyces were usually the dominant microorganisms in alcoholic fermentation, but recent discoveries also show the efficiency of Candida species such as C. tropicalis in the fermentation of glucose [25].This result is in line with reports of S. cerevisiae and C. tropicalis, which are capable of MG and CR decolourisation [6,26,27].Overall, the isolates SSCR1 and SSCR2 belonged to either Saccharomyces spp.and(or) Candida spp.group, except for them being C. albicans.Further molecular identifications, such as internal transcribed spacer (ITS) or ribosomal DNA (rDNA) sequencing, are required to fully determine the taxonomic origin of the isolates [28].

Conclusion
Both isolates, SSCR1 and SSCR2, demonstrated high potential as decolourisation agents against MG and CR, respectively.Notably, SSCR1 was able to decolourise both MG and CR with high effectiveness.The highest concentration of the CR dye decolourised by SSCR1 and SSCR2 with high effectivity was at 300 ppm.In contrast, the MG decolourisation capability of both isolates peaked at 50 ppm.Cell and colony morphology, as well as biochemical identification, revealed that both isolates were potentially in the genera Saccharomyces sp.and(or) Candida sp.

Figure 1
Figure 1 Streak culture of SSCR1 (A) and SSCR2 (B) on YDP media supplemented with 50 ppm of Congo Red at 30 o C for 2 days.

Figure 2
Figure 2 Decolourising ability toward different concentrations of CR.Isolates SSCR1 and SSCR2 were incubated in a stationary incubator at 30 o C for 2 days.Concentrations of CR are indicated with a monochrome colour scale from light to dark: 50, 150, 300, 600, and 800 ppm.

Figure 3
Figure 3 Decolourising ability towards different concentrations of malachite green (MG).Isolates SSCR1 and SSCR2 were incubated in a stationary incubator at 30°C for 2 days.Concentrations of MG are indicated with a monochromatic colour scale from light to dark: 50, 150, 600, and 800 ppm.

Table 1 .
Isolation of textile dye decolourising microorganisms of Tuak starter culture

Table 2 .
Isolates Colony and Cell Morphology