Decolorization of Two Dyes Using White Rot Fungus P. ostreatus (BWPH) Strain and Evaluation of Zootoxicity of Post Process Samples

Pleurotus ostreatus (strain BWPH) was chosen for this study, which were collected from the Fungal Strain Collection of Environmental Biotechnology Department, The Silesian University of Technology, Gliwice, Poland. For the multiplication of Pleurotus ostreatus mycelium, 4 cubes of mycelium from MEA plates were transferred to the flask containing the medium Glucose – 5g/L, Peptone – 1g/L, MgSO₄ x 7H₂O – 0.5g/L and KH₂PO₄ – 0.1g/L (pH 5.6). The media sterilization was performed at 121°C for 20 minutes in autoclave.

Two dyes belonging to anthraquinone and azo group were selected: Anthanthrone Red (C.I 59300; CAS 4378-61-4) and Disazo Red (C.I 20730; CAS 3905-19-9) respectively. The dyes were prepared in deionized water in concentrated solution form, autoclaved at 121°C and then added to the media containing 5 days old fungal biomass at the final concentration 0.08 g/L.

The BWPH strain's capacity to decolorize dyes belongs to different groups was checked by performing decolorization experiment. After approximately 5 days of growing fungal biomass on the media described above (100 ml of medium with 7 days cultured biomass), the biomass was broken up using a BagMixer and added to the reactor containing liquid organic medium having volume of 1500 ml. The reactors were left for 7 days on a shaker (150 rpm) in order to multiply the biomass. After growth of fungal biomass, Anthanthrone Red and Disazo Red were added separately to bioreactor with a final concentration of 0.08 g/L. After addition of dye, the reactor was operated in the static version. For 5 days a week, 200 ml of air was pumped through the filter using a syringe. The absorbance of the dye containing samples was measured spectrophotometrically by using Hitachi U-1900 UV-VIS spectrophotometer. The λ values were determined by measuring the absorbance at different wavelengths and determined that the Anthanthrone Red dye gave maximum absorbance at three wavelengths 538 nm, 510 nm and 462 nm and for Disazo Red dye the value of maximum absorbance was at 556 nm and 510 nm. The samples were collected, and absorbance was measured at mentioned time interval from the reactor after addition of dye; 0.25 h, 24 h, 48 h, 120 h, 144 h and 168 h. Changes in pH value were measured during the process by pH-meter. The percentage of decolorization (DP) of dye at fixed time interval was calculated using the below mentioned equation 1: (1) DP[%]=Ci−CtCi*100 DP[\%] = {{Ci - Ct} \over {Ci}}*100

Where, Ci is the initial concentration of dye (in milligram per Liter), and Ct is the concentration of dye in samples at time t (in milligram per Liter).

The total removal percentage was measured using equation 2: (2) Removal[%]=Ci−CfCi*100 Removal[\%] = \matrix{{Ci - Cf} \cr {Ci} \cr} *100

Where, Ci is the initial concentration of dye (in milligram per Liter), and Cf is the final concentration of dye in samples (in milligram per Liter).

For the assessment of toxicity, the acute immobilization test was conducted on crustacean Daphnia magna in accordance with OECD guideline 202. The test was performed in special 24-well plates. The range of the concentrations of the 5 tests sample arranged in a geometric series with a factor 2 for both dyes (3.125% – 100%) and the post process sample were collected from reactor. The same test was carried out for dyes solution in water. In each well, suitable concentration of test solution and post process sample were added followed by addition of five Daphnia magna organisms. After 24 hours of incubation at room temperature, the number of mobile and immobile test subjects was noted.

The concentration of the sample causing any change in 50% of the organisms in the population is considered as a measure of toxicity – EC₅₀. The EC₅₀ value was determined by the method of graphical interpolation of the obtained results. In order to classify the toxic substances, this value was converted into toxicity units according to the below mentioned Equation 2: (3) TUa=100/EC50 TUa = 100/E{C_{50}}

Based on the above calculations, the pure dye and its metabolites found in the post process sample were classified to appropriate toxicity group. Persoone toxicity classes has been described in the table below (Persoone et al., 2003).

The decolorization effectiveness of the dyes Anthanthrone Red and Disazo Red were measured spectrophotometrically at specific mentioned time interval. As soon as the dye was added to the reactor, the gradual decrease in the absorbance of dye was observed at particular wavelength which corelates with the commencement of dye degradation process by fungi. With reference to Fig. 1, the decrease in concentration of dye started just within 0.25 h after addition of dye. Starting decolorization may correspond to great biosorption capacity of WRF. As the homogenized biomass is utilized for the experiment, it serves for larger surface area for dye adsorption. The sorption of dye depends on the chemical nature of dye, interaction between functional group of dye and cell wall of fungi, pH of medium. The Anthanthrone dye contains halogen chromophore which could be absorbed by negative charges on fungal cell wall.

Figure 1

As mentioned, the DP was calculated for three absorbance maxima for Anthanthrone Red dye at 538 nm, 510 nm and 462 nm. The UV-VIS wavelength scan was utilized to monitor the changes in absorbance peaks at each wavelength which helps in estimation of changes in chemical structure of dye. The final decolorization measured at 168 h, was highest (93.4%) at 538 nm, followed by 92.9% at 510 nm and was lowest (86.8%) at 462 nm. It suggests that enzymatic degradation showed maximal effect at 538nm wavelength. In case of Disazo Red Dye, almost similar percentage of decolorization was observed at both wavelengths 556 nm (73.1%) and 510 nm (70.3%). The process of Anthanthrone Red decolorization was faster and more efficient than Disazo Red, as after 0.25 h, the decolorization percentage were observed 23.09% and 11.99% respectively (Fig. 1, Fig. 2). The results of decolorization experiment were as expected and it is found to be corelating with published literature as well [20].

Figure 2

The differential changes in absorbance maxima attributed to the possibility of removal of both dyes via biochemical transformation [21]. Because when the dye is removed via biosorption or bioaccumulation route, mostly it does not correspond to changes in wavelength peaks. Lu et al., 2016 [22] proposed removal of RBBR, an anthraquinone dye via Lignin modifying enzyme whereas Kunjadia et al., 2016 [23] mentioned enzymatic removal of recalcitrant azo dye with help of laccase as a major extracellular enzyme responsible for degradation of dye. The Lignin modifying enzymes and oxidases are responsible for degradation of pollutants. The enzyme activity is influenced by various parameters like pH, temperature, fungal species etc. Gahlout et al., 2013 has observed that the presence of glucose as carbon source and peptone as nitrogen source have major effect on laccase and Manganese Peroxidase MnP enzyme activity and apparently resulted in highest decolorization percentage [19]. So, it is evident that the medium composition utilized here for degradation studies had suitable components for efficient enzyme production. Jureczko et al., 2020 has measured laccase enzyme activity for P.ostreatus (BWPH) strain. It suggested that BWPH strain is potent producer of laccase enzyme which is responsible for degradation of recalcitrant pollutants [14]. The mechanism of microbial degradation of azo dyes involves the reductive cleavage of azo bonds (-N=N-) with help of azoreductase and results in formation of degraded products. The degradation pathway of anthraquinone dye was proposed and first reaction was hydrolysis of C-N bond in anthraquinone dye degradation [24]. And it is well documented that hydroxyl radicals play important role in degradation process by fungi.

In addition to the biochemical removal, the white rot fungi showcase great sorption capacity due to cell wall structure and remove dye efficiently via biosorption in living and dead biomass [25–26]. A regular increase in DP was found in case of the Anthanthrone Red dye whereas the Disazo Red showed almost a halt in decolorization between 24h and 48h. Here, shaking condition was utilized for growth of biomass and after addition of dye, the static condition was maintained. The proper aeration was provided after time intervals. It also corresponds to enhanced dye decolorization as it helped in oxygen transfer reaction. The parameters like pH and temperature also play important role in dye degradation mechanism. The pH of culture medium is critical for growth of fungi, metabolic activity, surface structure, enzyme production and hence, efficiency of recalcitrant compound degradation [10]. The charge present on surface of fungal biomass and ionic forms of dye pollutant can be controlled by pH of solution [27]. Here, pH of medium was set at 5.6 which generally imparts in good range of pH for effective dye degradation. But the pH of medium was dropped to 3.6 as compared to initial pH of medium. With time passed, a gradually the medium turned acidic. Overall, the set parameters for this experiment supported combinedly dye degradation mechanism.

The result of dye degradation is not always complete mineralization of dye, but it may produce toxic intermediates or final products of degradation which are sometime even more harmful than pure dye [28]. The ultimate goal of fungal bioremediation is not just decolorization but also toxicity of post process samples should be decreased as compared to start sample in order to utilize fungal bioremediation as a prime tool for cleaning wastewater effluent. The Ecological and Toxicological Association of Dyestuffs Manufacturing Industry (ETAD) was established with the aim of mitigating environmental damage, safeguard consumers and coordinating with government and public issues in relation to toxicological effect of their products [29–30].

The results of the toxicity test were obtained in form of percentage of immobilized D.magna. Based on the observation of immobile organism, the samples were distributed to toxicity class. In case of pure dye toxicity assessment, the Anthanthrone Red dye showed no toxicity but in contrast, Disazo Red Dye exhibited almost 20% immobilized organism at 100% concentration (0.08 g/L) of dye. At lower concentration of disazo dye (up to 25%), no mortality of organism was observed but as the concentration increases (50–100%), there was not harmful effect on the crustacean (data not shown here).

The post process samples were collected from two-time interval: in the beginning of dye decolorization process (0.25 h) and at the end of experiment (168 h). The purpose of evaluation of these samples was to compare the toxicity of metabolites in the starting and at the end stage. When the post processed sample of time period 0.25 h and 168 h were assessed for toxicity, they belonged to higher toxicity classes of Persoone as compared to pure dye. The Anthanthrone Red dye executed higher percentage of immobilization of test organism at all ranging concentration, starting from 100% immobilization at highest concentration to 25% inhibition at lowest (3.125%). Whereas Disazo Red showed harmful effect up to 50% concentration of dye in 168 h post process sample. The EC₅₀ value was determined using extrapolation. In case of Anthanthrone Red dye the EC₅₀ value was 9.37 and 4.25 respectively at 0.25h and 168 h whereas it was 37.5 and 38.2 for Disazo Red dye (Fig. 3). The difference between EC₅₀ value for post process samples with increasing time period showed the gradual accumulation of toxic metabolites which results in higher number of test organism mortality. As in case of Anthanthrone Red dye, the toxicity increased as the time passed (168 h). It corresponds to that fact that as enzymatic degradation proceeds, it resulted in conversion of chemical property of dye. Overall, Anthanthrone Red dye showed much higher toxicity to crustacean D.magna than Disazo Red. The aromatic nature of Anthanthrone Red dye proved to be more toxic. The data reported here is in contrast with existing literature as it resulted in increase in zoo toxicity as compared to pure dye [31]. Such results can confirm formation of toxic metabolites of dyes as well as production of toxic metabolites by fungi. Mostly post process samples obtained as a result of decolorization were classified as toxic by-products [32].

Figure 3