3.1 Screening of TPM dyes decolorization strain
In this study, the 14 strains of endophytic fungi were isolated from the roots of Sinosenecio oldhamianus. The decolorization capability of these endophytic isolate were assessed on MBM plates with 4 types of TPM dyes (CV, MV, MG and CB). The tested fungal strains on solid medium exhibited a different potential of TPM dyes decolourisation. It is worth noting that only one endophyte, isolate SWUSI4, was able to decolourise all four different TPM dyes and also demonstrated the highest decolorization potential (Table S1 and Fig. S1). This result conforms to most observations (Jasinska et al. 2012; Yang et al. 2016; Marcharchand and Ting 2017), suggesting that different fungi had different dye decolorization abilities. Simultaneously, the potential of decolorization is related to the adaptability and activity of the selected strains. Obviously, our results indicated that strain SWUSI4 had the capability to decolorize all tested four TPM dyes on solid culture (Table S1 and Fig. S1). Generally, strains isolated from contaminated areas have been reported to have a strong tolerance and potential for remediation to contaminants (Yang et al. 2009; Almeida and Corso 2018). This study is also the first to report the effectiveness of endophytic fungus from the dyes-contaminated Sinosenecio oldhamianus, and its potential in decolorizing TPM dyes. Hence, according to above mentioned that isolate SWUSI4 was selected for further taxonomical identification as well as more detailed decolorization study.
3.2 Identification of isolate SWUSI4
Morphologically, isolate SWUSI4 produces white fast-growing colonies with a diameter of 8.34 cm (±0.26) after 5 days of growth on PDA at a temperature of 28 ºC, aerial mycelium abundant, woolly, at first white but later becoming yellowish, reverse white (Supplementary Fig. S2A, B). Advancing hyphae dichotomously branched, 4.54 µm (±1.76) diameter; and air-borne hyphae dividing into one-celled arthroconidia which remain cylindrical or become ellipsoidal or slightly barrel-shaped, 4.08 (±0.81) × 6.20 (±2.16) µm (Supplementary Fig. S2C), which is consistent with the morphological description of B. adusta R59 in the published literature (Kornillowicz-Kowalska et al. 2006). For isolated SWUSI4, the 5.8SrDNA gene sequence was determined and classified in the genus Bjerkandera sp. on the basis neighbor-joining analysis compared to other similar fungi stains (Fig. 1). This identity was provided on the basis of the nucleotide sequence having a 99 % homology (E value of 0.0; 99 % query coverage) with that of B. adusta strain MJ01 deposited in GenBank (accession number HQ327995.1) and B. adusta strain SM27 deposited in GenBank (accession number KU055647.1). Based on molecular taxonomy investigation of the strain SWUSI4, this fungus was identified as B. adusta. And the nucleotides sequences were submitted to GenBank and provided a GenBank accession number MN640911.
3.3 Dye decolorization activities of adusta SWUSI4 in different conditions
3.3.1 Effects of biomass on dye removal
The effect of biomass dosage was evaluated at the concentration of 100 mg/L (100 mL) at 30 ℃ for 14 day. As revealed in Fig. 2A, application of 4 g of biomass was sufficient to achieve the decolorization efficiency (DE %) for CV (85 %) and MG (95 %), while CB and MV required 6 g to achieve maximum DE 89 % and 92 %, respectively. In terms of CV and MG, the maximum DE was observed when the biomass dosage was 6 g, but there were no conspicuous differences between biomass dosage of 6 g and 4 g (Fig. 2A). The benefit of using more biomass has also been reported in other studies Kaushik and Malik et al. (2009); Wang et al. (2017); Bankole et al. (2018); Almeida and Corso (2018), which primarily attributed to having more binding sites and enzymes give rise to biosorption and biodegradation, respectively.
In this study, TPM dyes were effectively decolorized by endophytic fungus (isolated SWUSI4) under non-nutritive conditions. Interestingly, successful decolorization TPM dyes by isolated SWUSI4 was similar to others environmental isolates, such as Trichoderma asperellum and Penicillium simplicissimum, revealing the nature of fungi as dye degraders (Chen and Ting 2015a, b; Marcharchand et al. 2017). Therefore, we further compared the decolorization rate of endophyticB. adusta SWUSI4 with other strains reported previously. For example, Marcharchand and Ting (2017) have reported that CV, MV, MG and CB (100 mg/L) was decolorized up to 11, 67, 76 and 57 % respectively, by Trichoderma asperellum within 336 h (14 days). Another non-white rot fungi Penicillium simplicissimum showed 76, 79, 54 and 64 % decolorization of CV, MV, MG and CB (100 mg/L) within 336 h (14 days), respectively. However, endophytic fungus SWUSI4 demonstrated strong decolorization efficiency for CV, MV, MG and CB (100 mg/L) within 14 days were 72 %, 81 %, 91 % and 70 %, respectively. By contrast, endophytic strain SWUSI4 decolorization efficiency to CV, MV, MG and CB is greater than above mentioned control strains.
In general, decolorization efficiency rose with the increase of biomass dosage at a certain dye concentration. However, in the present study, when biomass ranged from 6 to 8 g, DE value was significantly down to 84 %~74 % (CV), 92%~83 % (MV), 96 %~82 % (MG) and 89 %~77 % (CB) respectively, (Fig. 2A). This result showed that the higher biomass (6 or 8 g) suppressed decolorization efficiency, the reason of which was caused by the dye initial concentration in the culture medium rather than the dosage of inoculum (Chen et al. 2015a, b). Hence, the results obtained from this investigation demonstrated that biomass of endophytic fungus not only can be decolorized TPM dyes but also suitable biomass could be efficiently employed as a low-cost and eco-friendly biosorbent for TPM dye removal.
3.3.2 Influence of initial dye concentrations on decolorization
Effect of initial dye concentration on the decolorization ability of SWUSI4 was studied by adding the fungal biomass (2 g) to CV, MV, MG and CB solution (100 mL) with different initial concentrations (50, 100, 150, 200 and 250 mg/L, respectively). Decolorization efficiency (DE) was calculated after 14 day at 30 ℃. For four tested TPM dyes, it was evident that the DE declined when initial dye concentration increased. At the dye concentration of 50 mg/L, SWUSI4 allowed 85, 90, 94 and 80 % DE of CV, MV, MG and CB, respectively (Fig. 2B). Whereas, when the initial dye concentration was 250 mg/L, DE reached 34 %, 37 %, 67 % and 35 % for CV, MV, MG and CB, respectively (Fig. 2B). Similar results have also been reported by Lin et al. (2010), who found that the decolorization efficiency of Mucoromycotina sp. declined with increasing initial dye concentrations. The implications of high initial dye concentrations agreed to most investigations (Chen and Ting 2015a, b; Wang et al. 2017; Almeida and Corso 2018), thus suggesting as the toxicity of dye could be more pronounced at higher dye concentrations which may suppress the microbial growth. In order to improve the decolorization ability, 50 mg/L TPM dyes solution was chosen as the optimum dye concentration.
3.3.3 Effect of shaking andstationary conditions
To determine the effects of static and shaking conditions on decolorization, thus this effect was studied under two different conditions (0 rpm and 150 rpm, respectively), by treating 100 ml of dye solutions with fungal biomass 2.0 g, initial dye concentrations 100 mg/L as with other experimental conditions remained constant. Comparatively, SWUSI4 in shaking condition were more effective in decolorizing TPM dyes than static condition (Fig. 2C). Among them, the DE on CV, MV and CB were significantly higher in shaking condition with means 72 %, 81 % and 70 % as compared to 27 %, 47 % and 58 % in static condition, respectively (Fig. 2C). Nevertheless, the DE of MG dye under shaking condition is similar to static condition (90% vs. 91%) (Fig. 2C). According to the authors, the higher decolourization under shaking condition than a static condition is primarily dependent on the oxidative reactions by key enzymes such as LiP and Mnp (Shedbalkar et al. 2008; Zhuo et al. 2011). Besides, it has been reported that the shaking condition was better for faster and complete adsorption and decolourization of MV and CV by Coriolopsis sp., as well as CB by Penicillium simplicissimum KP713758 or Coriolopsis sp. as compared to static conditions (Chen and Ting 2015a, b). However, in another case, the process of decolorization does not require oxygen and most possibly involved reductive reactions by a different set of reductases. We assumed that the discrepancy between static and shaking incubation conditions decolorization seemed to be related to fungal species. All in all, our results demonstrated that shaking incubation conditions could be efficiently decolorization.
3.4 Analysis of decolorizing mechanism
3.4.1 Biomass absorption or adsorption and enzymolysis contributions of B. adusta on the removal of TPM dyes
To evaluate the effect of biodegradation and biosorption, the decolorization of TPM dyes was performed separately by live cells and dead cells under optimized conditions. As shown in Fig. 3, TPM dyes solution (50 mg/L) was mixed with fungal biomass (4 g) under shaking (150 rpm) conditions at 30 ℃ for 7 day, the decolorization efficiency of live cells for MG, MV, CB and CV were 97 %, 94 %, 94 % and 92 %, respectively. By contrast, dead cells showed decolorization capacities of 72 %, 71 %, 64 % and 53 % for MG, MV, CB and CV, respectively. Furthermore, the decolorization process by live cells of B. adusta SWUSI4 was rapid compared to dead cells. Live cells achieved DE for CV, MV, MG and CB allowed rapid decolorization within 24 h (91 %, 94 %, 96 % and 93 %, respectively). By contrast, dead cells achieved DE for CV, MV, MG and CB allowed 45 %, 63 %, 68 % and 55%, respectively, decolorization within 24 h (Fig. S3). In generally, the higher DE by live cells as opposed to dead cells has been reported in other studies as well Ting et al. (2016); Przystas et al. (2018); Chen et al. (2019). This has been primarily attributed to the biodegradation of live cells because they can produce the lignin enzymes, such as MnP, LiP and Lac (Srinivasan and Viraraghavan 2010; Marcharchand and Ting 2017; Munck et al. 2018).
It is well known that decolorization occurred by adsorption of the fungal mycelium firstly, and then followed by enzymes of the live cells (Parshetti et al. 2011). However, the decolorization merely depended on the absorption once the mycelia were dead (Wang et al. 2017). On the other hand, according to Casas et al. (2009) described, the occurrence of biosorption can be concluded by the dye-colored fungal cells after decolorization. In our study, after the decolorization the color of dead cells was same as corresponding TPM dyes, respectively, while the dye solution turned lighter after decolorization by live cells (Fig. S4). This result indicated that live cells may to degrade dyes to a certain extent by enzymes. Thus, absorption presumably played a major role in the decolorization as well as degradation also had a certain role when using live cells.
3.4.2 UV-Vis Analysis
As shown in Fig. 4, for both treatments with live and dead cells of B. adusta SWUSI4 for 7 days, these absorbance peaks were obviously observed to reduce or disappear after decolorization. According to the reports (Ting et al. 2016; Ortiz-Monsalve et al. 2019; Munck et al. 2018), the disappearance or reduction of peaks in dyes can be attributed to the enzymatic biodegradation or biosorption of biomass. In our results, the complete dissolution of maximum absorption peaks were clearly observed for CB and MG treated with live cells of B. adusta (Fig. 4a, c). For CV and MV treated with live cells, the corresponding maximum absorption peak dramatically decreased in intensity after application with SWUSI4 (Fig. 4b, d). For CV, MV, MG and CB treated with dead cells of B. adusta, the maximum absorption peaks remained detectable after 7 days (Fig. 4e, f, g and h). Furthermore, the decrease degree of the maximum absorption peaks seems to be proportional to the decolourization percentage of their corresponding dyes detected, especially from 0-1 day irrespective of whether live or dead cells of B. adusta SWUSI4 were used. Previously, it has been reported that a decrease in absorbance peaks and appearances of a new peak reflects the removal of dye via biodegradation leading to biodecolourisation, while dead cells destroyed the absorption peak via biosorption (Asad et al. 2007; Chen and Ting 2015). Association with decolorization percentage and UV-vis analysis, it can be stated that the decolorization of four tested TPM dyes carried out by live cells of isolate SWUSI4 were related to both biodegradation and biosorption, while dead cells only via biosorption.
3.4.3 FTIR analyses
In the present study, the FTIR analysis revealed there were no significant differences in the number of functional groups present on the cell wall of live cells and dead cells forms of SWUSI4. These primary functional groups include hydroxyl, amino, phosphoryl alkane, and ester-lipids groups (Table 1 and 2). For live cells, the treatments with CV, MV, MG and CB caused peaks to shift at 3394 cm−1 (representing O-H and N-H groups), 2368 cm−1 (C=C stretching of ester), 1654 cm−1 (C=O stretching and N-H deformation of amide I band), 1076 cm−1 (denoting C-C, C=C, C-O-C and C-O-P groups of polysaccharides) and 528 cm−1 (nitro compounds and disulphide groups) (Table 1). Interactions between phosphoryl group and TPM dyes may have occurred according to the shift of a peak at 1076 cm−1 and disappearance of a peak at 1149 cm−1. Furthermore, another peak at 1251 cm−1 (also denoting the phosphate group) was masked after CV treatment and shifted to 1261 cm−1 after MG treatment although no changes in this peak was not observed for CB and MV treated live cells. In additional, new peak at 1741 cm−1 were detected (C=O group) in all dye-treated live cells. On the contrary, the involvement of C-H stretching virations at 2926 and 2339 cm−1, and amide III group at 1456 cm−1 in dye adsorption were not prominent (Table 1). On the other hand, dye-treated dead cells displayed similar changes in vibrational frequencies as for live cells, though with was more new peaks appearing (Table 2). The peaks at 3415, 2368, 1404 and 1033 cm−1 shifted after exposure to the four dyes. In addition, a weak shift from 2926 to 2924 cm−1 (C-H asymmetric stretching) was observed for CV, MV and MG treated dead cells. All four dyes caused the disappearance of two peaks at 1327 and 775 cm−1, and the emergence of a new peak at 1741 cm−1(C=O stretching of ester). Further, new peak at 2857 cm−1 were detected (C-H stretching) in CB-treated dead cells, whereas peaks at 927 cm−1 masked in all-treated dead cells but MG. The MV and MG treatments result in the emergence of a new peak at 1342 cm−1 (Amide III), and shifting of existing peaks at 1456 and 1404 cm−1(C-N stretching) (Table 2). Differently, the CV and CB treatments led to the emergence of a new peak at 1344 cm−1, at 1456 cm−1 in dye adsorption were not prominent and an existing peak at 1404 cm−1 shifted to1415 cm−1.
The changes in vibrational frequencies recorded via FTIR analyses on the chemical surface composition of dye-treated B. adusta confirmed the involvement of biosorption in dye removal. For example, Yang et al. (2011) showed that the biosorption of Acid Blue 25 by dead (autoclaved) Penicillium YW 01 involved amine, amide and carboxyl groups. Another fungus Aspergillus fumigatus, removal of Acid Violet 49 by dead (through freezing) was attributed to amino, carboxyl, phosphate, and sulfonyl groups (Chaudhry et al. 2014). Similarly, Chen et al. (2019) also reported that removal of TPM dyes by live cells and dead cells (autoclaving) of Penicillium simplicissimum involved amino, hydroxyl, phosphoryl, and nitro groups etc. This is evidence that dead cells (autoclaving) did not have severe implications to the functional groups on the cell wall, as most of the major functional groups were detected. They may also be typical functional groups on cell walls of a variety of fungi (Marcharchand and Ting 2017; Chew and Ting 2016). Additionally, the number of functional groups does not necessary correlate to their potential in dye removal due to the number of functional groups is almost the same present on the cell wall of live cells and dead cells forms of SWUSI4. This further explained that the higher decolorization efficiency of live cells may be attributed to the role of enzymes secreted. However, positively-charged and negatively-charged groups are important to attract both basic and acidic dyes through electrostatic attraction which is the basis of the biosorption mechanism of dye removal (Marcharchand and Ting 2017). Therefore, the involvement of chemical groups of SWUSI4 in the removal of TPM dyes substantiated biosorption as part of the dye removal mechanisms.
3.5 Enzymatic activities
In order to get additional insight into the enzymolysis contributions of SWUSI4 on the removal of dye, the enzyme activities of Lac, MnP and LiP were monitored after 24 hours in the treatment and control groups. As shown in Table 3, our results demonstrated that SWUSI4 produced more LiP and MnP as their levels were significantly induced compared to controls in the presence of TPM dyes. Differed from LiP and MnP, Lac activities demonstrated to be significantly lower than in the control. The levels of MnP and Lip were significantly higher in all TPM dyes compared to the level of Lac levels, indicating SWUSI4 may rely more on LiP and MnP oxidase for biodegradation. Additionally, MnP and LiP activity in MG was higher than that in MV, indicating that the relative contribution of each enzyme to dye decolorization is different for SWUSI4.
Generally, the relative contributions of LiP, MnP and Lac to the decolorization of dyes may be different for each fungus (Srinivasan and Viraraghavan 2010; Wang et al. 2017). Previously, Phanerochaete chrysosporium have been reported as Lip producers able to decolorize CV (Bumpus and Brock 1988), Irpex lacteus as Mnp producers able to decolorize MG (Yang et al. 2016; Duan et al. 2018) and Pleurotus ostreatus as Lac producers able to decolorize MG and CV (Morales-Álvarez et al. 2018). Besides fungi, contribution of these enzymes in biodegradation process highly depends on type of dyes (Al Farraj et al. 2019). For instance, Chen and Ting (2019) revealed that enzymes the activities of manganese peroxidase were significantly enhanced activities in response to MG, whereas only tyrosinase activities were higher when inoculated into MV and CB. Similarly, Penicillium simplicissimum (isolate 10) has been showed as higher levels of LiP was detected in cultures supplemented with MV and CV (Chen and Ting 2015b). Therefore, according to the above mentioned this study was conducted to detect SWUSI4 may have a similar enzyme to degraded TPM dyes. For this reason, we studied the enzymes produced by endophytic isolate SWUSI4. As a whole, the strain SWUSI4 showed that the activity of MnP and Lip are higher than Lac in this study. As such, MnP presumably played an important role in the decolorization as well as Lip also had a certain role and therefore can regulate biodegradation of TPM dyes to some extent.
3.6 Phytotoxicity test
According to Parshetti (2010) reported that plants belong to the group of sensitive indicators of remediation Therefore, two common crops Vigna radiata and Zea mays were tested for toxicity in this study. The germination rate, shoot and root length of germinated seeds were observed and presented in Table 4. The seeds germination rate in Vigna radiata was not affected by CB (100 %), but was inhabited by CV, MV and MG (85, 90 and 80 %, respectively). Similarly, the inhibition of CV, MV, MG and CB on germination of Zea mays were 70, 80, 60 and 90 % respectively, compared with sterile distilled water (Table 4). Meanwhile, seed germination of Vigna radiata was enhanced and improved by 100 % for all tested four TPM dyes compared to untreated dye sample. Similarly, seed germination of Zea mays was also increased by 93, 95, 90 and 100 % for CV, MV, MG and CB of isolate SWUSI4 treatment, respectively. On the other hand, compared to untreated TPM dyes, the enhanced and significant growth of the shoot and root of Vigna radiata and Zea mays suggests reduced toxicity after treatment (p < 0.05) (Table 4). In additional, in case of plant, Vigna radiata was less affected than Zea mays, suggesting that Zea mays might have higher sensitivity towards dye toxicity compared to Vigna radiata. As a whole, the result suggests that the all four tested TPM dyes were toxic to both plants, while the metabolites formed after treatments were less toxic or nontoxic, which signifies the detoxification of TPM dyes by B. adusta SWUSI4 (Chen et al. 2019).