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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Synthetic dyes biodegradation by fungal ligninolytic enzymes: Processoptimization, metabolites evaluation and toxicity assessment

Jazel Doménica Sosa-Martíneza, Nagamani Balagurusamyb, Julio Montañeza,Rosely Aparecida Peraltac, Regina de Fátima Peralta Muniz Moreirad, Adelar Brachte,Rosane Marina Peraltae, Lourdes Morales-Oyervidesa,*a Department of Chemical Engineering, Faculty of Chemical Sciences, Autonomous University of Coahuila, Boulevard Venustiano Carranza SN, Saltillo, Coahuila, 25280,Mexicob Bioremediation Laboratory, Faculty of Biological Sciences, Autonomous University of Coahuila, LibramientoTorreón-Matamoros, Torreón, Coahuila, 27000, Mexicoc Department of Chemistry, Federal University of Santa Catarina, BrazildDepartment of Chemical Engineering, Federal University of Santa Catarina, Brazile Department of Biochemistry, State University of Maringá, Avenue Colombo 5790, Maringá, Paraná, 87020, Brazil

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O

Editor: R Teresa

Keywords:BiodecolorizationSynthetic dyeLigninolytic enzymesOptimization

A B S T R A C T

This work aimed to provide information that contributes to establishing environmental-friendly methods forsynthetic dyes’ degradation. The potential decolorization capacity of the crude enzymatic extract produced byPhanerochaete chrysosporium CDBB 686 using corncob as a substrate was evaluated on seven different dyes.Critical variables affecting the in-vitro decolorization process were further evaluated and results were comparedwith an in-vivo decolorization system. Decolorization with enzymatic extracts presented advantages over the in-vivo system (higher or similar decolorization within a shorter period). Under improved in-vitro process condi-tions, the dyes with higher decolorization were: Congo red (41.84 %), Poly R-478 (56.86 %), Methyl green(69.79 %). Attempts were made to confirm the transformation of the dyes after the in-vitro process as well as toestablish a molecular basis for interpreting changes in toxicity along with the degradation process. In-vitro de-gradation products of Methyl green presented a toxicity reduction compared with the original dye; however,increased toxicity was found for Congo red degradation products when compared with the original dyes. Thus,for future applications, it is crucial to evaluate the mechanisms of biodegradation of each target synthetic dye aswell as the toxicity of the products obtained after enzymatic oxidation.

https://doi.org/10.1016/j.jhazmat.2020.123254Received 27 March 2020; Received in revised form 3 June 2020; Accepted 17 June 2020

⁎ Corresponding author.E-mail address: lourdesmorales@uadec.edu.mx (L. Morales-Oyervides).

Journal of Hazardous Materials 400 (2020) 123254

Available online 20 June 20200304-3894/ © 2020 Elsevier B.V. All rights reserved.

T

1. Introduction

Synthetic dyes are substances considered recalcitrant xenobiotics(Enayatizamir et al., 2011). They are used in many industries (food andbeverages, paper, plastics, cosmetics) (Przystaś et al., 2018), but it is thetextile industry that mostly uses these compounds for dyeing yarns andfabrics (65 % of all dyes industrially used). Studies have indicated anindustrial use of dyes from 10 to 20 million tons, of which about15–20% goes to wastewater (Sarkar et al., 2017). The treatment ofthese sludges is difficult and it requires a high demand for oxygen.Furthermore, the sludges contain suspended solids and toxic com-pounds and their color makes them even more challenging (Deswalet al., 2012). Physical and chemical methods are the most widely used;however, they present some disadvantages such as elevated costs, theydemand high amounts of energy and the use of large quantities ofchemical products (Oliveira et al., 2020). Additionally, in order to in-crease the depth of shade, color strength and the fastness properties ofthe fabric, the dyes are modified with acid and alkaline groups, metalions, among others, to protect the chromophore group and make thegarment resist to factors such as detergents, abrasion, light, andchlorine. Such procedures make it more challenging to eliminate thecolor from the sludge (Przystaś et al., 2018). Furthermore, this type ofwater-intensive industry consumes a large volume of water in its pro-cesses, what generates colored and toxic industrial effluents that causedamage to the environment and human health (Rodríguez-Couto,2009). Thus, it is crucial to eliminate the harmful compounds beforedischarging the wastewater into the natural environment.

Biodegradation has emerged as an alternative for the treatment ofthe textile industry effluents (Unuofin, 2020; Merino-Restrepo et al.,2020). Treatment with white-rot fungi, for example, can achieve mi-neralization of the contaminated vessels thanks to the ligninolytic en-zymes of these microorganisms (Mostafa et al., 2019). One of the ad-vantages of these enzymes is that they have a high redox potential and abroad substrate specificity (Kuhar et al., 2015). There has been mucheffort in evaluating biodegradation, and consequently decolorization, ofvarious industrial dyes (Manai et al., 2016; Tychanowicz et al., 2004;Zeng et al., 2015). However, most of these studies have been carried outusing the cultivation system itself. This method may cause furtherproblems such as the accumulation of biomass, long adaptation time ofthe microorganism, or difficulties in establishing the optimum condi-tions for degradation (Murugesan et al., 2007). Therefore, the appli-cation of isolated enzymes or enzyme crude extracts could represent abetter alternative (Murugesan et al., 2007). The potential advantages ofenzymatic treatments are their application to ample amounts of dyesover wide pH ranges and the perspective of saving energy and materials(Hussain and Wahab, 2018). The task of optimizing a given biode-gradation process runs in parallel with an understanding of its under-lying mechanisms. The latter implies identifying the critical variablesaffecting the whole process and also the interaction between them-selves. Equally important is to identify the possible degradation pro-ducts and their toxicity in comparison with the original compounds(Dauda and Erkurt, 2020).

The present work was, thus, planned following these guiding prin-ciples. In specific terms, the aim was to evaluate the decolorizationcapacity of the ligninolytic enzymes produced by Phanerochaete chry-sosporium CDBB 686, to evaluate the critical factors affecting the de-colorization process and further attempts at identifying the degradationproducts and evaluating their toxicity potential were made. P. chry-sosporium was selected due to its capacity to produce extracellular en-zymes able to degrade a wide range of xenobiotics (Zeng et al., 2015; Liet al., 2015; Bosco et al., 2016; Yu et al., 2006). These enzymes are theperoxidases: lignin peroxidase (LiP; EC 1.11.1.14) and manganeseperoxidase (MnP; EC 1.11.1.13), and one the multicopper oxidase lac-case (Lac; EC 1.10.3.2) (Kuhar et al., 2015). Seven dyes of variouschemical structure categories were tested in order to get a broad view ofthe capabilities of the P. chrysosporium enzymes in degrading colored

contaminants. The study hopes to provide information that contributesto establishing environmental-friendly methods for the degradation ofpolluting agents.

2. Materials and methods

2.1. Microorganism and inoculum preparation

Phanerochaete chrysosporium CDBB 686 acquired from MicrobialType Culture Collection of CINVESTAV (Mexico City, Mexico) was usedfor the production of ligninolytic enzymes. The strain was maintainedat −20 °C in a cryoprotectant solution (glycerol-skimmed milk). Forinoculum preparation, the microorganism was inoculated in 250mLErlenmeyer flasks containing 50mL of potato-dextrose-agar (PDA)medium and incubated at 30 °C for 5 days. After this period, the sporeswere scrapped down from the PDA with sterilized water and counted ina Neubauer chamber.

2.2. Enzymes production

Enzyme production was carried out via solid-state fermentationusing corncob as a carbon source. The corncob was moistened with amineral medium previously reported to reach a humidity of 60 % andinoculated with spores at 1× 106 spores/mL (Tien and Kirk, 1988). Thefermentation was carried out in a 500mL packed-bed column bior-eactor for eight days. The column was connected to an air humidifier tosupply air (relative humidity of 95–100 %) at the bottom of the columnand to maintain the desired moisture content. The column was filledwith corncob at a packed density of 0.16 g/mL. The inlet and outlet airtemperature were continuously monitored, and the room temperaturewas adjusted to keep the culture temperature around 30 °C. After theend of cultivation, extraction was carried out in a glass beaker using4mL of distilled water per gram of fermented residue. The mixture wasstirred at 200 rpm for 60min and then filtered through 0.50 μm filterpapers. The filtered liquid was centrifuged at 3000 g for 30min to ob-tain the crude enzyme extract. The supernatant fluid was then lyophi-lized and used for ligninolytic enzymes activity analyses and syntheticdyes decolorization assays.

2.3. Enzymatic assays

LiP, MnP, and Lac activities were measured as described elsewhere(Tien and Kirk, 1988; Niku-Paavola et al., 1990; Kuwahara et al., 1984).One unit (U) of LiP activity was defined as the amount of the enzymerequired to produce 1mM veratryl aldehyde from the oxidation ofveratryl alcohol per minute. One unit (U) of MnP was defined as 1 μM2,6-dimethoxyphenol (2,6-DMP) oxidized per minute. One unit (U) oflaccase was defined as 1 μM ABTS (2,2-azinobi [ethyl-benzo-thiazolinesulfononate]) oxidized per minute.

2.4. In-vitro dye decolorization experiments

The potential decolorization capacity of the crude enzymatic ex-tracts (0.2 g/mL) was evaluated on 7 different dyes, one anthracenederivative, Remazol brilliant blue R (RBBR, reactive blue 19); threetriphenylmethane dyes, Ethyl violet (basic violet 4), Methyl violet, andMethyl green; one heterocyclic dye, Methylene blue; one polymericdye, Poly R-478; and one azo dye, Congo red. Reaction mixtures used toassay enzymatic activity were utilized to measure the decolorizationcapacity by replacing the substrate utilized in each reaction mixture bythe evaluated dyes at a concentration of 50 ppm. A reaction mixture inwhich the enzymatic extract was replaced by distilled water was used ascontrol (0.2 g/mL). The samples were incubated at 30 ℃. The capacityof decolorization was followed spectrophotometrically (UV-1601 PCShimadzu spectrophotometer) and expressed as a percentage.

The dyes with higher degradation were chosen and the effect of

J.D. Sosa-Martínez, et al. Journal of Hazardous Materials 400 (2020) 123254

2

critical variables on the degradation process was further studied.Central Composite Design (CCD) with three factors at three levels wasapplied to evaluate the effect of temperature (35, 40, 45 °C), time (12,24, 36 h), and H2O2 concentration (0.5,1.0, 1.5 mM) on dye degrada-tion. Assigned codes for minimum, intermediate, and maximum levelswere -1, 0, and 1, respectively. Two other levels, known as star points(± α), were considered. The α value was calculated as 2n/4, where nwas the number of factors.

2.5. In-vivo decolorization experiments in SSF

In-vivo decolorization experiments were carried out using P. chry-sosporium in solid culture. The same dyes tested for the in-vitro studieswere evaluated for the in-vivo decolorization experiments. Preliminarystudies were carried out in Petri dishes with PDA medium and 100 ppmof dye to select the colorants for evaluation in solid state culture usingcorncob. A mycelial plug (diameter 10mm) was placed in the center ofthe dish as an inoculum. Fungal growth in the presence of each dye wasqualitatively monitored for 8 days. In-vivo studies using corncob werecarried out in Erlenmeyer flasks (250mL) using a higher dye con-centration than in the in-vitro studies. Previously, it was observed thatthe corncob absorbed the dye at the concentration used for the in-vitroexperiments (Data not shown). Thus, the dyes were mixed with 5 g ofcorncob to obtain a final concentration of 200 ppm. The humidity of themedium was maintained at 70 % with the addition of the mineralmedium of Tien and Kirk (Kirk and Tien, 1984). The media were ster-ilized (15 psi, 121 ,℃ 15min). Three mycelial plugs (diameter 10mm)from an 8-day grown fungus on PDA, were used as inoculum. Two se-quential extractions were made to quantify the degraded dye, one withdistilled water and another one with a mixture of ethanol and acetone(1:1). The dye was quantified spectrophotometrically, and the total dyequantity was taken as the sum of the two extractions. LiP, MnP and Lacactivities were quantified in all in-vivo assays. A control (corncob+microorganism) was used to compare the enzymatic activities with andwithout the dye. An abiotic control (corncob+dye only) was used toestimate the decolorization percentage by the microorganism. The dyeextracted from the abiotic control was considered as 100 %.

2.6. Degradation products evaluation

Structure and presence of functional groups in selected colorantsand in their degradation products were assessed by Fourier TransformInfrared Spectroscopy (FTIR). Prior to analysis, samples from selectedcolorants (in-vitro and in-vivo) were treated and lyophilized as describedby Rodrigues et al. (Mota et al., 2015). Pellets composed of 200mg ofspectroscopic grade KBr plus 1mg of each lyophilized sample wereprepared at a pressure of about 1MPa. The spectra, in the range be-tween 400 and 4000 cm−1, were obtained in triplicates and had a re-solution of 2 cm−1. Mass spectrometry analysis was performed in po-sitive ion mode. Electrospray ionization was done by means of anAmazon-Ion Trap MS. In the apparatus, the following optimized para-meters were set: capillary voltage between −400 and −500 V andcapillary temperature between 150 and 200 °C; nebulizer gas flow was180 μL min−1 with a nebulizer pressure of 1 bar. The mobile phase was10 nM CH3CN. Trap Control 7.0 (Bruker) and Data Analysis 4.0(Bruker) software were used (Iark et al., 2019).

The acute toxicity test of dyes before and after decolorizing treat-ments was performed according to a methodology that uses the biolu-minescent bacteria Vibrio fischeri in a Microtox® Analyzer equipmentmodel M500 (ISO 11348-3, 2007). The lyophilized light-emitting bac-terial reagent was purchased from UmWelt, Biolux kit, and validated byZnSO4.7H2O with EC50= 4.73 ppm (between 3−10 ppm). The solutionsamples were adjusted to pH 7.0 ± 0.5 using 0.1 M H2SO4 or 0.1 MNaOH solutions (pHmeter Quimis model 400A). Vibrio fischeri was ex-posed to the solution samples for 30min. Toxicity was expressed as thebioluminescence inhibition rate (%).

2.7. Data analysis

The experimental results from CCD studies were analyzed using asecond-order polynomial model:

∑ ∑ ∑ ∑= + + += = = = +

R β β F β F β F Foi

n

i ii

n

ii ii

n

j i

n

ij i j1 1

2

1 1 (1)

where R is the target response (% decolorization); Fi represents thecoded levels of the factors; βo, βi, βii, and βij represent the intercept,linear, quadratic, and interactive effects, respectively. Coefficients ofthe polynomial model for each dye were obtained by minimizing thesum of squares of residuals (SSR) between the experimental data andthe values predicted by the model (Eq. 1). All the model fittings wereperformed using Microsoft Excel (Microsoft Excel version 2010,Microsoft Corporation). Results were confirmed with the softwareStatistica 8.0 (StatSoft, Tulsa, OK, USA) and the statistical analyseswere performed with this software as well. Optimum conditions wereobtained using Solver Excel function for maximizing the response (Eq.1) by modifying the levels of the factors. Response surface graphs wereused to avoid sub-optimal local regions.

3. Results and discussion

3.1. In- vitro decolorization

The enzymatic activity (U/L) of the crude enzymatic extracts using0.2 g/mL of lyophilized powder was 8.60 ± 0.72 for LiP, 5.87 ± 0.58for MnP and 1.57 ± 0.61 for laccase. Screening in-vitro decolorizationexperiments can be viewed in the supplementary material. The purposewas to evaluate the time dependences of all seven colorants degradationto select the level ranges for the evaluation of the process conditions.The process was monitored at 1 h intervals during the first 6 h.Although an apparent equilibrium seemed to have been reached at thistime, further prolongation of the incubation and analysis after 24 hrevealed variable increments in the decolorization percentage.

The various dyes were decolorized to different extents by each ofthe three enzymes, and the percentages of decolorization for most ofthem did not vary to a great extent irrespective of the enzyme used. Forexample, Methylene blue was quite poorly transformed by all threeenzymes, whereas Congo red was invariably well transformed by allthree enzymes. The transformation of Congo red and Methyl greenpresents a few interesting features. For the former, the decolorizationpercentage after six hours was clearly more pronounced when MnP wasused. For Methyl green, the decolorization percentage was also higherwhen MnP was used, but the most pronounced decolorization after sixhours was produced by LiP.

The percentages of decolorization of various dyes, using P. chry-sosporium crude extracts, that were reported in the literature as well asthe corresponding time dependences, present considerable variations. Apercentage of decolorization close to 90 % has been reported for Indigocarmine (30 ppm) after 6 h. However, in the culture, the activity of MnPwas induced, so that the extract presented high concentrations of thisenzyme, which could have caused a more efficient decolorization (Liet al., 2015). In another study, 56 % degradation of Congo red (50 ppm)was found after 30 h, a percentage that was reached with most of thedifferent mixtures of extracellular fluids that were investigated (Boscoet al., 2016). Yehia and Rodríguez-Couto reported 95 % degradation ofCongo red by a purified extract containing MnP mainly produced byPleurotus sajor caju after a reaction time of 4 h (Yehia and Rodriguez-Couto, 2017).

The percentages of oxidation of the dyes, as reported, are high whenthe ligninolytic activity is induced during the growth of the fungus.Common procedures are the induction of MnP by adding phenoliccompounds and Mn+2 to the medium and the increase in the activity ofLiP by the addition of veratryl alcohol. An increase in decolorization is

J.D. Sosa-Martínez, et al. Journal of Hazardous Materials 400 (2020) 123254

3

also observed when the crude extract is partially or completely purified.Thus, the time for reaching maximal decolorization can vary sig-nificantly depending on the extract’s nature (crude or purified), culti-vation conditions, microorganism, dye, degradation conditions, amongothers.

In any case, in the present work, the maximal decolorization per-centage reached was in the range of 10–30 %, which corresponded to5–15 ppm of dye removal. On average, for the three enzymatic pre-parations tested (LiP, MnP, and Lac), the lowest decolorization wasobtained for Methylene blue (11 %). Among the enzymatic prepara-tions, Lac was, on average, the least efficient for all colorants, whatcould be attributed to the low enzymatic activity (1.57 ± 0.61 U/L).Based on these results, thus, the colorants Methylene blue and Methylviolet and the enzymatic preparation Lac were not included in thesubsequent study.

3.2. Effect of process conditions for in-vitro decolorization

The evaluated factors, levels and assigned codes were presented insection 2.5. For temperature, the selected intermediate setting was thetemperature used for the screening studies (40 °C); maximum,minimum, and star point levels were chosen in order to avoid a tem-perature at which enzymes are degraded (< 50 °C). For the H2O2 con-centration, the intermediate level was the one used for the screeningstudy; minimum and maximum levels were selected in order to avoidnegative levels for the starting point. Respecting time, the intermediatelevel was the longest time evaluated in the screening studies since it ispossible that the decolorization process had not reached equilibrium.For time, the maximum and minimum levels were also defined to avoidnegative values for the start point.

Results for the experimental matrix runs with every colorant forenzymes LiP, and MnP are presented in Table 1. It can be seen thatdecolorization levels (%) ranged significantly from 2.2–49.51 for LiPand from 0.11–47.99 for MnP among the dyes. Table 2 summarizes thepolynomial model's coefficients, the goodness of fit, the optimum levels,the predicted response at optimum levels, and the observed responseduring the validation studies. Significant factor's effects (p < 0.05) arealso indicated in Table 2.

Overall, the polynomial model presented good to moderate corre-lations between predicted and experimental data (R2 = 0.46 – 0.90).Interestingly, the effects and type of effect of the evaluated factors weredifferent for each colorant and for LiP and MnP. The linear effect oftime was the only one that was significant for the five colorants for bothenzymes (MnP and LiP). However, the quadratic effect of time was alsosignificant except for Methyl green with both LiP and MnP and Congored with MnP. As for temperature, this factor was significant(p < 0.05) within all the colorants with the exception of Ethyl violetwith LiP. The quadratic effect of the temperature was higher than thelinear effect for all colorants but Congo red, where the quadratic effectwas negligible. The factor with the least effect was the concentration ofH2O2. It was not significant for Ethyl violet (LiP and MnP) and for PolyR-478 (MnP), and it presented a quadratic effect rather than a linearone for the colorants where it was significant. Surprisingly, almost nointeractive effects were significant. Just the interaction between tem-perature and time for Poly R-478 (LiP) and Congo red (MnP), the in-teraction between the concentration of H2O2 and time and temperaturefor Methyl green were significant. Moreover, the significant interactiveeffects were lower than the rest of the effects of the dyes in which theywere present. The fact that there are no interactive effects or that theseare minimum is a favorable outcome considering that a factor can befurther studied with confidence that the outcome will refer solely tothat factor and will not be due to the selected levels of the other twofactors.

Fig. 1 shows the individual effects of temperature, time, and H2O2

on each colorant with both LiP and MnP. The profiles for every factorare calculated with Eq. 1 and the coefficients included in Table 2, while Ta

ble1

Expe

rimen

talmatrix(C

CD)an

dde

colorization

%foreach

colorant

atallruns

andstud

iedco

nditions.

Run

Factors,

leve

lsRBB

REthy

lViolet

Poly

R47

8Methy

lGreen

Con

goRed

F1F2

F3LiP

MnP

LiP

MnP

LiP

MnP

LiP

MnP

LiP

MnP

11.00

1.00

1.00

17.48±

2.46

13.08±

2.09

8.88

±1.78

6.14

±1.39

9.85

±2.53

10.27±

0.53

23.18±

1.64

29.15±

1.46

20.84±

2.81

15.95±

0.43

21.00

1.00

1.00

25.33±

0.60

23.00±

2.93

26.14±

1.37

23.28±

1.06

29.06±

2.14

27.78±

1.30

39.61±

0.87

36.41±

1.42

30.41±

1.04

26.27±

1.00

31.00

1.00

1.00

15.74±

0.78

15.38±

1.90

10.20±

0.47

5.99

±0.42

6.12

±1.74

8.34

±1.84

29.27±

1.78

24.60±

2.68

29.38±

4.50

27.60±

0.94

41.00

1.00

1.00

25.77±

0.33

21.03±

1.79

29.08±

4.62

20.87±

3.50

33.67±

1.21

20.77±

2.24

43.24±

0.80

35.79±

2.23

38.29±

3.22

26.66±

0.72

5(C

)0.00

0.00

0.00

17.06±

1.08

12.68±

2.13

15.65±

0.64

17.15±

0.92

9.59

±1.65

18.84±

11.74

20.18±

3.28

24.81±

5.22

29.22±

1.29

21.16±

1.44

61.00

1.00

1.00

17.20±

2.03

12.35±

3.11

6.63

±1.35

6.15

±1.19

6.84

±0.18

7.05

±1.17

23.00±

4.57

21.31±

2.37

18.02±

1.01

18.71±

0.97

71.00

1.00

1.00

22.54±

1.73

17.81±

2.00

23.13±

3.76

25.71±

0.80

32.30±

1.92

21.34±

1.40

31.59±

5.26

36.10±

1.59

30.45±

1.70

25.49±

1.78

81.00

1.00

1.00

17.66±

2.10

4.23

±1.70

6.71

±0.45

7.04

±1.32

4.92

±0.88

5.22

±1.16

26.39±

2.58

25.85±

9.39

27.39±

0.61

23.57±

2.52

91.00

1.00

1.00

27.00±

1.56

21.72±

0.95

27.58±

0.78

24.72±

0.79

33.83±

1.05

21.45±

1.86

48.23±

1.51

46.59±

0.95

38.36±

0.52

32.50±

1.31

10(C

)0.00

0.00

0.00

17.20±

1.91

11.88±

2.17

14.82±

1.72

17.17±

1.57

10.06±

2.02

20.61±

2.27

21.54±

0.88

23.98±

2.06

27.41±

2.05

22.65±

3.60

111.67

0.00

0.00

17.27±

5.75

21.00±

1.85

7.80

±4.99

15.89±

2.24

5.72

±1.41

21.08±

1.16

26.51±

5.10

23.55±

6.44

24.36±

1.76

16.37±

1.13

121.67

0.00

0.00

15.81±

1.56

9.53

±3.80

9.22

±0.41

15.20±

0.38

10.81±

1.72

15.22±

1.95

27.86±

2.57

29.19±

3.05

29.65±

1.10

22.59±

4.18

130.00

1.67

0.00

4.21

±2.47

0.88

±0.60

8.46

±1.43

10.08±

1.53

6.61

±1.04

2.46

±1.56

8.27

±1.66

5.47

±1.28

12.63±

1.60

16.38±

0.98

140.00

1.67

0.00

8.64

±2.53

3.30

±1.11

19.23±

5.29

19.75±

1.65

36.52±

1.58

29.30±

4.60

30.69±

0.82

29.74±

3.84

28.48±

4.30

24.96±

1.12

150.00

0.00

1.67

14.90±

1.03

11.87±

1.98

12.70±

4.90

19.81±

1.39

8.87

±0.73

21.57±

1.12

27.12±

1.00

19.04±

5.12

31.87±

4.52

18.02±

0.81

160.00

0.00

1.67

16.58±

2.66

12.44±

1.47

8.27

±4.19

19.57±

2.24

11.86±

0.89

21.71±

0.97

28.98±

2.51

29.38±

0.92

28.35±

2.92

22.59±

1.99

17(C

)0.00

0.00

0.00

14.94±

0.80

10.07±

1.93

9.61

±3.81

19.79±

2.82

10.72±

1.66

23.20±

0.00

26.48±

2.79

23.68±

3.97

27.23±

1.50

21.64±

1.48

J.D. Sosa-Martínez, et al. Journal of Hazardous Materials 400 (2020) 123254

4

the remaining factors were set at optimum levels (also shown inTable 2). The information provided in Fig. 1 is quite extensive; thus, tobring order to the discussion the following comparisons will be con-sidered: i) comparison between the discoloration levels with LiP andMnP, ii) comparison of the discoloration levels obtained for each col-orant with LiP and MnP and, iii) comparison of the effect of each factorat all conditions.

Although it is challenging to delimitate the individual effect of LiPand MnP, it can be observed in Fig. 1 that the decolorization profiles ofall colorants obtained with LiP are mostly above all the decolorizationprofiles obtained with MnP. This might be due to the pH level and theuse of crude extracts. The optimum pH usually reported for MnP isbetween 4–5 but for a purified enzyme (Zeng et al., 2015; Yehia andRodriguez-Couto, 2017). Moreover, the existence of other metal ions inthe crude extract might be interfering with Mn2+ and affecting thereaction as previously shown for the degradation of an azo dye by acrude MnP (Yu et al., 2006). If it is desired to achieve higher decolor-ization levels with MnP, evaluation of other parameters such as pH andthe mediator concentration (MnSO4) could be the first approach. As forLiP, it has been established that low pH levels are required (Manaiet al., 2016; Chowdhary et al., 2019; Rodríguez-Couto et al., 2006).

As for the comparison between colorants, the influence of the fac-tors was remarkably contrasting among the various dyes. For RBBR, theprofiles are highly similar, that is to say, the effect of the evaluatedfactors was similar regardless of whether the enzymes LiP or MnP wereused. Both LiP and MnP oxidize RBBR because it is an anthracene-de-rived dye, structurally similar to the lignin molecule (Christian et al.,2005). Optimal conditions are also almost the same for LiP and MnP(except for time, see Table 2), and the higher decolorization expectedunder such conditions for RBBR is ≈ 30 % (15 ppm removal). A similardye removal capacity was shown by two purified extracts (MnP) pro-duced by Trametes pubescens (11.2–13.4 ppm removal) under conditionscomparable to those employed in this work (50 ℃, 24 h, and H2O2

0.4 mM) (Rekik et al., 2019). The decolorization levels achieved in thiswork are higher than those reported for the decolorization of RBBR byLiP produced by Ganoderma lucidum and Pleurotus ostreatus, removingonly 2.0–2.5 ppm under different conditions (10 ppm, 25 ℃, 24 h and0.2 mM H2O2) (Oliveira et al., 2018). In that same work, authorscompared their results with immobilized LiPs produced by the samestrains, which removed 2–4 ppm and 6–8 ppm during the first andsecond use, respectively. Higher decolorization levels have also beenobtained with a purified and immobilized LiP (Shaheen et al., 2017).However, the decolorization increment is not as high as to justify theenzyme purification costs.

For Ethyl violet and Methyl green, although the profiles are notentirely similar between each other with LiP and MnP, there was atendency for a higher decolorization at the maximum factor's settings(≈ 15–35 ppm removal, respectively). Both dyes are derived from tri-phenylmethane; however, it is reported that ligninolytic extracts de-grade more easily the Methyl green dye, reaching a dye removal of32 ppm from an initial dye concentration of 50 ppm (Cardoso et al.,2018; Boer et al., 2004). Nonetheless, lower decolorization levels havealso been reported for Methyl green; a dye removal of 18 ppm wasachieved with MnP, which reacted at 40 ℃, for 24 h with H2O2 at0.4 mM (Rekik et al., 2019). In the particular case of the effect of timeon the decolorization of Methyl green, the profiles with LiP and MnPwere most assuredly the same. This makes sense given the coefficientsfor time contribution that were obtained under both conditions (β0, β2and β22), which are not statistically different from each other.

In the case of Congo red, the profiles for all factors with LiP andMnP are notably different (linear for MnP, quadratic for LiP); however,the higher decolorization is obtained at the maximum settings for allthe cases. The fact that only linear effects were obtained with MnPimplies that different levels (higher) are required to optimize furtherthe factors studied in this work. Also, different parameters, such as pHand mediator concentration, can be assessed. As for the Congo redTa

ble2

Polyno

mialmod

elco

efficien

ts(Eq.

1),g

oodn

essof

fit,op

timum

leve

lsan

dpred

ictedan

dob

served

deco

lorization

(%)at

such

leve

lsforalldy

esan

dco

nditions.

Coloran

tRBB

REthy

lViolet

Poly

R47

8Methy

lGreen

Con

goRed

Enzyme

LiP

MnP

LiP

MnP

LiP

MnP

LiP

MnP

LiP

MnP

Coe

fficien

tseq

uation

1an

dgo

odne

ssof

fit

β 015

.77±

1.35

11.07±

1.34

12.87±

1.59

18.15±

1.07

9.68

±1.07

20.71±

1.20

22.14±

1.45

23.52±

1.67

27.70±

0.92

21.48±

0.82

β 10.09

±0.63

*-1.69

±0.63

0.86

±0.74

−0.22

±0.50

0.66

±0.50

*-1.5±

0.52

*2.35±

0.68

1.42

±0.79

*3.13±

0.43

*2.52±

0.38

β 11

*1.97±

0.70

*2.77±

0.70

−0.57

±0.83

*-1.24

±0.56

0.69

±0.56

*-1.55

±0.59

*3.41±

0.76

*2.75±

0.88

0.43

±0.48

0.20

±0.43

β 2*2

.94±

0.63

*3.13±

0.63

*6.92±

0.75

*6.52±

0.50

*11.12

±0.50

*7.75±

0.52

*7.23±

0.68

*6.45±

0.84

*5.03±

0.43

*2.90±

0.38

β 22

*-1.64

±0.70

*-1.93

±0.70

*1.66±

0.83

*-1.47

±0.56

*5.44±

0.56

*-2.36

±0.59

0.66

±0.76

0.35

±0.93

*-1.87

±0.48

0.63

±0.43

β 3*0

.34±

0.63

*1.27±

0.63

−0.53

±0.75

0.24

±0.50

0.01

±0.50

0.53

±0.52

*1.39±

0.68

1.42

±0.79

−0.49

±0.43

*1.55±

0.38

β 33

*1.68±

0.70

*1.66±

0.70

0.46

±0.83

0.24

±0.56

*1.44±

0.56

−0.30

±0.59

*3.72±

0.76

*1.98±

0.88

*1.54±

0.48

0.50

±0.43

β 12

0.77

±0.83

0.97

±0.82

1.07

±0.98

−0.11

±0.65

*1.47±

0.66

−0.39

±0.67

1.35

±0.89

1.24

±1.03

−0.27

±0.56

*-1.14

±0.50

β 13

−0.4±

0.83

0.92

±0.82

−0.16

±0.98

1.06

±0.65

0.95

±0.66

0.07

±0.67

0.00

±0.89

*2.13±

1.03

0.62

±0.56

0.79

±0.50

β 23

0.78

±0.83

−0.57

±0.82

0.35

±0.98

0.41

±0.65

−0.16

±0.66

0.90

±0.67

1.29

±0.89

*2.53±

1.03

0.11

±0.56

−0.02

±0.50

R2

0.46

0.57

0.62

0.76

0.90

0.80

0.73

0.61

0.79

0.65

Optim

umleve

lsF1

,oC

48.35

48.35

45.67

38.62

48.36

40.29

48.35

48.36

48.36

48.36

F2,h

44.04

35.80

44.04

44.04

44.07

41.42

44.04

44.07

39.47

44.07

F3,mM

1.83

1.83

1.83

1.83

0.16

1.44

1.83

1.83

1.83

1.83

Dec

olor

ization,

%Pr

edicted

30.26±

10.80

27.24±

1080

32.82±

10.30

27.53±

5.65

52.45±

8.61

25.96±

3.06

69.79±

11.60

69.79±

13.43

41.84±

7.39

35.13±

6.57

Obs

erve

d21

.36±

1.97

26.41±

0.86

31.46±

1.23

27.06±

0.60

48.75±

0.56

28.91±

1.15

69.23±

1.61

74.83±

0.71

50.35±

0.52

38.93±

1.97

*Ind

icates

sign

ificant

effect(p

<0.05

);ane

gative

numbe

rindicatesane

gative

effectof

thepa

rameter.

J.D. Sosa-Martínez, et al. Journal of Hazardous Materials 400 (2020) 123254

5

degradation with LiP, at the higher studied levels of temperature andH2O2, an optimum has been reached at 39.5 h (≈ 50 %, 25 ppm re-moval). In the literature, discoloration levels of 58 % have beenreached, but with a purified MnP at 30℃, 30 h and 0.2 mM H2O2 usingthe same initial dye concentration than in this study (50 ppm) (Boscoet al., 2016).

As to Poly R-478, the profiles for the temperature and H2O2 effects

with LiP and MnP were utterly opposite. A temperature of 40 °C and1.4 mM H2O2 were the optimum conditions for MnP, while for LiP, ahigher temperature and a lower H2O2 concentration were required.Also, a much higher decolorization can be obtained with LiP (≈ 52 %,26 ppm removal) than with MnP (≈ 23 %, 11.5 ppm removal). Thisdiffers from the other colorants, for which the % difference between thedecolorization achieved with MnP or LiP was under 10 %. The dye Poly

Fig. 1. Decolorization profiles obtained for each factor and dye at LiP (white circles) and MnP (gray circles) conditions (In-vitro).

J.D. Sosa-Martínez, et al. Journal of Hazardous Materials 400 (2020) 123254

6

R-478 is representative of the class of polycyclic aromatic hydro-carbons, and it is used in qualitative assays of lignin peroxidases orquantitative assays of laccase activities (Kameshwar and Qin, 2017).Most studies regarding the application of enzymatic extracts in Poly R-478 degradation use MnP or laccase extracts (Rodríguez-Couto et al.,2006; Moreira et al., 2001). The results with MnP are to some degreehigher than the previous findings obtained in in-vitro decolorizationexperiments of Poly R-478 by an extract produced by Phanerochaetechrysosporium, where 7.5 ppm were removed (20 % decolorization)with MnP (40 ℃, pH 4.5 and 0.4 mM H2O2) (Rodríguez-Couto et al.,2006). However, in the same study, a lower temperature (30 °C) wasselected due to the thermal stability of the enzyme. According to theresults, using a lower temperature than the optimum one such as 30 °Cwould imply a 5% decrement in degradation.

In regard to the comparison of each factor's effect under all condi-tions, a quadratic contribution of temperature is observed for all col-orants except Congo red, where a linear profile is observed. Higherlevels of temperature could be studied for this dye; however, a highertemperature (> 48 °C) could have a negative impact on the enzymaticactivity of the extracts. The optimum growth temperature for mostfungi is around 25–35 °C. However, this temperature can be increased ifan enzymatic extract is used for in-vitro degradation. Temperature isone of the main parameters that affect the enzymatic degradation ofdyes. For example, the Congo red decolorization ability of a partiallypurified MnP produced by Pleurotus sajor caju evaluated at temperaturesranging from 20 to 80 °C was optimal at 35 °C with an abrupt decline attemperatures above the optimum (Yehia and Rodriguez-Couto, 2017).Similarly, the best temperatures for ligninolytic activities and dye de-colorization for most white-rot fungi were between 35 and 45 °C(Rodríguez-Couto et al., 2006; Sen et al., 2016). Stability evaluation ofan enzymatic extract produced by Phanerochaete chrysosporium con-taining LiP and MnP revealed that the half-life time of both enzymesdecreased significantly above 37 °C, the MnP stability being the mostaffected by temperature changes (Rodríguez-Couto et al., 2006). Asexpected for enzyme-based biological systems, increasing the incuba-tion temperature would lead to denaturation of enzymes, and hence toan eventual decline in dye degradation efficiency. Most textile andother dye effluents are produced at relatively high temperatures, andthis will be an important factor in the real application of enzymaticbioremediation in the future (Hussain and Wahab, 2018).

Also, the time variable presented a similar trend in most of thestudied cases; there was a linear contribution at the minimum levels,but then an equilibrium seemed to have been reached (quadratic con-tribution). Aside from Methyl green (LiP and MnP), Ethyl violet (MnP),and Congo red (MnP), where the linear profile is evident, a higherdecolorization was obtained at longer times. In the case of further at-tempts at evaluating the decolorization over a more extended period,

special care is needed with the interactive effects. This should not re-present a problem for Ethyl violet because no interactive effects werefound for this dye (Table 2). As for Methyl green and Congo red, it isadvisable to look at the surface response graphs to analyze the timeprofile obtained at different levels of the other two factors. The timetaken to achieve maximum decolorization depends on many factors,such as purification of the enzyme, dye concentration, enzyme con-centration, the concentration of cofactors and mediators of the reaction.There are studies in which the maximum time of decolorization is re-ported to be 2 h (Tychanowicz et al., 2004), whereas other studies havereported up to 72 h (Shaheen et al., 2017).

As for the effect of H2O2, the prevailing one among all the condi-tions is a positive quadratic effect, meaning also that a minimum isobtained at an intermediate setting. This trend would be under-standable if interactive effects had also been significant. It is, to someextent, illogical to obtain a higher decolorization with the minimumlevel followed by a decrease and a subsequent increase when in-crementing the concentration of H2O2. This profile may be due only tothe nature of the curvature of Eq. 1, which is a drawback of using RSM.It is required to fit data to Eq. 1, where the data are forced to be de-scribed by a parabola. This could bring problems of being misled to asuboptimal region. As an example, one can look at the surface plots forRBBR (Fig. 2). For the decolorization as a function of temperature andH2O2, it can be observed that there are three sub-optimal regions. Inthis case, the optimum selected levels (Table 2) were the maximumsettings for both factors. According to the polynomial model obtained,similar decolorization levels can be obtained at the minimum settingsfor both factors. The surface plot of time versus temperature is also anexample of finding sub-optimum local regions. In this case, a saddle-type surface is obtained, although the selected optimum levels areroughly higher than the sub-optimal region. In any case, some aspectsof the effects of H2O2 are consistent with literature reports: i) H2O2 isrequired to initiate the dye degradation reaction by LiP and MnP(Chowdhary et al., 2019); ii) ligninolytic enzymes stabilities under highH2O2 concentration are essential for their application in a real systemsuch as in the textile dye wastewater treatment (Machado and Matheus,2006); iii) the H2O2 required concentrations and enzymes stabilitieswill depend on the source and process conditions (Hoshino et al., 2002).Therefore, the reported optimum levels for H2O2 concentration varysignificantly within a range of 0.01–2mM (Yu et al., 2006; Rekik et al.,2019; Moreira et al., 2001; Machado and Matheus, 2006). Nevertheless,a concentration lower than 2mM is recommended in order to preventenzyme inactivation (Zhang et al., 2012). Thus, even though the ob-tained trend might indicate that higher decolorization can be achievedat a higher H2O2 concentration, it is not recommended to evaluatehigher concentration levels further.

Despite the differences among dyes, from the above results, some

Fig. 2. Response surface plots showing the effect of a) temperature vs H2O2 concentration, b) temperature vs time, c) time vs H2O2 concentration on RBBR.

J.D. Sosa-Martínez, et al. Journal of Hazardous Materials 400 (2020) 123254

7

similarities can be identified that are important for selecting the opti-mization method, the factors and their levels for future optimization ofthe biodegradation of a different dye. The reaction time and tempera-ture were the factors that presented a significant effect on most of thedyes. Also, almost no interactive effects were observed among theevaluated factors, which implies that a method with lesser experimentalrequirements can be selected for the biodegradation of a distinct dye.

In any optimization-model approach, a validation of the optimumsettings is strictly recommended. Table 2 also shows the predicted va-lues at the optimum factor's levels. Validation trial experiments withmore than three replicates were conducted using such settings; resultsare shown in Table 2. Mostly, the results were within the confidenceinterval predicted by the model for all cases. Only in the case of RBBR(LiP) the decolorization percentage was appreciably lower; however,the obtained value was still within the confidence interval of the ex-pected response. For the most part, the obtained equations are capableof predicting an outcome as a function of the evaluated factors, and thedecolorization percentage of all colorants was improved. However, onlythe colorants Methyl green (LiP and MnP) and Congo red (LiP) weredoubtlessly transferred to a more favorable region of the solution spaceby incrementing the decolorization levels three and four times, re-spectively.

In this sense, it was shown that at improved conditions, high de-gradation of some dyes could be obtained with fungal ligninolytic en-zymes produced using an agro-industrial waste as substrate without theaddition of cofactors that could increase the process costs. Recently, ithas been highlighted that a crucial constraint for the application ofligninolytic enzymes is the production of large volumes of the enzymesat an affordable cost and that the application of cheap and availableresidues is an alternative to mitigate the enzyme production costs(Noman et al., 2020; Vats and Mishra, 2018).

3.3. In- vivo decolorization

For the in-vivo decolorization studies, the colorant Ethyl violet wasdiscarded because the microorganism was not able to grow on the PDAagar plates. The results for the in-vivo decolorization tests using corncobas support for the SSF are shown in Fig. 3. The graphic shows the de-colorization (in %) and the enzymatic activities present with andwithout the dyes. The study of in-vivo decolorization offers new data onthe induction of the enzymes involved. It has been reported that theactivity of these ligninolytic enzymes is generally enhanced by theaddition of exogenous stimulators (Li et al., 2015), which could lead toa higher decolorization when using the same system.

In this study, the MnP activity displayed in all evaluated colorantswas lower than the control, except for RBBR. As for LiP, only RBBR andMethyl green were the dyes that clearly induced a higher LiP activitythan the control. Similar LiP activities than the control were obtained

with Methyl violet, and Methylene blue and lower LiP activities wereobtained with Poly R-478 and Congo red. An important word of cautionmust be added; it has been reported that the LiP activity could beoverestimated in the presence of MnP, and vice versa, due to theirability in acting on both reaction substrates (Kudo et al., 2017).

For RBBR, both enzymatic activities were higher than the control;thus, its degradation could be equally attributed to the action of bothperoxidase enzymes (LiP and MnP). For this colorant, the % of deco-lorization obtained in-vitro was 5% higher than the higher one expectedusing the enzymatic extract under optimized conditions; though, it wasstill within the confidence range predicted by the model.

As for the solid culture with Methyl green, only 35 % decolorizationcompared with the abiotic control was reached despite the higher LiPactivity. This means that the decolorization of Methyl green using thecultivation system was lower than that one obtained using the enzy-matic extract, considering that corncob absorbed a high amount of dye(> 100 ppm). It is possible that the enzyme failed to achieve higheroxidation of the dye due to the absence of the synthesis of peroxide-producing enzymes. It has been reported that extracellular H2O2 pro-duction significantly impacts the fungi's decolorization process on solid-state fermentation cultures (Eichlerová et al., 2006). About the color-ants with similar LiP activities as the control (Methyl violet and Me-thylene blue), a low decolorization was obtained for Methyl violet (15%), and a high decolorization with Methylene blue (50 %). The lowdecolorization with Methyl violet, despite the high LiP activity, mightbe due to inhibition of the fungus’ growth by the dye. Higher deco-lorization has been obtained using dyes such as Methyl green andMethyl violet in in-vitro systems; however, the incubation time ex-ceeded 22 days (Deswal et al., 2012; Boer et al., 2004).

Concerning the decolorization of Poly R-478 and Congo red (lowenzymatic activities), only 20 % was reached with Poly R-478 and 50 %with Congo red. In another study carried out with Poly R-478, 70 %decolorization was reached after 22 days of P. chrysosporium culturingusing nylon sponges as support, and the activity of LiP was also high(Rodríguez Couto et al., 2000). Concerning Congo red, a high deco-lorization was achieved despite the low enzymatic activities. It is pos-sible that the microorganism was able to degrade Congo red via otherintracellular mechanisms (Sun et al., 2016), or the result could besimply attributed to biomass biosorption (Bosco et al., 2016).

In summary, it was possible to determine the conditions that allowthe highest decolorization rate of almost all the evaluated colorantsusing the enzymatic extracts. On the other hand, the conditions for thehighest decolorization using the in-vivo system were obtained solely forMethylene blue. This could be indicating that the dyes inhibit thegrowth of the microorganism. Although the literature reports higherdecolorization levels when using the cultivation system, long adapta-tion times of the microorganism are required (Moreira et al., 2001;Hassan and Carr, 2018; Hong et al., 2017).

3.4. Analysis of degradation products after in-vitro decolorization

FTIR and mass spectra analyses were made in order to confirm thetransformation of the dyes Methyl green and Congo red by the enzymesas well as to establish a molecular basis for interpreting changes intoxicity along with the degradation process. These two dyes were se-lected because both of them are pollutants representing high risks to thewater resources (Iark et al., 2019; Sharma et al., 2014) and becausethey were degraded to a considerable extent in our in-vitro experiments.FTIR spectra for both dyes are shown in Fig. 4 and mass spectra beforeand after the in-vitro treatment are shown in Figs. 5 and 6 for Methylgreen and Congo red, respectively.

The FTIR spectrum of untreated Methyl green exhibited bands at3041, 2918, and 2862 cm−1, which can be attributed to C–H aromaticand aliphatic stretching, respectively (Fig. 4A). The bands around1600−1400 cm−1 can be related to the stretching of the C=C and C]N bonds, respectively (Sharma et al., 2014). Bands at 1581, 1362, and

Fig. 3. Dye decolorization during the in-vivo studies and enzymatic activitiesobtained with and without the dyes (Control).

J.D. Sosa-Martínez, et al. Journal of Hazardous Materials 400 (2020) 123254

8

1120–1223 cm↓1 have been related to the=N+ immonium ion, C–Hbending and the vibration of the heterocyclic skeleton (Sharma et al.,2014). Earlier studies have related bands at 1586 cm↓l and 1371 cm↓1

with the phenyl ring vibration and -N stretching, respectively(Margulies and Rozen, 1986). The spectrum after the in-vitro degrada-tion process (Fig. 4B) showed significant diminution of the bandsaround 1400 and 1300 cm−1, suggesting that cleavage of the=N+

immonium ion bond. Compared to pure dye (Fig. 5A), the dye's massspectrum after in-vitro treatment shows different fragmentation pat-terns, and the main m/z in the positive mode peak for the treatedMethyl green was of 365.15 (Fig. 5B). The proposed structure shows theloss of the immonium=N+ bond and also of the C]C bond betweenthe phenyl groups. This is consistent with the decrease in absorption ofthe dye after the treatment with oxidative enzymes. The presence offormic acid is due to the addition of this acid before analysis. For thepure dye, the main peak 193.65 (m/z=+2) was found.

It is crucial to evaluate the toxicity of the resulting products and toassure that these compounds are not even more toxic than the originaldyes. For Methyl green, the original dye showed a 97.66 ± 0.33 %inhibition of the bioluminescence induced by Vibrio fischeri, whatconfirms this dye's high toxicity. After the in-vitro degradation process,the inhibition was totally eliminated. These results, in particular, re-inforce the success in using the crude extract produced byPhanerochaete chrysosporium for the degradation of Methyl green underoptimized conditions.

The FTIR spectra obtained before (Fig. 4C) and after (Fig. 4D) the in-vitro treatment of Congo red showed a decrease in the peaks around1600–1400 cm−1. This is consistent with the cleavage of the N]N azobond. As observed in previous studies, the peaks below 1000 cm↓1 alsodisappeared, suggesting the cleavage of the dye molecule (Mota et al.,2015; Iark et al., 2019). Congo red is abundant in the textile and paper

industries effluents, and it has been reported to be extremely carcino-genic and toxic to both the environment and humans (Iark et al., 2019).All these facts make its degradation and detoxification highly desirable.As for Congo red, the inhibition of the bioluminescence induced byVibrio fischeri caused by the dye was 79.72 ± 0.85 %, which is con-sistent with previous studies (Iark et al., 2019). However, toxicity afterin-vitro degradation experienced a relative increment of 22 % comparedto the original dye (98.03 ± 0.71 %). This suggests that the degrada-tion products are more toxic than the original dye. An explanation ofthis undesirable result can be derived from the mass spectra before andafter the in-vitro treatment of Congo red (Fig. 6A and B, respectively).The Congo red sample's mass spectrum after the in-vitro treatmentshows a different fragmentation pattern compared to the pure dye. Themain m/z peak for the treated Congo red was 203.12. One of the pro-posed structures is an asymmetrical cleavage of the Congo red mole-cule, followed by oxidation of the fragment. This is consistent with theresults found after the degradation of Congo red by a laccase of Ga-noderma lucidum (Mota et al., 2015). As a second possibility, however,there could have been the formation of benzidine ([1,1′-biphenyl]-4,4′-diamine), a well-known hazardous aromatic amine (Choudhary, 1996).In a recent work, benzidine was found as a metabolite produced in thedegradation of Congo red by the white-rot fungus Ceriporia lacerate(Wang et al., 2017). Interestingly, when Congo red was submitted to thein-vivo degradation process, the Microtox assay detected that toxicitywas almost entirely eliminated (0.83 ± 0.28 %), what demonstratesthat the degradation pathway of Congo red in-vivo was different andthat the resulting metabolites are less toxic than the original dye.

4. Conclusions

Optimum levels of in-vitro degradation conditions varied in most of

Fig. 4. FTIR spectra of (A) methyl green before, (B) methyl green after in vitro degradation, (C) Congo red before and (D) Congo red after in vitro degradation withcrude extract of P. chrysosporium.

J.D. Sosa-Martínez, et al. Journal of Hazardous Materials 400 (2020) 123254

9

the evaluated dyes. Under optimum conditions, the crude enzymaticextract, produced with an agro-industrial waste without the addition ofcofactors, was sufficient to achieve a high degradation percentage ofvarious dyes. These results contribute to establishing a cost-effectivebioprocess for the production of ligninolytic enzymes and its applica-tion in contaminants biodegradation by reducing the cost related to rawmaterials and by eliminating the high costs related to the requiredpurification steps.

The most successful result was obtained for the in-vitro degradationof Methyl green since a high degradation percentage was obtained;moreover, the degradation products presented a considerable reductionof toxicity when compared to the original dye. However, the byproductsand toxicity evaluation showed that at least for Congo red, the in-vitrodegradation products presented higher toxicity than the original dye.Therefore, biodegradation not always entails detoxification. Thus, it iscrucial to evaluate the biodegradation mechanism of each target con-taminant as well as the toxicity of the degradation products for futureapplication of ligninolytic enzymes in biodegradation.

Author contributions

Jazel Doménica Sosa-Martínez: Performed experiments, wrote in-itial draft. Nagamani Balagurusamy: Data analysis and manuscript re-vision. Julio Montañez: Funding acquisition and manuscript revision.Rosely Aparecida Peralta: Performed experiments and manuscript re-vision. Regina de Fátima Peralta-Muniz-Moreira: Performed experi-ments and manuscript revision. Adelar Bracht: Performed experimentsand manuscript revision. Rosane Marina Peralta: Provided resources forthe study, mentorship to the team, manuscript review and editing.Lourdes Morales-Oyervides: Conceptualization, data analysis and

manuscript review and editing.

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

Author J.D. Sosa-Martínez acknowledges CONACyT-México ConsejoNacional de Ciencia y Tecnología (National Council for Science andTechnology), scholarship number: 466181 for the financial supportprovided for conducting her master studies. R. A. Peralta, R.F. PeraltaMuniz Moreira, R.M. Peralta, and A. Bracht acknowledge CNPq-BrazilConselho Nacional de Desenvolvimento Científico e Tecnológico(National Council for Scientific and Technological Development) forgrants.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.jhazmat.2020.123254.

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