**3. Results and discussion**

#### **3.1. Enzyme characterization of fungal extracts**

Laccase activity and H2O2 production in the fungal co-culture (laccase = 18956.0 U/mg of protein and H2O2 = 6.2 mg/L) were significantly higher (*T* = 6.19, *P* = 0.0004) than in the *T. maxima* monoculture (laccase = 12866.2 U/mg of protein and H2O2 = 4.2 mg/L). Regarding MnP activity, we did not find significant differences between the fungal co-culture and the *T. maxima* monoculture (*T* = 0.27, *P* = 0.3957). Since *P. carneus* is a soil microfungus (Hyphomycete), it did not show laccase or MnP activity; only H2O2 production (0.9 mg/L) was detected, which was significantly lower (*F* = 126.4, *P* = 0.00001) than in the *T. maxima* monoculture (4.2 mg/L) and fungal co-culture (6.2 mg/L, **Table 1**).


Laccase and MnP were compared with the *t*-student test, and H2O2 content was compared using an ANOVA and LSD test for mean comparison. Means with different letters are significantly different from each other (*P* = 0.05). ND = No detected.

**Table 1.** Amount of enzymes in fungal extracts.

Laccase is an important enzyme in white-rot fungi; this enzyme is a defence mechanism against saprotrophic and parasitic microfungi. This phenomenon has been reported for *Lentinula edodes* [21], *Agaricus bisporus* [22] and *Pleurotus ostreatus* [23] when infected with *Trichoderma* sp. These macromycetes have been studied due to their importance as edible mushrooms, and *Trichoderma* is their naturally antagonistic fungus, especially in production systems. In particular, recent studies have sought solutions stemming from fungal interactions to obtain relevant biotechnological solutions and products. Thus, the interaction between white-rot fungi (Basidiomycetes) and other soil-borne micromycetes (hyphomycetes) has received greater interest in recent years [24, 25].

One of the principal applications of fungal co-cultures is to increase ligninolytic enzyme activity (laccase, MnP and LiP), and these may then be applied to resolve environmental problems, such as the contamination of soil and water with pesticides or the presence of endocrine disruptors, medical drugs, hydrocarbons, dyes or other emerging contaminants in the environment. Several studies have reported that soil-borne micromycetes enhance ligninolytic enzyme activity in white-rot fungi; for example, Baldrian [25] reported that *Sphaerospermum* sp., *Acremonium* sp., *Fusarium reticulatum*, *Humicola grisea* and *Penicillium rugulosum* enhanced laccase activity in *Trametes versicolor* and *Pleurotus ostreatus* when cocultivated. Dwivedi et al. [26] reported an increase in the laccase activity of *Pleurotus ostreatus* when co-cultured with *Penicillium oxalicum*. In addition, Chan-Cupul et al. [15] recently demonstrated that laccase and MnP activity in a specific co-culture may be increased if the culture media are optimized. In that study, a 1.8- and 2.9-fold increase in laccase and MnP activities, respectively, was recorded for the co-culture of *T. maxima* and *P. carneus*.

#### **3.2. Biodegradation studies**

**3. Results and discussion**

fungal co-culture (6.2 mg/L, **Table 1**).

**Table 1.** Amount of enzymes in fungal extracts.

greater interest in recent years [24, 25].

Laccase

MnP

H2O2 (mg/L)

detected.

(U/mg of protein)

(U/mg of protein)

**3.1. Enzyme characterization of fungal extracts**

198 Soil Contamination - Current Consequences and Further Solutions

Laccase activity and H2O2 production in the fungal co-culture (laccase = 18956.0 U/mg of protein and H2O2 = 6.2 mg/L) were significantly higher (*T* = 6.19, *P* = 0.0004) than in the *T. maxima* monoculture (laccase = 12866.2 U/mg of protein and H2O2 = 4.2 mg/L). Regarding MnP activity, we did not find significant differences between the fungal co-culture and the *T. maxima* monoculture (*T* = 0.27, *P* = 0.3957). Since *P. carneus* is a soil microfungus (Hyphomycete), it did not show laccase or MnP activity; only H2O2 production (0.9 mg/L) was detected, which was significantly lower (*F* = 126.4, *P* = 0.00001) than in the *T. maxima* monoculture (4.2 mg/L) and

**Fungal enzyme extracts**

ND 12866.2 ± 446.7 18956.0 ± 204.0 *t*-student

ND 572.4 ± 31.8 542.6 ± 43.5 *t*-student

0.9 ± 0.07 c 4.2 ± 0.10 b 6.2 ± 0.15 a Fisher

Laccase and MnP were compared with the *t*-student test, and H2O2 content was compared using an ANOVA and LSD test for mean comparison. Means with different letters are significantly different from each other (*P* = 0.05). ND = No

Laccase is an important enzyme in white-rot fungi; this enzyme is a defence mechanism against saprotrophic and parasitic microfungi. This phenomenon has been reported for *Lentinula edodes* [21], *Agaricus bisporus* [22] and *Pleurotus ostreatus* [23] when infected with *Trichoderma* sp. These macromycetes have been studied due to their importance as edible mushrooms, and *Trichoderma* is their naturally antagonistic fungus, especially in production systems. In particular, recent studies have sought solutions stemming from fungal interactions to obtain relevant biotechnological solutions and products. Thus, the interaction between white-rot fungi (Basidiomycetes) and other soil-borne micromycetes (hyphomycetes) has received

One of the principal applications of fungal co-cultures is to increase ligninolytic enzyme activity (laccase, MnP and LiP), and these may then be applied to resolve environmental problems, such as the contamination of soil and water with pesticides or the presence of endocrine disruptors, medical drugs, hydrocarbons, dyes or other emerging contaminants in the environment. Several studies have reported that soil-borne micromycetes enhance

**Comparison test**

[*T* = 6.19, *P* = 0.0004]

[*T* = 0.27, *P* = 0.3957]

[*F* = 126.4, *P* = 0.00001]

**Variable** *P. carneus T. maxima* **Co-culture Mean**

**Figure 1** shows atrazine degradation by fungal enzyme extracts (FEEs) from the monocultures of *T. maxima* and *P. carneus* and their co-culture. One hour after application, the co-culture enzyme extract degraded 100% of atrazine at a significantly higher rate (*F* = 331.31, *P* = 0.00001) than *T. maxima* and *P. carneus* extracts, which degraded 80.0% and 27.3% of atrazine, respectively (**Figure 1A**). At 3 h after application, the monoculture extract of *T. maxima* (84.5%) statistically achieved the same level of atrazine degradation as the co-culture extract (89.1%); however, both values were higher (*F* = 320.5, *P* = 0.0001) than atrazine degradation by the *P. carneus* enzyme extract (5.3%, **Figure 1B**).

**Figure 1.** Atrazine degradation at 1 h (A), 3 h (B), 6 h (C) and 12 h (D) after application of fungal enzyme extracts in a clay-loam soil. Bars (mean ± standard error) with different letters are statistically different from one another (LSD test *P* = 0.05).

At 6 h after application (**Figure 1C**), the relationship of *T. maxima* and its co-culture with *P. carneus* was inverted. Atrazine degradation by the co-culture enzyme extract decreased by 23.9% in comparison to its initial rate of degradation (at 1 h). This may be attributed to the absorption of atrazine by the soil, which motivated the investigation of the kinetic absorptiondesorption parameters of atrazine in the studied clay-loam soil. Meanwhile, degradation of atrazine by the fungal monoculture extract increased to 97.9% (6 h), and *P. carneus* showed the lowest percentage of degradation (8.9%; *F* = 20.79, *P* = 0.0004).

However, during evaluation the degradation of atrazine by the fungal co-culture enzyme extract increased once again (92.2% at 12 h). This may be due to a desorption effect of atrazine previously absorbed by soil particles, principally clay. Meanwhile, the *T. maxima* monoculture extract degraded 100% of atrazine by this time, and the *P. carneus* extract also reached its maximum level of atrazine degradation (40.7%). At the end of evaluation period (12 h), both the *T. maxima* extract and its co-culture with *P. carneus* degraded 100% of atrazine. However, the increase in degradation by the *P. carneus* extract was not significant and did not reach levels of greater than 25% (*F* = 671.05, *P* = 0.0001, **Figure 1D**).

During mycoremediation, a single strain is commonly used. The application of bioremedial fungi in the soil is often based on the inoculation of immobilized mycelium in organic substrates, such as pine sawdust, wood chips, peat, corn cobs, wheat straw, bark, rice grains, sugarcane bagasse, coffee pulp or sugar beet pulp [27–30]. However, this technology has several challenges to overcome, which are as follows: (i) the competition and proliferation of native soil microorganisms (microfungi, bacteria and actinomycetes) with bioremedial fungi [9]; (ii) the limited capacity of inoculated fungi in the soil to produce sufficient amounts of the ligninolytic enzymes responsible for degrading contaminants [31–33]; (iii) the adverse effects of environmental and edaphic conditions on the establishment or growth of bioremedial fungus [14] and (iv) the amount of contaminants in the soil, which in some cases may be toxic to the bioremedial fungi [14].

One alternative for overcoming these challenges is the use of fungal enzyme extracts produced in fungal co-culture systems, which may enhance the amount of ligninolytic enzymes [34]; these extracts may then be applied to soil through irrigation systems by drenching or by immobilizing ligninolytic enzymes in chitosan, alginate or nanoparticles [35]. In our study, we applied fungal enzyme extracts from a co-culture to degrade atrazine in a clay-loam soil and found efficiencies of 100% at 6 and 12 h. Other studies have reported the ability of white-rot fungi extracts to degrade atrazine. For example, *Phanerochaete chrysosporium* extract can degrade atrazine in the soil microcosm (38% at 8 days), although its volumetric enzyme activity is low (MnP = 77.6 U/L, LiP = 149 U/L), as this species has low or null laccase activity [32]. In batch studies, Pereira et al. [36] reported that 39% of atrazine was degraded using a broth culture of *Pleurotus ostreatus* INCQ40310; the rate of degradation was enhanced to 71% when the broth culture was optimized by manipulating the nutritional compounds of the culture medium.

Several additional studies have used fungal co-cultures or their products, such as ligninolytic enzymes, to degrade contaminants. Recently, Pan et al. [37] demonstrated the feasibility of the fungal co-culture extract between *Coprinopsis cinerea* and *Gongronella* sp. to decolorize indigo dye. However, the native laccase from the fungal extract did not degrade indigo dye, and it was necessary to add ABTS (2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)) as a redox mediator to degrade 75% of the dye. In another study, Qian and Chen [38] reported that the crude extract from the co-culture of *T. versicolor* and *Phanerochaete chrysosporium* degraded 20% more benzo-α-pyrene than the crude extracts of the monocultures of both fungi.

#### **3.3. Atrazine absorption-desorption in a clay-loam soil**

atrazine by the fungal monoculture extract increased to 97.9% (6 h), and *P. carneus* showed the

However, during evaluation the degradation of atrazine by the fungal co-culture enzyme extract increased once again (92.2% at 12 h). This may be due to a desorption effect of atrazine previously absorbed by soil particles, principally clay. Meanwhile, the *T. maxima* monoculture extract degraded 100% of atrazine by this time, and the *P. carneus* extract also reached its maximum level of atrazine degradation (40.7%). At the end of evaluation period (12 h), both the *T. maxima* extract and its co-culture with *P. carneus* degraded 100% of atrazine. However, the increase in degradation by the *P. carneus* extract was not significant and did not reach levels

During mycoremediation, a single strain is commonly used. The application of bioremedial fungi in the soil is often based on the inoculation of immobilized mycelium in organic substrates, such as pine sawdust, wood chips, peat, corn cobs, wheat straw, bark, rice grains, sugarcane bagasse, coffee pulp or sugar beet pulp [27–30]. However, this technology has several challenges to overcome, which are as follows: (i) the competition and proliferation of native soil microorganisms (microfungi, bacteria and actinomycetes) with bioremedial fungi [9]; (ii) the limited capacity of inoculated fungi in the soil to produce sufficient amounts of the ligninolytic enzymes responsible for degrading contaminants [31–33]; (iii) the adverse effects of environmental and edaphic conditions on the establishment or growth of bioremedial fungus [14] and (iv) the amount of contaminants in the soil, which in some cases may be toxic

One alternative for overcoming these challenges is the use of fungal enzyme extracts produced in fungal co-culture systems, which may enhance the amount of ligninolytic enzymes [34]; these extracts may then be applied to soil through irrigation systems by drenching or by immobilizing ligninolytic enzymes in chitosan, alginate or nanoparticles [35]. In our study, we applied fungal enzyme extracts from a co-culture to degrade atrazine in a clay-loam soil and found efficiencies of 100% at 6 and 12 h. Other studies have reported the ability of white-rot fungi extracts to degrade atrazine. For example, *Phanerochaete chrysosporium* extract can degrade atrazine in the soil microcosm (38% at 8 days), although its volumetric enzyme activity is low (MnP = 77.6 U/L, LiP = 149 U/L), as this species has low or null laccase activity [32]. In batch studies, Pereira et al. [36] reported that 39% of atrazine was degraded using a broth culture of *Pleurotus ostreatus* INCQ40310; the rate of degradation was enhanced to 71% when the broth culture was optimized by manipulating the nutritional compounds of the culture

Several additional studies have used fungal co-cultures or their products, such as ligninolytic enzymes, to degrade contaminants. Recently, Pan et al. [37] demonstrated the feasibility of the fungal co-culture extract between *Coprinopsis cinerea* and *Gongronella* sp. to decolorize indigo dye. However, the native laccase from the fungal extract did not degrade indigo dye, and it was necessary to add ABTS (2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)) as a redox mediator to degrade 75% of the dye. In another study, Qian and Chen [38] reported that the crude extract from the co-culture of *T. versicolor* and *Phanerochaete chrysosporium* degraded 20%

more benzo-α-pyrene than the crude extracts of the monocultures of both fungi.

lowest percentage of degradation (8.9%; *F* = 20.79, *P* = 0.0004).

200 Soil Contamination - Current Consequences and Further Solutions

of greater than 25% (*F* = 671.05, *P* = 0.0001, **Figure 1D**).

to the bioremedial fungi [14].

medium.

**Figure 2** shows the adsorption-desorption of atrazine in the studied clay-loam soil. Depending on the concentration of atrazine dissolved in soil, between 39% and 77% is absorbed (**Figure 2A**). More atrazine is adsorbed than desorbed, or in other words, the desorption of atrazine is slower than its adsorption given the studied the soil type and time period (24 h, **Figure 2B**). Atrazine desorption is slower when high concentrations are adsorbed by the soil; i.e., when 20 mg/L was absorbed, only 1% was desorbed at 24 h. In this sense, Davidchik et al. [39] suggest that the adsorption of atrazine may be irreversible if a high concentration is found in the soil; these authors consider that oxidative binding is the most probable mechanism of atrazine incorporation into the organic matter.

**Figure 2.** Adsorption (A) and desorption (B) of atrazine in a clay loam soil.

Adsorption and desorption values were linearized using the Freundlich equation (Eq. (1)), where *qe* is the amount adsorbed at equilibrium (mg of atrazine/g of soil) and *Ce* the equilibrium concentration of atrazine in the solution (mg of atrazine/L). **Figure 3** shows the linearized Freundlich isotherms for atrazine adsorption and desorption, while **Table 2** describes the Freundlich isotherm parameters and hysteresis index.

**Figure 3.** Linearized Freundlich isotherms for atrazine adsorption (A) and desorption (B) from a clay-loam soil.


**Table 2.** Freundlich isotherm parameters and hysteresis index values for atrazine adsorption-desorption in a clay loam soil.

$$
\log q\_{\boldsymbol{\epsilon}} = \log K\_{\mathbb{F}} + \mathbb{I} / n \log C\_{\boldsymbol{\epsilon}} \tag{1}
$$

The Freundlich constant for adsorption of atrazine was 8.2148, which was higher than that reported by Kulikova et al. [7], who studied the absorption of atrazine to three soils with different textures (silt-loam: sod-podzolic [*K*F = 4.51] and gray forest [*K*F = 0.81] and clay-loam: chernozem [*K*F = 5.54]). These authors suggest that clay-loam soil has high levels of organic carbon (organic matter), which leads to a high rate of atrazine absorption. In our study, the soil also possessed this characteristic, as a high organic matter content (4.35%) was detected in the soil analysis due to the incorporation of crop residues (sugarcane stalks) to the soil. In another study, Naga-Madhuri et al. [40] reported a lower *K*F (=2.66) for atrazine adsorption in a silty clay-loam soil; the authors suggested that this value is high and may be due to the high electric conductivity and organic matter content of the studied soil.

On the other hand, the Freundlich desorption constant for atrazine was lower (*K*F = 5.4992) than the adsorption constant (*K*F = 8.2148). This was reflected in the hysteresis value (*H* = 0.573), which has a maximum value of 1; in this case, values near 1 indicate that almost all adsorbed atrazine is readily desorbed [7]. In this study we found that the clay-loam soil used in mycoremediation experiments does not desorb the adsorbed atrazine to a great extent, due to the high organic carbon content of the soil. Future studies will need to further examine the effect of enzyme extracts from fungal co-cultures and the adsorption-desorption phenomenon of atrazine in contaminated and bioremediated soils.
