**2. Materials and methods**

**1. Introduction**

194 Soil Contamination - Current Consequences and Further Solutions

[7].

fungi [9].

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) is the most widely used herbicide around the world. In agricultural soils, approximately 29–34 million kg of atrazine are applied per year [1]. In Mexico, more than 45% of pesticides are categorized as herbicides, and atrazine is one of the most commonly used herbicides in Mexican agriculture [2]. Since 1975, atrazine has been applied to control broadleaf and grass weeds in agricultural crops, including corn, sorghum and sugar cane. Atrazine kills susceptible plants by binding to the quinone protein

Atrazine is a pre-emergent herbicide and is considered to have low persistence in soil (<12 months). However, its low mobility in soil and its physical and chemical properties contribute towards the contamination of ground and surface waters, which represents a risk to the environment and to human health [4]. In Mexico, atrazine levels in water often exceed the maximum permissible levels for drinking water (0.1 μg L−1) as established by Europe and by the health advisory board of the United States Environmental Protection Agency (EPA) [5, 6]. At a molecular level, atrazine has distinct fates in the environment and may be found in soil, water, biomass (plants) or air. In soil, atrazine is adsorbed by clay particles; however, other adsorption-desorption processes may be involved in its translocation in plants, movement in soil and mobility in aqueous systems, as well as its eventual abiotic or biological degradation

The clean-up of soils contaminated with atrazine is an ecological responsibility, and the discovery of a safe and economical method is a major priority for land management agencies [8]. One such possibility is mycoremediation, or the use of fungal organisms and their enzymes to degrade or transform environmental pollutants [9]. This strategy has been used to degrade pesticides [10], aromatic and polycyclic hydrocarbons [11] and endocrine disruptors [12]. The degradation of environmental pollutants by fungi, specifically by white-rot fungi, is due to their ability to synthesize ligninolytic enzymes, such as laccase, manganese peroxidase and

However, mycoremediation faces several challenges in order to improve the feasibility of this strategy. The following issues, for example, should be addressed: (i) the competition/proliferation of native soil microorganisms (actinomycetes and bacteria) may inhibit the growth of bioremedial fungi; (ii) bioremedial fungi have a limited capacity to produce ligninolytic enzymes. Enzyme production varies depending on the strain and species and is mainly influenced by the content and availability of nutrients (carbon, nitrogen, metal ions, etc.), which stimulate fungal growth and the synthesis of ligninolytic enzymes; finally, (iii) edaphic and environmental factors may adversely affect the establishment and growth of bioremedial

The use of fungal extracts with a proven high activity of ligninolytic enzymes is one means of improving the degradation of pollutants in soil, which may also address some of the aforementioned challenges. Ligninolytic enzymes in white-rot fungi, for example, may be enhanced through the use of fungal co-cultures, although the mechanism by which increased enzyme

lignin peroxidase, as well as their production of hydrogen peroxide [13, 14].

in photosystem II and inhibiting photosynthetic electron transport [3].

#### **2.1. Fungal source and molecular identification**

The white-rot fungi *T. maxima* was isolated from a carpophore collected in a rain forest (19°32′21.23″ N, 97°00′47.29″ W) near Vega del Pixquiac, San Ándres Tlalnelhuayocan, Veracruz, Mexico. To obtain the isolate, 0.5–1 cm fragments of the carpophore were cut and removed; these were washed in ethanol (70%) for 1 min, in sodium hypochlorite (50%) for 3 min and finally, in sterile, distilled water. The washed and disinfected fragments were placed on potato-dextrose agar plates (Bioxon®, Mexico) and supplemented with chloramphenicol (20 mg/L; Sigma-Aldrich, St. Louis, MO, USA) to prevent bacterial contamination and benomyl (3 mg/L; Biesterfeld Co., Mexico) to inhibit mold growth.

The soil-borne micromycete *P. carneus* Duché & R. Heim (Trichocomaceae: Ascomycota) was donated by the Micromycetes Laboratory of the Institute of Ecology (INECOL A.C.) located in Xalapa, Mexico. This strain was isolated from an andic acrisol soil (texture: loam-silt loam) from a coffee plantation in Huatusco, Veracruz, Mexico (location: 19°12′57″ N, 96°53′7″ W). The carpophores of *T. maxima* (Mont.) A. David & Rajchenb (Polyporaceae: Basidiomycota) and the *P. carneus* strain are stored in the herbarium (XAL) and Micromycetes Culture Collection of INECOL. Both strains were maintained and subcultured in potato dextrose agar.

#### **2.2. Soil sampling and characterization**

Soil samples were collected from the first horizon of <20 cm profundity at a sugar cane plantation in Mahuixtlan, Veracruz, Mexico (location: 19°23′21.3″ N, 96°53′34.9″ W). Plant residues and rocks were removed manually. Soil was sieved in 2 mm mesh in the laboratory and dried at 20°C for 5 days prior to use. The physical and chemical characteristics of the soil were determined using standard methods to establish texture (clay loam soil), soil organic matter (4.35%), pH (4.86), NH4-N (5.8 mg kg−1), soluble salts (5.38 S m−1), acidity (0.053 meq 100 g−1), cation exchange capacity (16.41 meq 100 g−1), water holding capacity (WHC) (53.6%) and electrical conductivity (53.75 μS cm−1).

#### **2.3. Production of ligninolytic enzymes through fungal co-culture**

Modified Sivakumar culture medium [16] was used to produce laccase, MnP and H2O2 for the monoculture of *T. maxima* and the co-culture of both *T. maxima* and *P. carneus*. To establish the co-culture, four agar plugs of *T. maxima* (7 days old) were deposited in a 250 mL Erlenmeyer flask with 120 mL of modified Sivakumar culture medium. After 3 days, four agar plugs of *P.* *carneus* (9 days old) were added. Monocultures of both fungi were established at the same time. Fungal cultures were incubated at 25°C and 120 rpm for 6 days. After this step, the fungal enzyme extracts (FEEs) were centrifuged at 7000 rpm during 10 min. The supernatant was filtered with a 0.2 mm nylon filter; this process allows a cell-free extract to be obtained, which was used to determine laccase and MnP activity and H2O2 content.
