Physical, chemical and microbiological characterization of the soil and residual substrate of A. bisporus

The physical and chemical characterization of the agricultural soil and the residual substrate of A. bisporus was carried out as described in NOM-021-SEMARNAT-2000 (^{DOF, 2002}). For the microbiological characterization of the soil and the RS, serial dilutions and the plate-counting technique were performed in differential culture media for the quantification of populations of bacteria, fungi, actinomycetes and bacteria tolerant to diesel expressed as log₁₀ CFU g_ss ^-1 (colony-forming units/gram of dry soil).

The mesophilic bacterial population was quantified in nutritive agar medium (Bioxon, Mexico), the fungi in the potato dextrose agar (PDA) medium, the actinomycetes in Czapek Dox Agar and the diesel-tolerant bacteria in the basal medium; the latter with the following preparation (g L^-1): NH₄NO₃, 1; K₂HPO₄, 1; KH₂PO₄, 1; MgSO₄7H₂O, 0.409; CaCl₂, 0.02; FeCl₃, 0.00005; bacteriological agar, 6.5; pH 7, supplemented with 100 μl of sterile diesel distributed on the gelled agar (^{Mauricio-Gutiérrez et al., 2014}). Mesophilic bacteria, tolerant bacteria and actinomycetes were incubated at 30 °C for 72 h, fungi were incubated at 25 °C for 72 h.

Diesel biodegradation at microcosm level using RS of A. bisporus

The soil used was sterilized and intentionally contaminated with 7 039 ppm of diesel and weathered for three months. A completely randomized experimental design was used in a microcosm system to evaluate the participation of microbial populations and laccase (Lac) activity present in the study system with four treatments with different soil: substrate ratios on a dry basis (T1 (95: 5), T2 (90: 10), T3 (85: 15) and T4 (80:20)) and the contaminated soil (T0₁) and RS (T0₂) were used as controls.

For each treatment, serological bottles of 120 ml were used, with 30 g of the soil-substrate mixture and were adjusted to the C: N: P ratio to 100: 10: 1 using sterile solutions of NH₄SO₄ 1N and K₂HPO₄ 1N. Moisture was maintained between 23.8-25.6% ±5.23. The atmosphere of the bottles was exchanged every third day with an airflow (1.8 ml air/sec); through a sterile membrane (0.22 μm), the amount of CO₂ produced was previously evaluated. All treatments were incubated for 28 days at two temperatures (25 and 37 ºC).

The percentage of diesel biodegradation (%), actual density by means of the NOM-021-SEMARNAT-2000, initial and final activity of laccases (Lac), initial and final population of diesel-tolerant bacteria and fungi (^{DOF, 2002}) were determined. All treatments were carried out in triplicate. The percentage of biodegradation was determined according to the following equation: percentage of biodegradation= [(A - B)/A]*100, A= initial concentration of diesel; B= final concentration of diesel

Basal respiration

The CO₂ produced by the soil for 24 h was quantified and absorbed by 5 ml of NaOH 0.1 N, calculated through titration with HCl 0.1 N using phenolphthalein, the excess of carbonates was previously precipitated with 5 ml of BaCl₂ 0.5 N (^{Rivera-Espinoza and Dendooven, 2004}).

Diesel quantification

The initial and residual diesel was quantified based on the EPA Method 8015 C (Nonhalogenated Organics by Gas Chromatography) using a GC-MS with an HP-5 capillary column (30 m x 0.25 mm x 0.25 μm), whose initial flow was 1 ml min^-1. The conditions of analysis of the furnace were: initial temperature 50 ºC, maximum temperature 300 ºC.

Enzyme quantification

An extraction was carried out at the beginning and end of the experiment of the sample of the RS of A. bisporus and of the different treatments in soil: substrate ratios (5 g) with 20 ml of sodium acetate buffer (50 mM, pH 5) for 2 h at 80 rev min^-1 and at 4 °C, then filtered through Whatman No. 1 paper. The extracts were stored at 4 °C for the determination of Laccases (^{Isikhuemhen and Mikiashvilli, 2009}). The activity was determined by oxidation of 2.2’-azinobis, 3-ethylbenzothiazoline-6-sulphonate (ABTS) at 25 °C and read at 420 nm (ε₄₂₀= 36 000 mM^-1 cm^-1), enzymatic activity was reported as specific activity (U g soil^-1) (^{Bollag and Leonowicz, 1984}; ^{Gayosso-Canales et al., 2011}).

Microbiological analysis

The initial and final population of diesel-tolerant bacteria and fungi was quantified by direct plate counting. The determination of bacteria was performed for treatments incubated at 37 °C grown in basal medium mentioned above. The determination of fungi was for treatments incubated at 25 °C and the basal medium with the following chemical composition (g L^-1) was used: (NH₄)₂SO₄, 7; K₂HPO₄, 1; KH₂PO₄, 1; MgSO₄7H₂O, 0.409; dextrose, 0.1; Sol. E 100x, 100 μl; rose bengal, 0.05; streptomycin sulfate, 0.05; pH 5.5. Diesel previously sterilized by filtration was used as a source of carbon, adding 100 μl on the petri dish (^{Mauricio-Gutiérrez et al., 2014}). The microorganisms were reported as log ₁₀ CFU g_ss ^-1 (colony-forming units/gram of dry soil).

Statistical analysis

An analysis of variance to compare the different treatments and a Tukey multiple comparison test of means (p< 0.05) were performed using the Minitab Version 17.3 statistical package.

Physical, chemical and microbiological characterization of soil and RS

According to the physical and chemical characteristics (Table 1) of the agricultural soil of Acatzingo, Puebla, Mexico, it was classified as loam-sandy, with low values of moisture (17.35%), organic matter (2.04%), total nitrogen (0.032%) and phosphorus (0.003%) compared to the parameters of the RS (190.32%, 57.62%, 1.46% and 0.79% respectively). In addition, it presented a moderately alkaline pH value (pH= 8.01). The RSs of Agaricus bisporus are characterized by high concentrations of nutrients, these properties have been used as biostimulant agents for the bioremediation of xenobiotic since they have high doses of nitrogen (2.16%), phosphorus (0.69%) and carbon (54.3%) (^{Corral-Bobadilla et al., 2019}).

With respect to the quantification of cultivable microorganisms in Table 1, the bacterial population had the highest values in the soil (6.021 log CFU g_ss ^-1) and the RS (7.708 log CFU g_ss ^-1). The size of the other microbial populations varied between the samples, with the agricultural soil where there was a greater number of hydrocarbonoclastic bacteria (5.037 log CFU g_ss ^-1), followed by the fungal population (3.455 log CFU g_ss ^-1) and finally actinomycetes (2.895 log CFU g_ss ^-1). However, in the RS there was a greater number of fungi (5.554 log CFU g_ss ^-1) and the groups of actinomycetes and hydrocarbonoclastic bacteria remained at the same order of magnitude (3.559 and 3.828 log CFU g_ss ^-1). The soil and RS harbor a native microbial population, the latter have been used as sources of bioaugmentation for bioremediation processes (^{Wang et al., 2016}; ^{Leong et al., 2022}).

Diesel biodegradation at microcosm level using RS of A. bisporus

Diesel biodegradation experiments at the microcosm level (Table 2) showed a higher biodegradation at 37 ºC; treatment T4 was the one that obtained the highest removal (68.747%) followed by T4 at 25 ºC (61.261%) and T3 at 37 ºC (60.14%). On the contrary, the treatments with the lowest percentage of removal were T0₁ (contaminated soil) at 25 ºC with a removal of 20.603%, followed by T1 (27.034%) and T2 (29.791%) at 25 ºC, presenting highly significant statistical differences (p= 0.001).

The cumulative and quantified CO₂ generation in the study system indicated that treatments incubated at 37 ºC produced a higher concentration (mmol CO₂ m^-3 day) (Table 2). The treatments that obtained the highest CO₂ production were T0₂ (RS) (6.173 x 10^-4 mmol CO₂ m^-3 day) at 25 ºC, T4 (6.144 x 10^-4 mmol CO₂ m^-3 day) and T3 (6.014 x 10^-4 mmol CO₂ m^-3 day) at 37 ºC. And the treatments: T1 (4.319 x 10^-4 mmol CO₂ m^-3 day) at 25 ºC, T2 (4.343 x 10^-4 mmol CO₂ m^-3 day), T3 (4.808 x 10^-4 mmol CO₂ m^-3 day) and T0₁ (contaminated soil) (4.902 x 10^-4 mmol CO₂ m^-3 day) at 25 ºC presented a lower CO₂ production rate. ^{Margesin et al. (2007}) positively correlated hydrocarbon degradation with microbial activity and biomass, results similar to those obtained in the present investigation.

A 4:1 ratio of soil: substrate (T4) biodegraded 68.747% of diesel in 28 days, which allows treating 6.92 t m^-3 of soil, as mentioned by ^{Sasek et al. (2003}), who added RS of A. bisporus to a 4:1 ratio (soil: substrate) and obtained a degradation of 68.8% of PAH (polyaromatic hydrocarbons) at the end of 154 days in 2.8 m³ of soil. While ^{Mohammadi-Sichani et al. (2019}) reported a degradation of 71.5% in three months of TPH (total petroleum hydrocarbons) in soil, applying 10% of RS of A. bisporus. In addition, the actual density of each treatment was determined in order to assess the RS of A. bisporus as a texturing agent for application in bioremediation of soils contaminated with hydrocarbons.

The results obtained indicated that there is no statistically significant difference in the actual density between the treatments, where the values are in the range of 2.36 to 2.536 g mL^-1 (Table 3). The percentages of biodegradation obtained can be due to the physical and chemical properties that RS provides to the soil such as: texture, water retention capacity, porosity and contribution of essential nutrients such as nitrogen and phosphorus (Table 1), which allow stimulating degradative microbial activity, helping in soil bioremediation (^{Stabnikova et al., 2010}; ^{García-Delgado et al., 2013}).

The population dynamics of diesel-tolerant bacteria in the study system indicated a highly significant statistical increase (p= 0.001) at the end of the experiment for all treatments: T1 (7.83 log CFU g_ss ^-1), T2 (8.9 log CFU g_ss ^-1), T3 (7.7 log CFU g_ss-1) and T4 (7.6 log CFU g_ss ^-1). However, the population count of tolerant fungi showed a highly significant statistical decrease (p= 0.001) with population sizes of 4.8 to 5.6 log CFU g_ss ^-1 (Figure 1).

Figure 1 a) dynamics of the bacterial population of the treatments incubated at 37 °C; and b) of the fungal population of the treatments incubated at 25 °C tolerant to diesel. At the beginning (light bars) and end (dark bars) of the experiment with different soil: RS of A. bisporus ratios on a dry basis. Each value represents the average of triplicates. Different letters indicate statistically significant differences between treatments (p= 0.05) according to the Tukey test. 

The quantification of Lac decreased significantly (p= 0) in the treatments incubated at 25 °C, quantifying specific enzymatic activities from 0.083 to 0.48 U g^-1 (Figure 2). Treatment T4 (80:20) had the highest enzymatic activity (0.48 U g^-1) unlike the control group T0₂ (RS) of A. bisporus (2.46 U g^-1). In the treatments incubated at 37 °C, Lac activity was not detected according to the established methodology.

Figure 2 Laccases activity at the microcosm level with different soil: RS of A. bisporus ratios on a dry basis. Each value represents the average of triplicates of the treatments at the beginning (light bars) and end (dark bars) of the experiment. *= different letters indicate statistically significant differences between treatments (p= 0.05) according to the Tukey test. 

The group of microorganisms studied in the biodegradation of diesel were the tolerant bacteria native to the RS and the soil, since they presented an effectiveness in the biodegradation and contributed to the bioremediation process (^{Gallego et al., 2001}). On the contrary, treatments incubated at 25 ºC showed a decrease in the population of fungi since they were affected by the conditions of the microcosm (environment) and the type of contaminant used, as well as in the production of enzymes such as Lac. ^{Bento et al. (2005}) mention that microorganisms in a hostile environment decrease their metabolic activity and limit the growth of their population.

Although there are different reports of extracellular enzymes secreted by A. bisporus in RS that can be used in bioremediation processes (^{García-Delgado et al., 2013}; ^{Chatterjee et al., 2017}). It is important to mention that the control treatment T0₁ (soil) presented a diesel biodegradation of 37.357%, this value may be associated with the loss due to volatilization since it can be 35-40% in soil (^{Rhykerd et al., 1999}; ^{Saviozzi et al., 2009}) and the presence of reproductive structures of the native microbiota that resisted soil sterilization (^{Sylvia et al., 1999}), since in the present study, T0₁ (soil) presented populations of bacteria and fungi of 5.3 and 4.4 log CFU g_ss ^-1.

The RS of A. bisporus was characterized as a biostimulant agent for presenting organic nutrients, it also increased the porosity of the contaminated soil, improving oxygen diffusion and water retention capacity. It also presented bioaugmentation characteristics since it provided microbial load as a degrading source of diesel with a potential effect on the biodegradation of hydrocarbons. As established by ^{Chiu et al. (1998}), where they mention that RSs have the advantage of degrading xenobiotic compounds by the consortium of enzymes of various microorganisms, in addition to being relatively rich in nutrients that stimulate these microorganisms in the secretion of enzymes without having any nutritional limitation.

In this context, there are several studies where different RSs are evaluated, such as: activated sludge, sugarcane bagasse, sugarcane filter cake and corn residues for purposes of bioremediation of soils contaminated with diesel with biodegradation percentages of 61 to 90% in a time of 15 to 109 days, results similar to those obtained in the present study (^{Gallego et al., 2001}; ^{Molina-Barahona et al., 2004}; ^{Rivera-Espinoza and Dendooven, 2004}; ^{Bento et al., 2005}; ^{García-Torres et al., 2011}).