Biochar-immobilized Fungal Consortia for Enhanced Soil Bioremediation

doi:10.21203/rs.3.rs-7545057/v1

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Biochar-immobilized Fungal Consortia for Enhanced Soil Bioremediation

https://doi.org/10.21203/rs.3.rs-7545057/v1

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Excessive pesticide use in agriculture has led to significant soil degradation, highlighting the need for effective bioremediation strategies. This study investigates the potential of a fungal consortium—including Clonostachys rosea CCMIBA_R018, Purpureocillium lilacinum CCMIBA_R014, and Bjerkandera sp. CCMIBA_R111—as a bioinoculant for soil restoration. The research optimized growth conditions and evaluated the fungi’s ability to degrade atrazine, a common pesticide. The consortium showed increased biomass when cultivated with biochar. Despite each fungus showing individual ligninolytic potential, significant lignin peroxidase activity was observed when combined, suggesting synergistic effects. After confirming joint immobilization, the consortium’s atrazine degradation capacity was assessed in soil microcosms. The results revealed a 34% reduction in the atrazine concentration in sterilized soil and a substantial 66% reduction in non-sterilized soil. Additionally, significant changes were observed in soil parameters, notably increased phosphorus and sulfur levels, contributing to improved soil fertility. The fungal consortium combined with biochar presents an innovative bioremediation approach with the potential to enhance both soil health and productivity. This strategy is particularly novel, as most bioinoculants do not directly address pollutant degradation.

Bioinoculant

Fungal consortium

Soil restoration

Atrazine degradation

Soil—composed of minerals, organic matter, and living organisms—is essential for global food and raw material production. However, the excessive use of chemical pesticides in agriculture is degrading its properties, undermining its ability to supply nutrients to the ecosystem (Gouda et al., 2018; Kopittke et al., 2019). Estimates suggest that 25% of soils are already severely degraded, and this number continues to grow despite efforts to preserve and restore them (Crossland et al., 2018). The use of pesticides in agricultural practices affects soil quality, leading to increased leaching, erosion, and diminished retention of greenhouse gases (Tang and Maggi, 2021).

Atrazine (ATZ) is a widely used pesticide in agriculture (Henn et al., 2020), applied in both pre- and post-emergence stages to control weeds and other pests, particularly in corn and soybean cultivation (Aquino et al., 2013). Primarily because of its low volatility, high leaching potential, high solubility, and strong mobility in soils (Henn et al., 2020), ATZ has low adsorption in soil and poses a significant risk of contaminating water bodies. It also negatively affects soil health and the overall functionality of the soil ecosystem (Dos Santos et al., 2023; Hu et al., 2023).

To mitigate the effects of ATZ and other pesticides, bioremediation strategies are being developed to restore severely degraded aquatic and terrestrial ecosystems. Fungi—particularly white rot fungi (phylum Basidiomycota)—have shown strong potential for the bioremediation of ATZ-contaminated soils (Sista Kameshwar and Qin, 2018). However, achieving healthy soil, which is essential for enhancing agricultural productivity, requires more than just reducing or eliminating contaminants; it also involves managing phytopathogens and improving the availability of nutritional resources. In this context, fungi have emerged as a promising tool for both biological control and nutrient recycling.

The efficiency of soil bioremediation can be enhanced by using specific conditioners, such as biochar—a bio-stimulant that promotes microbiological activity in the soil. Biochar is produced from biological waste that has been carbonized under low-oxygen conditions at high temperatures, resulting in a porous, carbon-rich material (Beesley et al., 2011). When added to soil, biochar offers several benefits, including increased microbial activity, enhanced degradation of pollutants, improved nutrient release, and better aeration and porosity (Gregory et al., 2015).

Combining the properties of biochar with the benefits of filamentous fungi that hold biotechnological potential for agricultural soil bioremediation represents a promising strategy for developing a fungal inoculant. An inoculant is a biological input containing microorganisms that carry out beneficial activities essential for plant development (Owen et al., 2015). The use of fungus-based inoculants in agriculture is considered the most cost-effective approach for ensuring both short- and long-term crop sustainability. In this context, the present study evaluated associations between fungi capable of degrading ATZ and those with potential for biological control, using biochar as an immobilizing agent.

Microorganisms

The fungi Clonostachys rosea CCMIBA_R018, Purpureocillium lilacinum CCMIBA_R014, and Bjerkandera sp. CCMIBA_R111 were obtained from the Culture Collection of Microorganisms of Biotechnological and Environmental Importance (CCMIBA) at the Federal University for Latin American Integration, Brazil. All three fungi were isolated from agricultural soil and taxonomically identified (GenBank: MT757802.1, MT757800.1, and MT757835.1, respectively). To assess growth kinetics and determine the logarithmic phase for biomass optimization prior to immobilization, three 5-mm mycelial discs were incubated in various liquid culture media (see supplementary material) at 28°C and 140 rpm for different durations (4, 7, 9, 10, 11, and 17 days).

Assessment of Fungi for ATZ Degradation

Three 5-mm mycelial discs of the fungi, previously cultured on 2% malt extract agar (MA2), were transferred to 100 mL of MA2 liquid medium containing 10 mg/L of commercial ATZ solution. As a positive control, fungi were inoculated without ATZ; the negative control consisted of MA2 with 10 mg/L of ATZ but without fungal inoculation. All tests were performed in triplicate and incubated at 140 rpm and 28°C for 10 days. After incubation, the samples were centrifuged at 10,000 rpm for 30 minutes at 4°C and filtered through filter paper. The supernatant was collected for chromatography and toxicity analysis.

ATZ degradation was analyzed using liquid chromatography coupled with tandem mass spectrometry, following validated methods at the Pesticide Residue Analysis Laboratory of the Federal University of Santa Maria, where the analyses were performed as a service.

Phytotoxicity analysis of ATZ post-fungal degradation was conducted using Cucumis sativus, following the method described by Wang et al. (2001). Cucumber seeds were pretreated with a 0.1% sodium hypochlorite solution for 20 minutes, then immersed in deionized water and rinsed to remove any residues. Toxicity assays were carried out in Petri dishes containing 15 seeds and 5 mL of each fungal treatment, with the supernatant filtered to eliminate any fragments or spores. Three replicates were prepared for each treatment, including fungi cultivated with and without ATZ, along with three replicates of the negative control (growth medium with ATZ only). The plates were kept in the dark at room temperature for 9 days. Distilled water and a 0.05 M ZnSO₄ solution were used as the positive and negative controls, respectively. After the incubation period, radicle and hypocotyl lengths were measured, and the percentage of inhibition was calculated using the equation provided by Wang et al. (2001). Mean differences were analyzed using one-way analysis of variance, followed by Tukey’s test. Values were considered significant at p < 0.05.

Individual Fungal Cultivation on Biochar

Some fungi may not sporulate, and optical density analysis is not feasible for filamentous fungi. Therefore, to standardize biomass, a pre-inoculum was prepared by transferring three 5-mm mycelial discs into Sabouraud Dextrose Broth (SDB) for C. rosea CCMIBA_R018 and P. lilacinum CCMIBA_R014, and into a malt medium enriched with peptone and yeast extract (containing 20 g/L dextrose, 10 g/L peptone, and 5 g/L yeast extract) for Bjerkandera sp. CCMIBA_R111. The pre-inocula were incubated for 10 days at 28°C and 140 rpm. To determine the amount of biomass added for each immobilization condition, a separate batch of pre-inoculum was prepared for each fungus. After cultivation, the biomass was filtered and dried in an oven at 90°C to obtain the dry weight.

For cultivation, the biomass produced in the pre-inoculum was filtered through filter paper and added individually to the biochar under four different conditions:

Cultivation test 1 (C1): 15 g of autoclaved biochar
Cultivation test 2 (C2): 15 g of autoclaved biochar with the addition of 3 mL of 1% buffered peptone water (pH 7.0) at the start of cultivation (day of inoculation)
Cultivation test 3 (C3): 15 g of autoclaved biochar with the addition of 3 mL of 1% buffered peptone water (pH 7.0) on the day of inoculation and every 4 days of cultivation
Cultivation test 4 (C4): 15 g of autoclaved biochar with the addition of 3 mL of 1% unbuffered peptone water on the day of inoculation and every 4 days of cultivation

Biochar Brasil commercial biochar was used for cultivation (components: nitrogen, phosphorus, iron, copper, potassium, calcium, zinc, manganese, magnesium, sulfur, boron, and sodium; non-flammable and free of ammonium nitrate).

All tests were incubated at 28°C with a photoperiod for 21 days and conducted in triplicate, each with its respective negative controls (cultivated components without the fungal biomass). Following incubation, the cultivation assays were washed and homogenized with a phosphate solution (7 g/L Na₂HPO₄), filtered through filter paper, and then separated into supernatant for enzymatic evaluation and biomass, which was dried in an oven at 90°C to obtain the final dry weight.

Bioinoculant: Fungal Consortium Immobilized on Biochar

The inoculant consisted of immobilizing the three fungi together in biochar. The fungi were first cultivated individually under two conditions: with and without biochar. The individually evaluated cultivation without biochar (referred to as inoculant treatment 1, IT1) was prepared by adding three fungal mycelial discs to 25 mL of the respective liquid culture medium for each fungus. In the biochar-containing cultivation (referred to as inoculant treatment 2, IT2), three mycelial discs were also added to 25 mL of the corresponding medium, along with 5 g of autoclaved biochar. Triplicate cultures of each fungus were incubated under agitation (28°C, 140 rpm) for 10 days. After incubation, the cultures were filtered through filter paper, and the resulting biomass was used to inoculate 15 g of autoclaved biochar mixed with 3 mL of 1% buffered peptone water (pH 7.0). This condition was selected based on the results of previous trials. In other words, the biomass obtained from the pre-inocula of the three fungi was combined and added to each 15-g portion of hydrated biochar, resulting in three replicates each of the IT1 and IT2 consortia.

The IT1 and IT2 consortia were incubated under a photoperiod at 28°C for 21 days. Following incubation, the treatments were washed with a phosphate solution (7 g/L Na₂HPO₄) and filtered through filter paper. The resulting biomass was dried and weighed to determine the dry weight, which was used to identify the consortium with the highest fungal growth. The supernatant obtained from each treatment was used to evaluate the ligninolytic enzyme activity.

Ligninolytic enzyme activity

During the experiments, the supernatants obtained from the tests were separated to evaluate the enzymes manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac), following the methods described by Kuwahara et al. (1984), Arora and Gill (2001) and Baldrian (2009), respectively. Enzyme activities were calculated according to the procedure outlined by Baltierra-Trejo et al. (2015). One unit of enzyme activity was defined as the amount of enzyme that oxidizes 1 mol of substrate per minute.

Evaluation of the degradation capacity of ATZ by the bioinoculant and analysis of soil quality in agricultural soil microcosms

The IT2 consortium was selected for evaluation within a microcosm system. The soil used was collected from an agricultural crop in the region of Foz do Iguaçu, Paraná, Brazil (25°26'38.5" S 54°31'01.8" W) during the off-season. The microcosms were structured following the protocols described by Wang et al. (2018) and dos Santos et al. (2025), with some modifications: the soil was evenly distributed into polystyrene trays measuring 29.6 × 43.5 cm. In all treatments, 3 kg of agricultural soil was used, and the IT2 consortium was added at a concentration of 4.29 g per kg of soil. The microcosms were contaminated with 10 mg/ L of commercial ATZ, with the soil condition varying between previously sterilized in an autoclave (referred to as ME1) and not sterilized (referred to as ME2). Controls for the microcosms (referred to as ME1C and ME2C) were maintained under the same conditions, except for the absence of the fungal consortium.

All trays were placed in a greenhouse and incubated for 24 days under stable conditions of 30°C and a 12-hour photoperiod. After the incubation period, samples were collected and analyzed for the ATZ concentration and the physical and chemical properties of the soil. These included pH, organic matter content, total nitrogen, total organic carbon, total phosphorus, carbon-to-nitrogen ratio, calcium, magnesium, potassium, nitrate, and ammonia levels. Analyses were performed as a service, with ATZ measured using gas chromatography coupled with mass spectrometry.

Assessment of Fungi for ATZ Degradation

In the individual assessment of ATZ degradation potential (Fig. 1), the negative controls (without fungal addition) yielded an average ATZ concentration of 8.8 mg/L. This suggests non-biological ATZ degradation or losses during analysis, despite the initial addition of 10 mg/L. In the biological treatment tests, the average ATZ concentrations observed were 3.25 mg/L for Bjerkandera sp. CCMIBA_R111, 2.83 mg/L for C. rosea CCMIBA_R018, and 4.71 mg/L for P. lilacinum CCMIBA_R014. These results correspond to 62.7%, 67.7%, and 46.2% ATZ degradation, respectively, highlighting the varying effectiveness of the selected fungi in ATZ removal. C. rosea CCMIBA_R018 showed the highest degradation potential, followed by Bjerkandera sp. CCMIBA_R111, with P. lilacinum CCMIBA_R014 showing the lowest.

INSERT Fig. 1

Toxicity tests were performed using C. sativus as a phytotoxicity model after ATZ treatment. Despite the lower percentage of ATZ degradation, the supernatant from the test with P. lilacinum CCMIBA_R014 reduced toxicity by 100%, allowing the model seeds to grow when exposed to the supernatant. By contrast, cultivation of C. sativus with the supernatants from Bjerkandera sp. CCMIBA_R111 and C. rosea CCMIBA_R018 indicated toxicity levels of 85% and 81%, respectively, reducing ATZ toxicity by only 17% on average despite their higher degradation rates.

Individual Fungal Cultivation on Biochar

For the fungal cultures (supplementary material), SDB was selected as the most suitable medium for producing biomass of the fungi C. rosea CCMIBA_R014 and P. lilacinum CCMIBA_R018. Enriched malt broth was chosen for Bjerkandera sp. CCMIBA_R111. The incubation time was set at 10 days to allow subsequent tests to proceed.

To assess tolerance and growth on biochar, the fungi were cultivated in their specific culture media with the addition of biochar under the four proposed conditions (Fig. 2). Condition C2, which included the use of buffered peptone water added only at the start of cultivation, yielded the greatest amount of fungal biomass. This approach maintained viable growth for all fungi; consequently, condition C2 was selected for consortium assembly.

INSERT Fig. 2

Bioinoculant: Fungal Consortium Immobilized on Biochar

For the consortium, individual incubation was carried out in two ways: pre-inoculum prepared without prior addition of biochar (IT1) and pre-inoculum with the addition of biochar (IT2), followed by co-incubation of the fungi in 15 g of biochar. The dry weight of the fungal biomass (excluding biochar) was 1.05 mg for IT1 and 7.19 mg for IT2. The individual addition of biochar before fungal association (condition IT2) led to an 85% increase in consortium biomass, which was significantly higher than in condition IT1 (p < 0.05).

Ligninolytic enzyme activity

All fungi cultivated in the simple pre-inoculum (in the selected media and without biochar) were able to produce the Lac and MnP enzymes (supplementary material), with Bjerkandera sp. CCMIBA_R111 standing out for the highest expression of both. In individual fungal cultivation on biochar, the highest enzyme activity was observed when the biochar was not humidified. However, after the addition of peptone water—buffered or not—significant variations in enzyme activity were noted. Continuous wetting had a direct impact on Lac activity, regardless of whether the peptone water was buffered.

Finally, enzymatic activity of the bioinoculant was evaluated after associating the three fungi in a consortium system with biochar (21 days of incubation). LiP was produced only in the consortium using pre-inoculum obtained by incubating the isolates with biochar (the treatment with the highest biomass production, T2), reaching 295.13 U/L. None of the other enzymes were produced in any of the treatments.

3.5 Evaluation of the degradation capacity of ATZ by the bioinoculant and analysis of soil quality in agricultural soil microcosms

After confirming joint immobilization, the next step was to evaluate the degradation of the pesticide ATZ in the microcosm system after 24 days of incubation. In the microcosm with sterilized soil, a 34% reduction in ATZ concentration was observed (ME1: 29 mg/kg of ATZ; ME1c, negative control: 44.6 mg /kg of ATZ). In the system with non-sterilized soil, the addition of the consortium led to a 66% reduction in ATZ compared with the control (ME2: 73 mg/ kg of ATZ; ME2c: 216.60 mg/kg of ATZ).

In addition to the ATZ concentration, more than 30 physical and chemical soil parameters were analyzed (supplementary material), including pH, phosphorus, magnesium, calcium, organic matter, and total organic carbon. These indicators are commonly used to assess soil fertility (Nguemezi et al., 2020). Statistically significant differences (p < 0.05) were observed for several parameters following consortium application. Soils treated with the consortium showed a marked reduction in potential acidity (H⁺Al), decreasing from 2.8 to 1.9 cmolc/dm³ in ME1 and from 2.5 to 2.1 cmolc /dm³ in ME2. Available phosphorus increased significantly, from 2 to 7 mg/dm³ in ME1 and from 1.4 to 6 mg /dm³ in ME2. Iron concentrations also rose significantly in ME1 (60 mg/dm³) compared with its control (45 mg/dm³). Base saturation (V%) improved, increasing from 73% to 81% in ME1, with a similar trend observed in ME2. Conversely, organic matter and total organic carbon content decreased significantly in ME1 following treatment (from 10 to 7 g/kg and from 0.58% to 0.41%, respectively), while remaining relatively stable in ME2.

In this study, the initial culture medium tested was Malt Extract Broth, selected after the fungi were reactivated on MA2 agar plates. However, the fungi did not exhibit substantial biomass growth under these conditions. As a result, experiments were conducted using different culture broths to promote the formation of mycelial pellets. The optimal condition for the fungi belonging to the phylum Ascomycota was found to be SDB. Developed by Sabouraud, this medium is specifically designed for cultivating fungi, particularly those of clinical relevance. It is considered enriched due to its high concentration of dextrose as a carbon source and peptone as a nitrogen source. Its low pH favors morphological identification by stimulating spore and pigment production, making it especially suitable for fungal isolation.

By contrast, Bjerkandera sp. CCMIBA_R111, a member of the Basidiomycota phylum, demonstrated a higher mycelial mass concentration when cultured in malt extract enriched with peptone and yeast extract. Culture media enriched with complex substances such as meat, malt, and yeast extracts support the growth of a wide range of microorganisms. A study by Korniłowicz-Kowalska and Rybczyńska-Tkaczyk (2021) investigating the growth conditions of Bjerkandera sp. found that media rich in natural organic components were particularly effective, with carbon and organic nitrogen identified as key nutritional requirements.

The results of the assessment of ATZ degradation potential reveal intriguing findings regarding both the efficiency of the selected fungi in degrading ATZ and their subsequent impact on toxicity levels. Toxicity assessments using C. sativus as a phytotoxicity model showed nuanced outcomes. Despite a lower percentage of ATZ degradation, the supernatant from the test with P. lilacinum CCMIBA_R014 exhibited a remarkable 100% reduction in toxicity. This suggests that although this fungus may not degrade ATZ as efficiently as others, its metabolic byproducts or other mechanisms may effectively mitigate toxicity, allowing for healthy growth of the model seed.

Purpureocillium lilacinum has attracted significant interest for its potential in biocontrol, as it is an entomopathogenic species effective in the biological control of nematodes. It is classified as an opportunistic parasite of nematode eggs, cysts, and adults, producing extracellular enzymes that degrade the proteins and chitin present in the eggs and bodies of these nematodes (Khan et al., 2003). Spinelli et al. (2021) reported that P. lilacinum is an excellent candidate for the remediation of the herbicide glyphosate because it was able to degrade 80% of the initial concentration of glyphosate and use it as a source of phosphorus. Additionally, P. lilacinum has been reported to be tolerant to several other herbicides with active ingredients such as pendimethalin, pethoxamid, clomazone, chlortoluron, and imazamox (Ondráčková et al., 2019). It also has the ability to biosorb and remediate heavy metals such as cadmium, chromium, and lead (Sharma and Adholeya, 2011).

Conversely, cultivation of C. sativus with the supernatants of Bjerkandera sp. CCMIBA_R111 and C. rosea CCMIBA_R018 indicated 85% and 81% toxicity, respectively, reducing only 17% of ATZ toxicity on average despite exhibiting higher degradation rates. This highlights a discrepancy between ATZ degradation and toxicity reduction, suggesting that factors beyond simple degradation rates may influence overall toxicity levels. The variation in toxicity after ATZ degradation is due to the generation of metabolites (Vryzas et al., 2007). According to Ralston-Hooper et al. (2009), using a different toxicity model, the overall ranking of acute and chronic toxicity classified ATZ as the most toxic compound, followed by its derivatives desethylatrazine and desisopropylatrazine. Currently, assessing degradation alone is insufficient to indicate bioremediation potential. Therefore, studying toxicity is the primary method for monitoring the treatment process. Because the project’s initial aim was to evaluate degradation potential, and considering that the ongoing studies aim to co-cultivate the fungi, associating these fungi with eliminating toxic compounds becomes an even more compelling strategy.

Clonostachys rosea was selected for co-cultivate in the bioinoculant because of its characteristics in the biological control of mycoparasites, acting against fungal plant pathogens, nematodes, and insects (Sun et al., 2020). However, the present work presented interesting results in the degradation of ATZ.

With respect to the genus Bjerkandera, Dhiman et al. (2020) demonstrated the efficacy of Bjerkandera adusta in removing ATZ by up to 92% in batch liquid cultures under various pH, temperature, and concentration conditions. Additionally, B. adusta exhibits proficiency in degrading other herbicides such as chlortoluron, isoproturon, and diuron. Furthermore, Bjerkandera sp. showcases the capability to bioremediate soils contaminated with polycyclic aromatic hydrocarbons (Kotterman et al., 1998). These studies collectively highlight the variability in the ability of fungi from the Bjerkandera genus to degrade ATZ or other toxic compounds, contingent upon experimental conditions such as temperature, pH, culture medium, contaminant concentration, and incubation duration. Such findings underscore the importance of optimizing environmental parameters to enhance fungal remediation efficacy in diverse pollution scenarios.

Biochar was selected as a substrate for the fungi because it is also a promising organic material for remedying soil contamination. Several studies have demonstrated biochar’s effectiveness in remediating environments contaminated with organic pollutants (Guo et al., 2020; Haider et al., 2022). The immobilization of microorganisms in biochar is regarded as an optimization of bioremediation. Bacteria immobilized in biochar and applied to soil have been shown to enhance soil quality and increase the degradation rate of polycyclic aromatic hydrocarbons (Bonaglia et al., 2020; Song et al., 2021), as well as petroleum hydrocarbons (Guo et al., 2022). Similarly, immobilizing arbuscular mycorrhizal fungi on biochar in chicory cultivation serves as an organic fertilizer, augmenting soil microbiota and acting as a soil remediator for cadmium contamination, thereby reducing cadmium absorption by the soil (Zhao et al., 2021). In another study, it was observed that biochar, when combined with fungus, can decrease cadmium levels in soil by up to 62%, consequently increasing the yield of kangkong plants (freshwater spinach) by more than 25% (Hu et al., 2014).

Considering the results, we found that beyond identifying the optimal growth medium for each fungus, a more effective strategy involved pre-producing biomass using biochar. This approach provided the fungi with a nutrient-rich culture broth while also allowing them to adapt to the biochar as a support material. This innovative method, not previously identified in our literature review, emerged as a novel alternative proposed in this study.

Several studies have linked the ability of fungi to break down ATZ and its derivatives to non-specific extracellular enzymes such as Lac, MnP, and LiP (Parenti et al., 2013; Lin et al., 2014; Knop et al., 2015). Nonetheless, other research suggests that the primary enzymes involved in the degradation of this herbicide are intracellular, particularly those in the cytochrome P450 complex (Verma et al., 2014). Given this context, we evaluated the ligninolytic potential of the fungi as part of the consortium’s bioremediation strategies. Although ascomycetes are not typically known for strong production of this enzyme group, they still demonstrated enzymatic potential. However, the presence of biochar was found to negatively affect their activity.

Biochar can affect the activity of ligninolytic enzymes in fungi through various mechanisms. Its high surface area and strong adsorptive properties may lead to the adsorption of enzymes such as Lac, MnP, and LiP (Baldrian, 2006; Wong, 2009). Additionally, biochar may interfere with enzyme production by sequestering essential nutrients or inducers required for the synthesis of ligninolytic enzymes (Elisashvili and Kachlishvili, 2009; Novotný and Svobodová, 2010). Chemical interactions may also reduce the availability of substrates necessary for enzyme activity (Elisashvili and Kachlishvili, 2009; Chandra and Chowdhary, 2015). Furthermore, changes in pH and other environmental conditions induced by biochar can affect the optimal functioning of these enzymes (Ren D, Wang Z, Jiang S et al., 2020; Elisashvili and Kachlishvili, 2009). The presence of biochar might also trigger metabolic shifts in fungi, diverting resources away from ligninolytic enzyme production (Pointing and Hyde, 2000). Together, these factors contribute to the observed reduction in enzyme activity following exposure to biochar. However, once the bioinoculant is added to the soil, there is no longer an impediment for the fungi—now isolated from the consortium environment—to resume enzymatic activity. Notably, within the consortium, the activity of LiP, an important enzyme for ATZ treatment, was maintained.

After obtaining the bioinoculant, the initial phase of product evaluation focused on assessing ATZ degradation. These results underscore the crucial role microbial consortia play in the degradation of pesticides in soil. A 34% reduction in the ATZ concentration in the sterilized soil microcosm suggests that even in the absence of native soil microbiota, the bioinoculant consortium can effectively degrade ATZ. This finding aligns with existing literature, which highlights the potential of bioinoculants to enhance the biodegradation of persistent organic pollutants in various environments (Awasthi et al., 2014).

The more substantial 66% reduction in ATZ concentration in the non-sterilized soil microcosm indicates that native soil microbiota, when combined with the bioinoculant consortium, produces a synergistic effect that enhances the degradation process. This observation aligns with studies showing that microbial consortia can work synergistically with native microbes to break down complex compounds more efficiently than single strains alone (Cao et al., 2022).

The difference in ATZ degradation between sterilized and non-sterilized soils underscores the importance of microbial diversity and community interactions in maintaining soil health and supporting pollutant degradation. Non-sterilized soils typically harbor a rich microbial community capable of contributing to contaminant breakdown through multiple metabolic pathways (Torsvik and Øvreås, 2002).

The physical and chemical parameters evaluated revealed significant differences, notably an increase in phosphorus and sulfur levels, as well as a reduction in organic matter compared with the controls. Phosphorus is recognized as one of the essential nutrients for promoting soil fertility, playing a key role in the storage and transfer of energy produced during photosynthesis (Johan et al., 2021). Previous studies have shown that incorporating biochar into soil can enhance phosphorus availability (Ng et al., 2022). In our study, the introduction of the bioinoculant may have further contributed to this effect, complementing the known benefits of biochar.

Several studies have investigated strategies to improve sulfur and phosphate levels in soil, including the use of fungal bioinoculants and biochar. Fungal bioinoculants, in particular, have received attention for their ability to mobilize and solubilize nutrients, including sulfur and phosphate. For instance, mycorrhizal fungi form symbiotic relationships with plant roots, enhancing the uptake of these nutrients and thereby improving overall plant health (Smith and Read, 2008).

Additionally, biochar has been explored as a soil amendment to increase sulfur and phosphate availability. Because of its large surface area and high adsorption capacity, biochar supports the retention and gradual release of nutrients—sulfur and phosphate among them—to plants (Lehmann, 2015).

While evidence suggests that both fungal bioinoculants and biochar can positively influence sulfur and phosphate levels in soil, it is important to recognize that their effectiveness may vary depending on factors such as soil composition, fungal species, and the specific type of biochar used. Further research is needed to fully understand the efficacy of these approaches and their broader implications for plant health and agricultural productivity.

Moreover, the ability of the bioinoculant consortium to significantly reduce ATZ levels in agricultural soils carries important implications for improving soil quality and ensuring crop safety.

This study presents an innovative approach to soil bioremediation through the use of a fungal consortium immobilized on biochar. It not only opens new avenues for the application of fungal bioinoculants but also raises important questions about soil treatment processes and microbial interactions. Based on these findings, future research should focus on evaluating the interactions among the three fungi during immobilization, analyzing ligninolytic enzyme activity, investigating the mechanisms of ATZ degradation, and conducting greenhouse experiments with plant species. This work highlights the potential of fungal bioinoculants in soil treatment and underscores the importance of understanding microbial dynamics for the development of effective bioremediation strategies.

Funding

This work was financially supported by UNILA and CAPES

Competing Interests

The authors declare that they have no competing interests.

Authors’ contribution

Adriana H. Siqueira: Formal analysis, Methodology, Funding acquisition, Project administration, Writing - original draft. Giuliana Bonassa, Alessandra Andreta and Dayana Lunkes Colaço: Formal analysis, Methodology. Rafaella Costa Bonugli-Santos: formal analysis, Funding acquisition, Project administration, Supervision, Visualization, Writing - original draft, Writing - review & editing.

Data Availability

Not applicable.

Ethics approval

Not applicable.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publication

The authors declare to participate in the work, and they agree with the publication.

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Biochar-immobilized Fungal Consortia for Enhanced Soil Bioremediation

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