Biodegradation of Atrazine And Ligninolytic Enzyme Production By Basidiomycete Strains

doi:10.21203/rs.3.rs-19578/v1

Atrazine is one of the most widespread chlorinated herbicides, leaving large bulks in soils and groundwater. Some bacterial strains can degrade atrazine, but little is known if fungal species have the same ability. Thus, we investigated the toxicity and degradation of atrazine by 13 basidiomycete strains. In liquid medium, P. cubensis SXS320, Gloelophyllum striatum MCA7, and Agaricales MCA17 removed 30, 37, and 38%, respectively, of initial 25 mg.L^-1of herbicide within 20 days. Deficiency of nitrogen drove atrazine degradation by Pluteus cubensis SXS320 and also induced synthesis and secretion of extracellular laccases by Datronia caperata MCA5, Pycnoporus sanguineus MCA16, and P. tenuiculus MCA11. Enzyme levels in contaminated medium of P. tenuiculus MCA11 were 13.3 fold superior than in control. However, we found no relationship between laccase production and the ability to metabolize atrazine. Laccase from P. tenuiculus MCA11 was incubated under optimal physico-chemical conditions for catalysis (pH 2.5 and 40º C), with an atrazine solution. Although 88% of initial laccase activity remained after 6h, no evidence of in vitro degradation was observed, even though ABTS was present as mediator.

Plant Molecular Biology and Genetics

fungal metabolism

bioremediation

laccase

atrazine

biodegradation

co-metabolism

Agricultural production worldwide is strongly dependent on agrochemicals. Atrazine is one of the most employed herbicides to control broadleaf weed in sugarcane and maize plantations. It has considerable persistence, mobility and solubility in soils, with a half-life of around 140 days. This herbicide can also cause groundwater contamination. As a result, atrazine may enter the food chain, affecting human health (Abate, Masini, 2005; Armas et al., 2005).

Atrazine (2-chloro-4-ethylamino-6-isopropylamino)-1,3,5-triazine) belongs to the chemical class of chlorinated triazines and is formed by a heterocyclic aromatic ring alternating carbon and nitrogen atoms, besides chlorine substituent (Barid, 2002; Donnelly et al. 1993; Mougin et al. 1994). Like other halogenated aromatic compounds, atrazine and its dealkylated and deaminated metabolites are toxic and carcinogenic (Ralebitso et al., 2002). Indigenous microorganisms can degrade and detoxify this kind of compounds. Thus, they can potentially be used in bioremediation of contaminated areas, if the physiological routes involved in these processes are known (Gianfreda et al., 2006).

White-rot fungi produce non-specific oxidases that can act upon a wide range of substrates. Laccases, lignin peroxidases, and manganese peroxidases can degrade pollutants, such as PAHs, polychlorinated phenols, polymers, dioxins, pesticides, and dyes (Cameron et al., 2000; Gomes et al., 2009; Rabinovich et al., 2004). Fungi are promising organisms for use in bioremediation (Harms, Schlosser, Wick, 2011). Besides producing inoculum in large scales, they synthesize and secrete enzymes responsible for translocating essential factors throughout the hyphae, allowing them to colonize new environments. Here, we screened fungal strains, isolated from tropical forest fragments, for their capacity of growing in the presence of atrazine and metabolizing it. We also evaluated ligninolytic enzyme production of these strains.

Microorganisms

We used basidiomycete strains isolated from decaying wood from Atlantic Semi-deciduous forest fragments at “Noroeste Paulista Ecological Station”, Northwestern São Paulo, Brazil (Santos et al., 2004; Abrahão et al., 2008). Fungi were preserved in sterile distilled water (Castellani, 1969).

Fungal tolerance to atrazine

A commercial formulation of atrazine (Nortox® 500SC, 50% atrazine m/v) was used to test fungal resistance and biodegradation. The herbicide was added to liquid and warm PDA medium, to reach concentrations of 6.25 g.L^-1 and 10.0 g.L^-1. The medium was homogenized and placed in Petri dishes. Plates were inoculated with 6 mm agar plugs from actively growing cultures in PDA and maintained at 28º C, in the dark, for 15 days. Colony diameters were measured daily and results were expressed as percentage of inhibition related to control (PDA cultures without herbicide). Mycelium density was visually measured and categorized as high, medium, or weak. Results were expressed as mean of three replicates for each treatment.

Inoculum for liquid cultures

To standardize inoculum size in liquid culture experiments, a cell suspension was produced. Mycelia fragments were scraped from growing cultures of each strain in PDA medium and transferred to 250 mL Erlenmeyer flasks containing 50 mL of culture medium (see Kirk et al., 1978). The medium was supplemented with 1 % of glucose and NH₄NO₃to supply 25 mM of nitrogen. After 6 days at 28º C, cultures were drained and mycelium was washed twice with sterile Knapp buffer (Tixier et al., 2000), being grinded on a sterile mixer with the same buffer. The resultant cell suspension was diluted in buffer to reach an optical density of 0.5 at 550 nm (DU Spectrophotometer, Beckmann), and then used immediately to inoculate test cultures.

Atrazine degradation and enzyme production

An atrazine stock solution was prepared by homogenizing the commercial formulation in methanol, followed by two sonication pulses of 15 min and centrifugation at 10,000 g for 10 min. Supernatant was filtered through a Millipore membrane of 0.2 µm pore size. Aliquots were dispensed into sterile 125 mL Erlenmeyer flasks to reach final concentrations of 0 to 50 mg.L^-1. After complete methanol evaporation, 25 mL of sterile culture medium (Kirk et al. 1978) were added. The medium was supplemented with 1% glucose and NH₄NO₃ (to reach 2.5 mM of N for a nitrogen-deficient medium, or 25 mM of N for nitrogen sufficiency). Each flask was inoculated with 1 mL of the cell suspension. Cultures were vacuum filtered after incubation for 20 or 40 days, under static conditions at 28º C. Supernatants, referred as crude extracts, were kept in ice bath for enzyme assays, and aliquots were frozen. Mycelia were dried at 70º C to evaluate biomass and fragments were reserved to measure fungal atrazine sorption. Sterile controls were included. Results are express as mean of three replicates under each condition.

Atrazine biotransformation HPLC analysis

Frozen samples from cultures were melted, centrifuged at 10,000 g for 10 minutes and filtered in a 0.22 µm pore size filter. Then, 20 µL of each sample were injected into a Jasco HPLC, by using a C18 Perkin Elmer column contained in an oven at 40ºC. Pesticides and metabolites were eluted by using a mobile phase of acetonitrile:water (55/45 v/v) with a flow rate of 1 mL.min^-1, and detected by UV absorbance at 220 nm. Analytical standard was purchased from Sigma (atrazine, 98% of purity). To evaluate sorption of atrazine to fungal mycelium, the biomass (approximately 150 mg) was grinded in a mortar and extracted twice with 500 µL of n-hexane. Extracts were combined and air dried. The residue was re-suspended into 1 mL of mobile phase, filtered through 0.22 µm pore filter, and analyzed in chromatograph.

Enzyme assays

Laccase activity was measured throughout the reaction using 0.9 mL of sodium acetate buffer (10 mM, pH 3.5) with 0.03% of ABTS (Fluka, Switzerland), and 0.1 mL of diluted enzyme crude extract. After incubation for 1 minute at 40º C, the absorbance was read at 420 nm (Buswell et al. 1995). For the reaction blanks, water replaced enzyme extract. Reaction mixtures of controls lacked ABTS. One unit of activity represents the amount of enzyme that oxidizes 1 µmol of ABTS per minute, considering ε₄₂₀= of 3,6.10⁴ M^-1cm^-1 for oxidized ABTS. Activity was expressed as U.g^-1 of dry biomass.

Analysis for manganese-dependent peroxidases were carried out by adding 0.1 mL of crude enzyme extract into a mixture containing 0.8 mL of sodium lactate buffer (50 mM, pH 4.5), 0.1 mL of MnSO₄ solution (400 mM) and 4 µL of a 10 mM H₂O₂solution. After 10 minutes incubated at 40º C, readings were made at 240 nm (Gianfreda et al. 2006). In the blanks, enzyme was replaced by distilled water, while controls lacked MnSO₄. One unit of enzyme activity represented the enzyme necessary to create 1 µmol of complexes lactate-Mn³⁺ per minute, considering ε₂₄₀= 8,1.10³ M^-1cm^-1 (Aitken, Irvine, 1990).

Reaction for lignin peroxidases was based on the oxidization of veratryl alcohol into veratraldehyde. Enzymatic extract was added to a mixture containing 0.8 mL of sodium tartarate buffer (pH 3.0, 50 mM), 0.1 mL of a 40 mM veratryl alcohol (Fluka, Switzerland), and 4 µL of 100 mM H₂O₂ (Tien, Kirk, 1988). After 10 minutes at 40º C, absorbance was read at 310 nm. In the blanks, distilled water replaced the enzyme; in controls, veratryl alcohol was replaced by water. One unit of enzyme activity represents the amount of enzyme that oxidizes 1 µmol of veratraldehyde per minute, using ε_310nm = 9.10³ M^-1cm^-1.

Effects of laccase upon atrazine in vitro

A reaction mixture was incubated at 40º C for 24 h containing 0.8 mL of a 25 mg.L^-1 atrazine solution, 0.1 mL of a 500 mM sodium acetate buffer (pH 2.5) with 0.03% of ABTS and 0.1 mL of P. tenuiculus MCA11 enzyme crude extract. Periodically, aliquots were taken and immediately frozen in liquid nitrogen. For the analysis of atrazine content, melted samples were filtered through a 0.22 µm membrane and evaluated by HPLC. Reactions employing pH 7.0 (McIlvaine buffer) or omitting ABTS, enzyme or atrazine were included as controls.

Data analysis

One-way ANOVA, followed by Tukey post-hoc tests, was used to test for the influence of fungal strains on atrazine levels, growth, and enzyme production. Analysis was conducted in the software PAST v. 2.17 (Oslo University).

Tolerance of basidiomycetes strains to atrazine in solid medium

Although all microorganisms were sensitive to atrazine to some degree, all could grow. Additionally, inhibition rates were very similar for the two atrazine concentrations (Tables 1 and 2), but distinct from the control. We found minor inhibitory effect at 6.25 mg.L^-1for Polyporus tenuiculus MCA9 (42%) and Pluteus cubensis SXS320 (44%). Conversely, the most affected strains were Polyporus sp. MCA128 and Datronia stereoides MCA167, which showed inhibition around 72 and 73%, respectively. However, the sensitivity of individual strains did not differ. Many strains also differed in pigmentation and mycelium density, when compared to control, probably in response to chemical stress created by the herbicide (Carlile et al., 2001). Interestingly, the fungal mycelium of some strains was much denser in contaminated medium, when compared to control, like Gloellophylum striatum MCA7. All these responses must be considered if strains are to be selected for bioremediation, since the success of environmental decontamination depend on morphological and physiological properties that regulate colonization and dispersion abilities (Pedra, Marino, 2006; Tien, Kirk, 1988).

Mycelia growth and atrazine degradation in liquid medium

In 20 day cultures containing atrazine, the biomass production decreased for the majority of strains, demonstrating the toxic effects of the herbicide, but this inhibition had been clearly lower than in solid medium (Table 2). There was no correlation between inhibition of mycelia growth and atrazine degradation.

Many recent studies have pointed out the feasibility of pesticide degradation by fungi in surface cultivation system. For example, dissipation rates of atrazine has been reported as being as high as 50 times after 40 days (Fan, Song, 2014). In this study, at least 11 strains degraded the herbicide in levels varying from 2.9 to 38.7%. The degradation potential of 3 groups was significant (Table 2). This happened despite growth inhibition. The growth of some strains, such as Gloelophyllum striatum MCA7 and unidentified Agaricales MCA17 was not inhibited by atrazine in surface cultivation medium. These microorganisms achieved 37.3 and 38.7% of degradation, respectively. However, Polyporus sp. MCA128, Datronia stereoides MCA167, Pluteus cubensis SXS320, and D. caperata MCA5 were slightly inhibited by atrazine and had a very variable xenobiotic metabolism (Table 2). Abiotic disappearance of herbicide was not detected.

A complete catabolic pathway for atrazine biodegradation is known only for bacteria (e.g., Pseudomonas sp. ADP). The plasmidial genes atzA, atzB, and atzC encode hydrolytic enzymes, responsible for atrazine mineralization (Govantes et al. 2009). Conversely, fungal biodegradation is based on oxidative-hydrolytic mechanisms and no strain is capable of mineralize atrazine completely. Fungi usually modify atrazine by sequentially removing aromatic ring substituent, with dealkylation as first step (Mougin et al., 1997; Mougin et al., 1994). The most studied basidiomicete strains, Pleurotus pulmonarius and Phanerochaete chrysosporium, change atrazine to metabolites retaining the aromatic ring, such as de-ethyl-atrazine, de-isopropylatrazine, de-ethyl-isopropylatrazine, and hydroxyatrazine (Mougin et al., 1994; Masaphy et al., 1996).

Atrazine metabolism and nitrogen availability

Herbicide metabolites appeared in culture extracts in which occurred some atrazine dissipation (data not shown). We investigated atrazine biodegradation in more detail using Polyporus tenuiculus MCA11 and Pluteus cubensis SXS320, because they had high tolerance to the herbicide, degradation rates, and laccase production. The influence of nitrogen availability on atrazine metabolism was also evaluated. Both strains were capable of reducing atrazine concentration in the media, by converting it to more polar metabolites (Figure 1-peaks 5). Pluteus cubensis SXS320 did not produce any ligninolytic enzyme, but we found a single profile of biotransformation, closely related to nitrogen availability. A more intense degradation of atrazine was achieved by fungi in medium lacking nitrogen, reaching 30% of removal after 20 days of cultivation (with 2.85 % adsorbed to biomass). Conversely, only 21% was removed after 40 days in nitrogen-rich medium (reaching a biomass adsorption of 4.46%). Biomass was smaller in nitrogen-deficient cultures (0.943 g.L^-1, Table 2), while cultures with sufficient nitrogen produced 1.6 g.L^-1of biomass on the 20^th day of cultivation (data not shown). Otherwise evidences found to Phanerochaete chrysosporium, inferring only a proportional dependence of biomass and associated enzymes for atrazine degradation (Mougin et al., 1997; Molugin et al., 1994), metabolism of P. cubensis SXS320 showed more complex nitrogen-dependent mechanisms. Mineralization could not be estimated because degradation produces aromatic metabolites which lacks standards to be measured (peaks #5, Figure 1).

The degradation of xenobiotics by fungi depend primary on carbon and nitrogen sources, sicne biotransformation of aromatic compounds occurs as part of its secondary metabolism (Gadd et al., 2004). However, recent studies revealed a fungal strain (tentatively identified as Penicillium sp.) that uses atrazine as nitrogen source (Singh et al., 2008). Some fungi could have degradation routes that are target of catabolic repression. Also, nitrogen amendments in growth medium often reduce xenobiotics’ dissipation by inhibiting transmembrane transport, as well as regulating enzymatic systems involved with degradation of the herbicide (Gianfreda et al., 2006; Govantes et al., 2009; Mandelbaum et al., 1995).

The influence of nitrogen availability upon atrazine degradation was not clear in P. tenuiculus MCA11. This strain removed 39.1% of initial atrazine after 40 days in culture medium with 25 mM of nitrogen, while in a culture containing 2.5 mM, degradation reached 13.9%, in 20 days of culture. The profile of metabolites after 40 days in high-nitrogen medium was similar to that of P. cubensis SXS320 under the same conditions, but only one peak appeared in low-nitrogen medium (peak 5, retention time of 3.16 minutes, Figure 1C). Two different peaks appeared in cultures of P. cubensis SXS 320, with retention times of 2.99 and 3.40 minutes (Figure 1b), but three peaks appeared in cultures with high nitrogen, with retention times of 2.98, 3.6 and 3.40 minutes (Figure 1A).

We cultured P. tenuiculus MCA11 with atrazine (from 0 to 50 mg.L^-1), with high N concentration (25 mM) to further explore the relationship among nitrogen availability, atrazine degradation, and laccase biosynthesis. Laccase was slightly induced by increasing atrazine levels, reaching a 4-fold activity level in 50 mgL^-1 atrazine medium, in comparison with control without the herbicide (Figure 2). This induction was lower than in the presence of 2.5 mM of N, in the medium without the herbicide that was 13-fold (Table 2). This pattern suggests that high nitrogen availability repress laccase synthesis. Taken together, these results suggest that laccase biosynthesis is induced by atrazine, repressed by nitrogen, while herbicide degradation rates are more influenced by nitrogen availability than laccase levels.

Correlation between lacase production and atrazine metabolism

Tests of ligninolytic activity are often used for selecting microorganisms for bioremediation of aromatics (Bending et al., 2002). In fact, enzymes of the laccase group, lignin peroxidases and manganese peroxidases can co-metabolyze pollutants like PAHs, phosphothyolates, polychlorinated phenols, polymers, dioxins, and pesticides (Cameron et al., 2000; Rabinovich et al., 2004; Amitai et al., 1998; Keum, Li, 2004; Johannes, Majcherczyk, 2000). In this study, only laccases could be detected in culture extracts with four strains. Maybe surface fermentation did not provide adequate conditions for the synthesis and secretion of these enzymes, since basidiomycetes are common producers. When one of the strains produced laccase, enzyme synthesis increased in presence of the xenobiotic (Table 2). Similar behavior is known for Cerrena maxima, Coriolus hirsutus (Gorbatova et al., 2006), Phanerochaete chrysosporium BKM-F-1767 (Fratila-Apachitei et al., 1999), and Trametes versicolor (Mougin et al., 2002). Additionally, previous studies reported a link between laccase biosynthesis and detoxification potential (Gorbatova et al., 2006). However, we found no evidences for a relationship between ligninolytic enzyme production and herbicide degradation (Table 2). This lack of connection also occurred for Pleurotus ostreatus, Bjerkandera adusta and Phanerochaete chrysosporium (Novotný et al., 2004; Singh et al., 2008), whose degradation is via cytochrome P450 monooxygenase system. Atrazine degradation rates for Pleurotus ostreatus INCQS 40310 were highly variable under different culture conditions. Recent studies pointed out that levels of FeSO₄ and MnSO₄ were key macronutrients for herbicide catabolism, but found no correlation between laccase levels and atrazine dissipation (Pereira et al., 2013). These aspects of xenobiotic catabolism by basidiomycetes remain unclear.

Although laccase had retained 88% of initial activity after 6 hours of incubation, no atrazine disappearance occurred. A recent study employing crude enzyme extract from Trametes versicolor exhibiting laccase activity reports a poor performance in atrazine removal (less than 5% after 24h), unless a mediator is used. When 1 mM of HBT (1-hydroxy benzotriazole) was provided, degradation rates improved by 93% (Nguyen et al., 2013). In this study, ABTS was employed as mediator, but was unable to oxidize chloride groups in the molecular structure of the herbicide. Therefore, atrazine removal depends on cell-bounded enzymes and/or cofactors that could act synergistically with laccase. The role of laccase in this process requires a deeper evaluation.

In conclusion, the basidiomycete strains evaluated showed a high potential for bioremediation. However, metabolites resultant from fungal activity upon pesticides and their toxicity must be identified before field application. The next step towards using these microorganisms in field bioremediation involved a better understanding of nitrogen influence on fungal metabolism and the profile of metabolites associated.

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Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

Not applicable

Availability of data and materials

The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

Conflicts of interest/Competing interests

The authors declare that they have no competing interests

Funding

This study was founded by FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo (GRANT N.º 06 /05411-).

Author’s Contribution

C.H. performed the laboratory assays and was a major contributor in writing the manuscript. D.A.M. was responsible by the chromatographic analysis. M. B., R. S. and E. G. oriented he general word and reviewed the manuscript.

Acknowledgements

Authors are grateful to CNPq – Conselho Nacional de Desenvolvimento Científico e Tecnológico and FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo for financial support, as well as to Maira C. Abrahão for the fungal strains employed. Diogo Borges Provete reviewed the English language.

Table 1. Growth inhibition of fungal strains in PDA medium containing atrazine. M.D.: mycelium density. Weak (1); medium (2); high (3).

Strain	Control		6.25mg.L^-1			10.0mg.L^-1
Strain	Diameter growth (cm/day)	M.D.	Diameter growth (cm/day)	Inhibition	M.D.	Diameter growth (cm/day)	Inhibition	M.D.
Gloelophyllum striatum MCA2	0.694	2	0.359	48%	3	0.326	53%	3
Datronia caperata MCA5	0.735	2	0.239	67%	1	0.241	67%	1
Trametes modesta MCA6	1.076	2	0.429	60%	3	0.367	66%	3
Gloelophyllum striatum MCA7	0.988	2	0.386	61%	3	0.378	61%	3
Polyporus tenuiculus MCA9	0.418	3	0.244	42%	3	0.218	42%	3
Polyporus tenuiculus MCA11	0.818	3	0.413	49%	3	0.326	60%	3
Pycnoporus sanguineus MCA16	1.408	3	0.456	67%	3	0.437	69%	3
Agaricales MCA17	0.848	3	0.405	52%	2	0.433	49%	2
Polyporus sp. MCA128	1.566	3	0.439	72%	3	0.404	74%	3
Hexagonia hirta MCA131	2.998	1	1.245	58%	1	1.164	61%	1
Datronia stereoides MCA167	1.524	3	0.407	73%	3	0.379	75%	3
Dacryopinax elegans SXS323	3.635	3	1.558	57%	3	0.572	57%	3
Pluteus cubensis SXS320	1.906	3	1.069	44%	3	0.729	62%	3

Table 2. Fungal biomass, laccase activity and percents of herbicide disappearance after 20 days in liquid medium, with 25mg.L^-1 of initial atrazine.

Strain	Control		Culture in presence of 25 mg.L^-1 of atrazine
	Biomass (g.L^-1)	Lac (U.g^-1)	Biomass (g.L^-1)	Inhibition of growth (%)	Lac (U.g^-1)	Degradation (%)
Gloelophyllum striatum MCA2	0.928	-	0.517	45	-	0.0
Datronia caperata MCA5	0.794	25.0	0.640	19	75.7	10.5
Trametes modesta MCA6	1.706	-	0.931	45	-	14.1
Gloelophyllum striatum MCA7	1.300	-	1.377	0	-	37.3
Polyporus tenuiculus MCA9	0.976	-	0.623	37	-	4.42
Polyporus tenuiculus MCA11	1.716	2.41	0.851	51	32.18	13.9
Pycnoporus sanguineus MCA16	1.468	9.89	1.008	32	15.18	11.4
Agaricales MCA17	0.781	4.03	0.715	0	-	38.7
Polyporus sp. MCA128	5.074	-	4.622	19	-	0.0
Hexagonia hirta MCA131	2.108	-	1.352	36	-	8.6
Datronia stereoides MCA167	5.522	-	5.039	18	-	2.9
Pluteus cubensis SXS320	1.046	-	0.943	10	-	30.0
Dacryopinax elegans SXS323	2.394	-	1.433	40	-	3.8
Not inoculated medium (abiotic control	0	0	0	0	0	0

Biodegradation of Atrazine And Ligninolytic Enzyme Production By Basidiomycete Strains

Status:

Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

Results And Discussion

References

Declarations

Tables

Status:

Journal Publication

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