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Article

Heavy Metal Biosorption Ability of EPS Obtained from Cultures of Fusarium culmorum Strains with Different Effects on Cereals

by
Jolanta Jaroszuk-Ściseł
1,*,
Artur Nowak
1,
Małgorzata Pac-Sosińska
2,3,
Dorota Kołodyńska
4 and
Iwona Komaniecka
5,6,*
1
Department of Industrial and Environmental Microbiology, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Akademicka St. 19, 20-033 Lublin, Poland
2
Department of Virology and Immunology, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Akademicka St. 19, 20-033 Lublin, Poland
3
Laboratory of Bioinformatics and Biostatistics, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Akademicka St. 19, 20-033 Lublin, Poland
4
Department of Inorganic Chemistry, Institute of Chemical Sciences, Faculty of Chemistry, Maria Curie-Sklodowska University, Maria Curie-Sklodowska Sq.2., 20-031 Lublin, Poland
5
Department of Genetics and Microbiology, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Akademicka St. 19, 20-033 Lublin, Poland
6
Laboratory of Structural Analyses, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Akademicka St. 19, 20-033 Lublin, Poland
*
Authors to whom correspondence should be addressed.
Sustainability 2025, 17(8), 3744; https://doi.org/10.3390/su17083744
Submission received: 12 March 2025 / Revised: 10 April 2025 / Accepted: 18 April 2025 / Published: 21 April 2025
(This article belongs to the Section Environmental Sustainability and Applications)
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Versions Notes

Abstract

:
To develop a strategy for sustainable bioremediation of heavy metal-contaminated environments, it is necessary to understand the mechanisms of remediation using microorganisms. A huge bioremediation potential is possessed by fungi. Fusarium culmorum, with their wide range of plant hosts, can be the basis for creating sustainable phytoremediation technologies and for creating sustainable agriculture methods. Exopolymers (EPSs) produced by F. culmorum can be excellent metal sorbents and basic factors in the biosorption mechanism. The sorption capacities of zinc, lead, and cadmium by the EPS of a pathogenic DEMFc37 strain and two non-pathogenic strains (PGPF-DEMFc2 and DRMO-DEMFc5) were compared, and the effects of these metals on EPS synthesis by the three strains was determined. EPS samples were chemically characterised in regards to their sugar, protein, and phenolic compound contents and used to study metal binding. The concentrations of metals bound/adsorbed to EPS were determined by Atomic Absorption Spectroscopy. The EPSs of all the strains bound more than 80% of Zn, as well as 64–84% of Cd and 74–79% of Pb. Thus, it has been clearly shown that the use of F. culmorum EPSs can be the basis for creating sustainable bioremediation, including phytoremediation.

1. Introduction

Microbial extracellular polymers, furnished with many functional groups, can act as excellent adsorbents of heavy metals (HMs), and can play an important role in the acquisition of resistance in metal-contaminated environments.
Extracellular polymeric substances (EPSs) produced by microorganisms (bacteria and fungi) are characterised by diverse properties and great plasticity, allowing their versatile use. First and foremost, they can protect microorganisms from various abiotic and biotic stresses [1,2] and can shape the soil environment, as well as the interactions occurring in the environment among microorganisms and between microorganisms and plants [3]. The type of fungal endophytic strains’ effect on plants can be strongly modified by heavy metals present in the plant growth environment, whereby contact with metals can increase the resistance of fungi to other factors regulating their growth and development [4]. The toxicity of metals to filamentous fungi is manifested at the morphological level by inhibition of growth of the filaments, their decolourisation, and the disruption of sporulation and spore germination. At the cellular level, fungi respond with the induction of antioxidant processes, and the hormesis phenomenon plays an important role in enhancing adaptation to diverse plant host micro-niches [5].
Effective plant protection agents against phytopathogens include HMs such as Zn and Cu, which are present in numerous fungicides, and the toxic effect of such zinc-based fungicides as zinc pyrithione is due to an increase in the cellular binding of this metal [6]. It is important to consider that most heavy metals (Cd, Cu, Hg, Pb, etc.) are antifungal agents. Cu and Zn are essential for growth and are physiologically important but most HMs are toxic, with the toxicity of physiological elements being concentration-dependent [7].
Filamentous fungi, including Ascomycota of the genus Fusarium, show a high tolerance to the presence of heavy metals in the environment [8]. The problem of HM accumulation in the fruiting bodies of saprophytic and ectomycorrhizal Basidiomycota fungi, which is species-specific and concentration-dependent, is well known [9,10]. These fungi have been shown to differ in their ability to accumulate metals: Agaricus campestris, Macrolepiota procera, and Boletus edulis strongly bound Cd, Pb, and Hg, respectively [10,11].
Heavy metals affect not only the physiology but also the morphology of fungi by affecting both vegetative filaments and sporulation and asexual and sexual reproduction, but there are relatively few studies on this topic [5,12].
EPSs primarily alter the surface area of both soil particles and the plants that colonise it. They also provide a source of carbon and other biogenic elements (oxygen, hydrogen, and nitrogen) and, thanks to numerous ligands, also cations such as sodium, potassium, magnesium, calcium, and iron. The mechanisms of immobilisation (biosorption) of metals by EPSs are still quite poorly understood [4,13]. These properties of EPS may result from physicochemical interaction processes occurring simultaneously and complementing each other, such as mechanical, physical, and chemical sorption, complexation, precipitation, or ion exchange. The gel-forming capacity, as well as the capacity to undergo denaturation and renaturation processes, during which the number of helices making up the EPS changes, e.g., from triple to single, are important to this process. It should be denoted that, lastly, Ding and co-workers elaborated on an advanced mathematical model for pollutant migration in porous matrices that could be very useful for mathematic description of the chemical sorption of metals by EPS and applicable to predicting the diffusion of heavy metals in EPS structures [14].
The metal complexation ability of EPS is mainly influenced by the presence of at least 10 functional chemical groups: amine -NH2, cyclic nitrogen =N-, carboxyl -COOH, amide -CONH2, carbonyl =CO, hydroxyl -OH, sulfonic -SO3H, sulfhydryl (thiol) -SH, phosphate -PO(OH)2, =POOH, and phosphodiester [15,16,17]. It can be assumed that the relatively high tolerance of fungi to heavy metals is precisely related to the great diversity of functional groups in the wall polymers and especially in the exopolymers, which, by binding heavy metal ions, contribute to the resistance of fungi to these metals. However, most studies so far focus on the use of endophytic bacteria in the bioremediation of metal-contaminated soils [18,19]. Fungi, on the other hand, tend to be used in the bioremediation of petroleum-polluted sites, and mycoremediation mainly uses their enzymes [20].
Among numerous metabolites, phytopathogenic fungal strains, e.g., Plenodomus (Leptosphaeria) biglobosus and P. lingam infecting winter oilseed rape (Brassica napus L.), have been already shown to commonly produce EPSs, and the level of these polymers is species-dependent [21].
The ability to produce EPSs with antioxidant properties was demonstrated for three endophytic Fusarium culmorum fungal strains with different effects on cereal plant growth, i.e., two non-pathogenic strains, Plant Growth Promoting (PGPF)-DEMFc2 and Deleterious Rhizosphere Microorganism (DRMO) DEMFc5, and one pathogenic strain, DEMFc37, colonising the rhizosphere, the border cell zone and, with various intensity, cereal plant tissues [22,23,24]. These strains have a strong environmental impact as they are potent producers of numerous CWDEs (Cell Wall Degrading Enzymes) capable of degrading the plant and fungal cell wall [25] and a number of phytohormones [26], as well as affecting plants indirectly through the induction of resistance. The EPSs of these strains are not only important in supporting the colonisation of soil and plant roots, but also in increasing their tolerance to different stress conditions, including the presence of HMs. These strains, and the EPSs they produce, could play a significant role in the rhizosphere and endosphere of cereal plants in heavy metal-contaminated areas by assisting in the phytoremediation of soils.
The objective of this research was to evaluate the metal (Zn, Pb, and Cd) absorption capacity of the EPSs of these three endophytic F. culmorum strains. Another objective was to demonstrate the impact of the HMs tested on the productivity of the EPSs.

2. Materials and Methods

2.1. Fungal Strains

Experiments were carried out on three strains of F. culmorum (W.G. Smith) with different effects on cereal plants [21]: (1) the PGPF (plant growth promoting fungi) DEMFc2 (CBS 120098, NCBI DQ453700), (2) the non-pathogenic growth damaging strain DRMO (deleterious rhizo-sphere microorganism) DEMFc5 (CBS 120101, DQ450880), and (3) the pathogenic DEMFc37 (CBS 120103, DQ450878). The non-pathogenic strains were from the rhizosphere of healthy rye (Secale cereale L.), and the pathogenic one from winter wheat (Triticum aestivum L.) with symptoms of fusariosis [27]. Plants were collected from a field crop grown in the Lublin Upland, Lublin Province, Poland (51.15 °N; 22.34 °E) in a temperate continental climate (−20 °C to +30 °C). The tested strains were deposited in the Centraalbureau voor Schimmelcultures Collections (CBS), Utrecht, the Netherlands and are also in the Fungal Collection of the Department of Environmental Microbiology (DEM) and Industrial Microbiology. Meanwhile, nucleotide sequences of these strains (18S rRNA, internal transcribed spacer 1; 5.8S rRNA, internal transcribed spacer 2; and 28S rRNA gene [partial sequence]) have been deposited in the NCBI GenBank.

2.2. Growth of Strains on a Culture Medium with the Addition of Heavy Metals

The medium used for the cultivation of fungal strains was Czapek–Dox [28,29], which contained sucrose (30 g), peptone (7.5 g), dipotassium phosphate (1 g), magnesium sulphate heptahydrate (0.5 g), potassium chloride (0.5 g), and ferrous sulphate (0.01 g) (all salts were from POCh, Gliwice, Poland). The initial pH was 7.0. Cultures were grown until three days of growth at 20 °C. The medium was sterilised by heating to 121 °C for 30 min. The optimal medium composition and cultivation time were determined in the previously published studies [24]. After sterilisation, lead, zinc, and cadmium ions in the form of nitrates (NO3−) were added to the culture medium to obtain final concentrations of 2, 4, 6, 8, and 10 ug/mL.

2.3. EPS Isolation and Chemical Characterisation

EPS was extracted from the tested strains grown in liquid cultures using Czapek–Dox medium [24]. After removing the mycelia, ethyl alcohol (Linegal Chemicals, Blizne Łaszczyńskiego, Poland) was added to the post-culture liquid at a 1:2 volume ratio. This mixture was stored at 4 °C for 48 h. The EPS was isolated by centrifugation at 10,000 rpm for 15 min at 4 °C (MPW 350-R, Warsaw, Poland), and then freeze-dried using Freezone 6 (Labconco, Kansas City, MO, USA). The lyophilised product served as the initial crude EPS (C). A portion of the crude EPS was purified. For every 100 mg of crude EPS (C), 30 mL of 50 mM phosphate buffer (pH 7.5) was added, along with 0.01% NaN3 and 1 mg of proteinase K (Sigma Aldrich, Saint Luis, MO, USA). The mixture was deproteinised at 37 °C for 72 h with continuous stirring. After this period, the enzyme was inactivated by heating to 80 °C for 15 min. The preparations were dialysed in distilled water using a 12 kDa cellulose membrane (Sigma Aldrich, Saint Luis, MO, USA) for 7 days, with water changed every 12 h. The final EPS preparations were freeze-dried (Freezone 6; Labconco, Kansas City, MO, USA) and referred to as proteolysed EPS (P).
The total sugar content in EPS preparations (both C and P) was measured using the Dubois method [30], the protein content was determined using Bradford [31], and the phenolic compounds were estimated using Folin–Ciocalteau reagent [32], as was designated previously in Jaroszuk-Sciseł et al. [24]. Additionally, the FTIR spectra of these EPSs shown in this work indicated the presence of numerous chemical groups: -OH, -COOH, NH2, and others [24].
For sugar analysis, the EPS samples (C and P) were acid hydrolysed with 2 M trifluoroacetic acid (TFA, Sigma-Aldrich, Saint Luis, MO, USA) (100 °C, 4 h). The liberated sugar monomers were converted into alditol acetates by sequential N-acetylation, reduction with sodium borodeuteride (NaBD4, Sigma-Aldrich, Saint Luis, MO, USA) (room temp., 18 h), and acetylation with a mixture of acetic anhydride/pyridine (Sigma-Aldrich, Saint Luis, MO, USA) (Ac2O:Pyr; 1:1, v/v; 85 °C, 30 min) [33].
Methylation of the proteolysed samples (P) was performed according to the method of Hakomori [34]. The proteolysed (P) sample (~2 mg) was dissolved in 0.5 mL dry dimethylsulfoxide (DMSO, Sigma-Aldrich, Saint Luis, MO, USA), then 0.5 mL of dimsyl Na was added, and samples were methylated using 1 mL of methyl iodide (MeJ, Sigma-Aldrich, Saint Luis, MO, USA). Methylated polysaccharides were extracted with chloroform. Then, the resulting partly methylated polymers were hydrolysed in 2 M TFA (100 °C, 4 h), reduced with NaBD4, and acetylated, as above.
All obtained sugar derivatives were analysed using a 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled to a 5975C MSD detector (inert XL EI/CI, Agilent Technologies, Santa Clara, CA, USA). The chromatograph was equipped with an HP-5MS capillary column (30 m × 0.25 mm) (Agilent, Santa Clara, CA, USA). Helium was a carrier gas, with a flow rate of 1 mL min−1. The temperature program was as follows: 150 °C for 5 min, raised to 310 °C at a rate of 5 °C min−1, and the final temperature was kept for 10 min. All components were identified based on their retention parameters and characteristic mass spectra.

2.4. Estimation of the Sugar Polymer Masses by Gel-Permeation Chromatography (GPC)

The molecular mass of the EPSs was estimated using GPC at a column filled out with Sepharose CL-6B (0.7 cm × 80 cm) (Sigma-Aldrich, Saint Luis, MO, USA). EPS samples after proteolysis (P) (~5 mg) were dissolved in 1 mL of 0.5 M sodium hydroxide water solution (NaOH, POCh, Gliwice, Poland), and eluted with the same solvent, at a flow rate of 0.2 mL min−1. Fractions containing 1 mL of eluate (40 drops) were subjected to carbohydrate estimation using the Dubois method [30]. The column was calibrated using dextrans of 2 mln Da and 10 kDa molecular masses.

2.5. Binding of Metal Ions

The tested EPS lyophilisates were weighed in an amount of 1 mg/mL into an aqueous solution of lead, zinc, and cadmium ions at a final concentration of 30 mg/mL. The resulting suspension (5 mL) was placed in cellulose dialysis tubes with a 12 kDa pore diameter (Sigma-Aldrich, Saint Luis, MO, USA) and then shaken in flasks containing 30 mL of deionised water for a period of 24 and 48 h. After this incubation time, the concentration of non-binding to EPS ions was determined in the solution outside the dialysis tube by Atomic Absorption Spectroscopy (ASA), using an Agilent AAS-240 AA spectrometer (Agilent, Santa Clara, CA, USA). The percentage of ion binding was determined by the following formula [10]:
% ion removal from solution
R = 100% × [(Ci − Cf)/Ci]
R—percentage of ion binding
Ci—initial concentration
Cf—final concentration

2.6. Statistical Analysis

For all experiments, 3 biological replicates were performed. Data are expressed as mean values with standard deviation (SD). PCA was conducted using the open-source software RStudio for Windows version 2024.12.0 + 467 (Posit, PBC, GNU Affero General Public Licence v3).

3. Results

3.1. Isolation and Chemical Characterisation of F. culmorum EPS Preparations

EPS preparations obtained in the metal-free medium were used to study metal binding after 24 and 48 h incubation of lyophilised preparations at 20 °C—(1) crude [C] and (2) proteolysed [P]—for which the general composition and detailed characterisation of sugar components were determined (Table 1 and Table 2).
The crude EPS preparation of the F. culmorum DEMFc2 strain differed significantly from the C preparation produced by the other two strains, by having a 2- to 4-fold higher protein content and a slightly higher content of phenolic compounds. In contrast, the C preparation of EPS of strain DRMO DEMFc5 was characterised by a lower sugar content (Table 1). On the other hand, the proteolysed EPS of strain DEMFc2 was 4-fold lower than the P EPS of strain DEMFc5 and 5-fold lower than that of the pathogenic strain DEMFc37, and also had a lower phenolic compound content than the P preparations of the other two investigated strains.
Sugar analysis of crude, lyophilised EPS preparations (Table 2) revealed that glucose (Glc) was the main component only in EPS from the DEMFc2 strain, whereas crude EPS derived from the DEMFc5 and DEMFc37 strains contained mainly Glc and mannose (Man) (Table 2). Galactose (Gal) comprised the minor structural component of all crude preparations. Samples after proteolysis showed slightly different compositions, with equivalent amounts of Man and Glc in the cases of the DEMFc2 and DEMFc5 EPS preparations, whereas EPS from DEMFc37 contained mainly Man. Small amounts of glucosamine (GlcN) were present only in proteolysed EPS from the DEMFc2 strain. These results are in general agreement with our previous findings [24].
Analysis of the molecular mass distribution of EPSs after proteolysis showed the presence of three fractions in the preparation from the DEMFc37 strain: high molecular weight (HMW)—around 1 MDa, medium molecular weight (MMW)—around 2 KDa, and low molecular weight (LMW)—around 5 Da fractions. EPS from DEMFc2 contained only HMW and MMW fractions, whereas DEMFc5 contained only a MMW fraction (Table 2).
Linkage analysis of EPS polymers (Table 2) showed the presence of terminal hexoses (Man and Gal), →2)-linked, →4)-linked, as well as →3,4)- and →3,6)-branched sugars in the proteolysed preparation from the DEMFc2 strain. EPS from the DEMFc5 strain additionally contained → 2,3,4,6)-branched sugar residues and a lack of → 3,4)- and → 3,6)-branched sugar units. Moreover, methylation analysis of the EPS from the DEMFc37 strain showed the presence of terminal Man as the main component, →2)-, →4)- and →6)-linked sugar units, and branched → 3,6)-, as well as → 2,3,4,6)-branched hexose units. This suggests that the analysed preparation from the DEMFc37 strain was highly branched.

3.2. F. culmorum Strains’ Growth and EPS Synthesis on a Media Free of Heavy Metals and with the Addition of Heavy Metals

3.2.1. Growth of the Fungal Strains

A strong inhibition of the growth of all the three F. culmorum strains was observed in cultures with 2, 4, 6, 8, and 10 µg/mL of Cd and Pb, with a significant decrease already observed at the lowest applied concentration of 2 µg/mL (Figure 1 and Figure 2).
In contrast, the presence of Zn ions introduced at the same concentrations as Cd and Pb even caused an increase in the biomass of the strains DEMFc5 (by about 20%) and DEMFc37 (usually by 70%). In contrast, in the PGPF strain DEMFc2, at a concentration of 2 µg Zn/mL, the biomass was 82% of the control (in metal-free culture) and dropped to 41% at 10 µg/mL (Figure 2).

3.2.2. EPS Synthesis by F. culmorum Strains

EPS was obtained in cultures of F. culmorum strains on Czapek–Dox medium without metals and in the presence of individual metals at concentrations of 2, 4, 6, 8, and 10 µg/mL.
The strongest inhibition of EPS production was found in cultures supplemented with lead ions (Figure 1 and Figure 2). It has been shown that the presence of lead had a strong effect on limiting EPS synthesis by the three investigated F. culmorum strains, while the effects of cadmium and zinc on EPS production by the individual F. culmorum strains were different (Figure 1 and Figure 2). Already at 2 µg/mL, a 90% decrease in exopolymer concentration was observed, but even at a concentration of 10 µg/mL Pb, EPS was obtained (at ~0.05 g/L).
A positive correlation of the EPS concentration of the PGPF strain (DEMFc2) with the cadmium concentration (2–10 μg/L) in the medium was observed (Figure 1). In cultures of the strain DEMFc2 with Cd and Zn ions, EPS concentrations were up to 60% higher than in cultures without metals (Figure 2).
In contrast, the EPS concentration of the DRMO strain (DEMFc5) was 2-fold lower in the presence of Cd than in the control, although no correlation was observed with the Cd concentration. The DEMFc5 strain in cultures with Cd and Zn produced EPS at 60–80% of the level in control cultures.
The concentration of EPS of the pathogenic DEMFc37 strain in the presence of Cd was strongly negatively correlated with the metal concentration. In the presence of Cd ions in the DEMFc37 strain cultures, the decrease in EPS concentration was correlated with an increase in Cd concentration, and at a dose of 10 µg Cd/mL, a 10-fold lower EPS concentration was observed vs. the metal-free cultures. A similar relationship was observed when introducing Zn ions into the culture (Figure 1 and Figure 2).
In cultures of PGPR DEMFc2 with Cd ions, a 2-fold increase in the efficiency (mg EPS/g biomass) of EPS production was recorded, and in cultures of this strain with zinc ions, this efficiency was even higher, reaching a value of 455% at the highest dose of 10 µg Zn/mL (Figure 1 and Figure 2). In the case of the DRMO strain DEMFc5, a huge increase (up to 490%) in EPS generation efficiency was recorded in cultures with Cd ions (Figure 2).

3.3. Binding of Metal Ions—Chelating Properties of F. culmorum EPS

F. culmorum EPS obtained in the metal-free medium was used to study metal binding after 24 and 48 h incubation of lyophilised preparations at 20 °C—(1) proteolysed and (2) crude—for which the general composition and detailed characterisation of sugar components were determined. The concentration of the metals bound/adsorbed to EPS placed in cellulose dialysis tubes was determined by Atomic Absorption Spectroscopy (ASA). The EPSs of all the strains bound more than 80% of Zn as well as 64, 78, and 84% of Cd and 74, 77, and 79% of Pb, respectively, after 24 h of shaking EPS in the metal salt solution.
Crude EPSs of F. culmorum strains strongly sorbed metal ions after their 24 h incubation in the metal solution. The degree of immobilisation of the metals by the tested EPS after the longer incubation time (48 h) was several percent lower than after 24 h. After 48 h of shaking, the degree of immobilisation of the metals by these tested EPSs was reduced compared to the immobilisation noted after 24 h, and as after 24 h, differences were noted for EPSs derived from cultured fluids of individual strains of F. culmorum. The EPSs of the DEMFc2, DEMFc5, and DEMFc37 strains bound 30, 60, and 76% of zinc, 60, 70, and 70% of cadmium, and 45, 47, and 37% of lead, respectively.
Crude EPS preparations of the strains DEMFc5 and DEMFc37 sorbed 80–85% of cadmium, lead, and zinc ions from solution. Crude EPS of the strain DEMFc2 showed greater variation in the level of biosorption of HM ions, with the weakest sorption of Cd ions (64%) and stronger sorption of Pb and Zn ions, at 74% and 81%, respectively (Figure 3).
The deproteinised EPS sorbed metals more weakly than crude EPS after the 24 h incubation with metal ion solutions, while after 48 h incubation, the crude and deproteinised preparations were able to sorb at similar levels for Pb ions, but in the deproteinised EPS preparations of strain DEMFc2, they sorbed more strongly than crude EPS, and even the sorption level increased relative to the 24 h incubation.
It was reported that the sorption of Cd by the deproteinised EPS of all three strains increased with time, and by the crude EPS, slightly decreased. In contrast, Pb and Zn sorption decreased with incubation time, with the exception of Pb sorption by the deproteinised EPS preparation produced by DEMFc37.
The strongest (by 50%) decrease in sorption with incubation time was observed for the crude EPS of the strain DEMFc2.

3.4. Relationship Between Metal Biosorption and Type and Composition of EPS

Through PCA, it has been clearly demonstrated that the process of biosorption of metals (Cd, Zn, and Pb) by EPS DEMFc2 occurs differently than by the two remaining strains (DEMFc5 and DEMFc37) (Figure 4A). Biosorption by EPS DEMFc2 does not depend on the presence of specific sugar components, phenolic compounds, or proteins (Figure 4A,C), while in the case of the DEMFc5 and DEMFc37 strains, this process depend on the total concentration of sugars (Figure 4C), and among monomers, depends on the presence of mannose, galactose, and glucosamine in the EPS preparations (Figure 4A). No clear correlation can be observed between the biosorption of Cd and the presence of specific sugar monomers (Figure 4B), nor with the total sugar content of the EPS (Figure 4D).
Crude (C) EPS carried out a stronger biosorption process than proteolysed (P) EPS. The biosorption process by proteolysed EPS preparations was more closely related to the total sugar content (Figure 5B), and among the sugar components, it was most strongly associated with the presence of galactose and glucose in EPS preparations (Figure 5A). In the case of crude EPS, this process is strongly correlated with the presence of phenolic compounds and proteins, not with the presence of specific sugar monomers (Figure 5B).
The relationship between the EPS concentration, mycelial biomass, and final medium pH in culture was completely different for the PGPF strain DEMFc2 than for the DRMO and pathogenic strains (Figure 6A). In the case of the DEMFc2 strain, there was a correlation between the Cd concentration and EPS mycelial biomass, and the effects of Pb and Zn were strongly involved with the pH value and less strongly influenced the EPS concentration produced by this strain (Figure 6B). Clearly, the effects of the individual heavy metals in the case of DRMO DEMFc5 were different: Zn had a strong positive effect on EPS concentrations and mycelial biomass, and Pb sorption was influenced by the pH value, while Cd had no clear effect on these parameters (Figure 6C). It was noticeable that, in the pathogen, the individual metals influenced the same parameters as in the DRMO strain, but the direction of the relationship between the Zn concentration and biomass and EPS concentration was opposite (Figure 6D).
The analysis presented in Figure 7 focuses on the impact of parameters such as the EPS, biomass, and pH in culture on the adsorption of individual heavy metals. Zn had the strongest effect on the biomass and EPS concentration produced by the F. culmorum strains, and sorption of Pb was correlated with changes in the final pH value (Figure 7A). However, Cd had a different effect on the pathogen strain than on the biomass and EPS concentration of the DEMFc2 and DEMFc5 strains (Figure 7B). The dependence of the PGPF strain DEMFc2 on the lead concentration was quite different from that of the DRMO and pathogen strains, in which Pb strongly affected the biomass and EPS concentration, and sorption of this metal was strongly affected by pH (Figure 7C). Zn had a clear effect on the biomass and pH values in the DRMO strain culture and on the EPS concentration in the pathogen culture, while Zn had a completely different effect on the PGPF strain (Figure 7D).

4. Discussion

A huge bioremediation potential is possessed by fungi inhabiting different environments, including the soil bulk, rhizosphere and plant endosphere, which are very poorly studied so far compared to bacteria. The F. culmorum fungi, with their wide range of plant hosts, on which they can act phytopathogenically but also non-pathogenically by promoting (PGPF) or weakening growth as deleterious microorganisms (DRMOs), can be the basis for creating sustainable phytoremediation technologies and, at the same time, by making plants resistant not only to abiotic but also biotic stresses, for creating sustainable agriculture methods. Particularly valuable in these methods is the possibility of using the exopolymers (EPSs) produced by these fungi, which, thanks to their numerous functional groups, can be excellent metal sorbents and basic factors in the biosorption mechanism. In order to gain a detailed understanding of these mechanisms, the sorption capacities of Zn, Pb, and Cd by the EPSs of a pathogenic DEMFc37 strain and two non-pathogenic strains, PGPF-DEMFc2 and DRMO-DEMFc5, were compared, and the effects of these metals on EPS synthesis by the three strains was determined.
EPSs were obtained in cultures of these strains on Czapek–Dox medium without metals and in the presence of individual metals at concentrations of 2, 4, 6, 8, and 10 µg/mL. EPSs obtained in the metal-free medium were used to study metal binding after 24 and 48 h incubation of lyophilised preparations at 20 °C—(1) proteolysed and (2) crude—for which the general composition and detailed characterisation of sugar components were determined. The concentration of metals bound/adsorbed to EPS placed in cellulose dialysis tubes was determined using the ASA technique. It has been shown that the presence of lead had a strong effect on limiting EPS synthesis by the three investigated F. culmorum strains, while the effects of cadmium and zinc on EPS production by the individual strains were different. EPS concentrations of the PGPF strain (DEMFc2) were significantly positively correlated with cadmium and zinc concentrations in the medium. In contrast, the concentration of EPS of the DRMO strain (DEMFc5) and the pathogenic strain DEMFc37 in the culture with these metals decreased two-fold and five-fold, respectively. A strong toxic effect on the mycelial biomass was observed in all strains tested (higher metal concentrations caused a decrease in fungal biomass) following the application of increased amounts of Cd and Pb, and only in the presence of zinc was an increase in the mycelial biomass of the pathogenic strain Fc37 observed. It was observed that the PGPF strain Fc2 in cultures with Cd and Zn corresponded to an increased production of EPS, while in the case of the DRMO strain Fc5 in cultures with Cd and Zn, the production remained at a high level.
The high resistance of fungi to heavy metals is related to the high osmotic pressure in the cell [35,36], the production of survival forms [37], the induction of the antioxidant machinery [38], and the ability to export metal ions by HM ATPases [6,39], but above all, to the production of compounds, such as EPSs, that bind metals in the extracellular space as well as promote intracellular sequestration and immobilisation [40].
The EPSs of all the F. culmorum strains tested sorbed the metals in solution rapidly (after 24 h incubation) and to a very high (70–80%) degree. The crude EPSs of all the strains bound more than 80% of Zn as well as 64, 78, and 84% of Cd and 74, 77, and 79% of Pb by those of DEMFc2, DEMFc5, and DEMFc37, respectively, after 24 h of shaking EPS in the metal salt solution. However, the degree of immobilisation of the metals by the tested EPS after the longer incubation time (48 h) was several percent lower than after 24 h. However, it was noted that this process is metal-dependent as well as dependent on the presence or not of protein components, as in the case of Cd bound by proteolysed EPS, where an increase in cadmium biosorption was observed after 48 h of incubation.
It should be emphasised that the crude EPS preparation of the F. culmorum DEMFc2 strain did not bind more metals than the crude EPS of the other strains despite significantly (2- to 4-fold) higher protein content and also phenolic compounds. It is most likely that the biosorption of the three metals tested in this study by the crude EPS of DEMFc2 was mainly mediated by the polysaccharide part, in which glucose predominates, while in the crude EPSs of the other strains, the predominant sugar is mannose.
A similar relationship was described by da Silva et al. [41] for EPS obtained from cultures of Colletotrichum sp. The polymers of this strain were able to bind 79% of cadmium ions and 98% of lead ions. Similarly, a high affinity for metal ions was described by Vimalnat and Subramanian [42] for EPS obtained from a culture of Pseudomonas aeruginosa, which at high polymer concentrations in solution bound lead ions at 90–95%.
In the case of the benthic fungus Ascomycota Aspergillus penicillioides (F12), its ability to biosorb lead very rapidly (5.74 h) was demonstrated, and optimisation of the ecological parameters [pH, time (h), and temperature (°C)] showed that the EPS of A. penicillioides under optimal conditions of pH (8.85) and temperature (32 °C) could sorb 73.14% of lead [43]. Paria et al. [43] also noted that at an EPS concentration of 0.5 mg/L of the fungus A. penicillioides, the highest (88.4%) flocculation capacity and as much as 50% emulsifying activity of this fungal exopolymer was recorded.
The metals tested (Zn, Pb, and Cd) were bound by–OH, -COOH, and -NH2 groups in the exopolymers of the fungus Ascomycota Pestolatiopsis sp. KCTC8637 [44] and Zygomycota Mucor rouxii [45]. We used the FTIR technique and performed an analysis of the EPSs produced by these strains, showing numerous chemical groups such as -COOH, -OH, and -NH2, as was shown in Jaroszuk-Ściseł et al. 2020 [24]. The literature emphasises that the identification of the biosorption mechanism can be obtained not only by comparing the FTIR spectra of potential natural and metal-loaded biosorbents, but also simply by identifying groups in potential sorbents such as EPS [10,46,47,48,49].
In the case of pullulan, an EPS produced by another Ascomycota representative Aureobasidium pullulans on a metal-containing substrate, the ability of pullulan to sorb metals such as Cd, Cu, Cr, Fe, Mn, Ni, and Pb from the substrate was established, but at very low concentrations of hundredths of a ppm, while most of the metals from the substrate were sorbed by the biomass of this fungus [50]. In contrast, purified pullulan devoid of uronic acids and their carboxyl groups did not bind any of these metals.
Examination of the EPS biosorption capacity of F. culmorum strains showed that the biosorption capacity of the three metals tested (Cd, Zn, and Pb) was present in both the crude preparations and EPS after deproteinisation. In most cases, a 20–30% decrease in the amount of bound metal ions was observed. Only the EPS of the PGPF strain DEMFc2 showed an increase in the affinity for cadmium ions, observed in the deproteinised EPS.
The binding of Cd, Co, and Cu by Saccharomyces cerevisiae was mainly mediated by mannan hydroxyl and protein thiol groups [51]. PCA indicated a relationship between heavy metal biosorption and mannan content in the proteolysed EPS preparations of F. culmorum analysed in this study.
An alkali-soluble polysaccharide obtained from the cell wall of Boletus edulis capable of biosorbing Ni, Zn and, to the greatest extent, Cd and Pb was described as a (1 → 3)-linked α-d-glucan decorated by α-(1 → 3)- d-mannose chains [10]. The amide, phosphate, and carboxyl groups of the EPS of the bacteria Bacillus subtilis and Pseudomonas putida were responsible for Zn binding [52], and thiol groups complexed especially strongly with Cd, but also Zn and Pb as well as other metals (e.g., Co and Ni) in the envelope polymers of Shewanella oneidensis [53]. During biosorption of heavy metal cations (e.g., Cd, Zn, and Pb) occurring by ion exchange sorption, there is a non-specific exchange between the heavy metal cations present in solution and cation (H, Ca, and K) functional groups by the living and dead fungal cells of Saccharomyces cerevisiae and Rhizopus arrhizus [14].
Therefore, the ability of biosorption of metals by the F. culmorum EPS was found and, although the concentration of immobilised metals decreased with time, the degree of immobilisation was significant, especially in the case of cadmium and the EPS of the pathogenic DEMFc37 strain.
The observed differences in metal biosorption between the EPSs of F. culmorum strains are due to differences in the total sugar content of the EPS, individual sugar monomers, proteins, and phenolic compounds, glycosidic bonds, and the number of functional groupings such as COOH, OH, or NH2.
PCA indicates a very clear and strong biosorption capacity for metals by crude EPS and indicates that this process depends most strongly on the protein components and phenolic compounds of the exopolymer. In contrast, the biosorption process of the tested HMs by the proteolysed EPS preparations depends mainly on the sugar content, being influenced by the presence of galactose and mannose monomers. In the case of crude EPS, the biosorption process was not dependent on the specific sugar monomers, but rather on protein and phenolic components.
The biosorption process of metals (Cd, Zn, and Pb) by EPS DEMFc2 occurs differently than by the two remaining strains (DEMFc5 and DEMFc37). Biosorption by EPS DEMFc2 depends on phenolic compounds and proteins as well as glucose, while EPS DEMFc5 and DEMFc37 depend on the presence of sugars (specifically, mannose and galactose).
Therefore, the use of plant inoculation with the PGPF strain as well as the use of the EPS of this strain and other F. culmorum strains can be the basis for creating sustainable bioremediation, including phytoremediation and, at the same time, stimulating plant protection against phytopathogens through the induction of resistance.

5. Conclusions

F. culmorum strains with different plant effects (beneficial, harmful, and pathogenic) were able to grow and produce EPS in the presence of heavy metals (Cd, Zn, and Pb). The EPSs produced by these F. culmorum strains sorbed Cd, Zn, and Pb, irrespective of the type of their interaction with the plants. Differences could be observed between the PGPF strain DEMFc2 and the DRMO strains DEMFc5 and pathogenic DEMFc37, both in terms of the effects of heavy metals on the fungal biomass and EPS production, as well as the biosorption of heavy metals by the EPS of the PGPF strain versus the biosorption of HMs by the EPS of the DRMO strain and the pathogen. The results of our study on the influence of metals on EPS production and the biosorption capacity of EPS obtained from F. culmorum strain cultures contribute a great deal to the knowledge of plant interaction with the rhizosphere and endophytic fungi, and also open up new possibilities for the development of alternative methods for the bioremediation of soil contaminated by heavy metals, not only by the bioaugmentation technique, but also by combining this technique with phytoremediation, which gives hope for the restoration of contaminated soils for agricultural use.

Author Contributions

Conceptualisation, J.J.-Ś. and A.N.; methodology J.J.-Ś., I.K., M.P.-S., D.K. and A.N.; software, M.P.-S.; validation, A.N., I.K. and J.J.-Ś.; formal analysis, M.P.-S.; investigation, A.N., I.K., M.P.-S. and J.J.-Ś.; resources, D.K.; data curation, A.N., I.K. and J.J.-Ś.; writing—original draft preparation, J.J.-Ś., A.N. and I.K.; writing—review and editing, J.J.-Ś., A.N. and I.K.; visualisation, J.J.-Ś., M.P.-S. and A.N.; supervision, J.J.-Ś. and I.K.; project administration, J.J.-Ś. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data of this article will be made available by the authors upon request.

Acknowledgments

The authors want to acknowledge Marzena Gęca from the Department of Inorganic Chemistry, Faculty of Chemistry at M. C.-S. University, Poland for technical support in the ASA experiments.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Fungal dry biomass [g/L], EPS yield: EPS concentration [g/L], EPS concentration converted into fungal biomass dry weight [mg/g], and final pH of the post-culture liquid on Czapek–Dox medium with different concentrations of (A) cadmium, (B) lead, and (C) zinc (2, 4, 6, 8, and 10 µg/mL). Standard deviations are shown as deviation bars (n = 9).
Figure 1. Fungal dry biomass [g/L], EPS yield: EPS concentration [g/L], EPS concentration converted into fungal biomass dry weight [mg/g], and final pH of the post-culture liquid on Czapek–Dox medium with different concentrations of (A) cadmium, (B) lead, and (C) zinc (2, 4, 6, 8, and 10 µg/mL). Standard deviations are shown as deviation bars (n = 9).
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Figure 2. Percentage against control of fungal dry biomass [g/L], EPS yield: EPS concentration [g/L], EPS concentration converted into fungal biomass dry weight [mg/g] on Czapek–Dox medium with different concentrations of (A) cadmium, (B) lead, and (C) zinc (2, 4, 6, 8, and 10 µg/mL). Standard deviations are shown as deviation bars (n = 9).
Figure 2. Percentage against control of fungal dry biomass [g/L], EPS yield: EPS concentration [g/L], EPS concentration converted into fungal biomass dry weight [mg/g] on Czapek–Dox medium with different concentrations of (A) cadmium, (B) lead, and (C) zinc (2, 4, 6, 8, and 10 µg/mL). Standard deviations are shown as deviation bars (n = 9).
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Figure 3. Percentage of (A) cadmium, (B) lead, and (C) zinc ion binding after 24 and 48 h of incubation by crude (C) and proteolysed (P) EPSs obtained from cultures of three F. culmorum strains: DEMFc2, DEMFc5, and DEMFc37.
Figure 3. Percentage of (A) cadmium, (B) lead, and (C) zinc ion binding after 24 and 48 h of incubation by crude (C) and proteolysed (P) EPSs obtained from cultures of three F. culmorum strains: DEMFc2, DEMFc5, and DEMFc37.
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Figure 4. Biplot of the Principal Component Analysis (PCA) describing corelations between the (A) metal binding capacity (biosorption) and EPS-boosting monomers (Glc, Man, Gal, and GlcN) within the tested strains (DEMFc2, DEMFc5, and DEMFc37); (B) metal binding capacity (biosorption) and EPS-bubbling monomers (Glc, Man, Gal, and GlcN) within the tested metals (Cd, Pb, and Zn); (C) metal binding capacity (biosorption), EPS building components (protein, total sugar, and phenolic compounds) within the tested strains (DEMFc2, DEMFc5, and DEMFc37); and (D) metal binding capacity (biosorption), growth components, and obtained EPS (protein, total sugar, and phenolic compounds) in the range of the tested metals (Cd, Pb, and Zn).
Figure 4. Biplot of the Principal Component Analysis (PCA) describing corelations between the (A) metal binding capacity (biosorption) and EPS-boosting monomers (Glc, Man, Gal, and GlcN) within the tested strains (DEMFc2, DEMFc5, and DEMFc37); (B) metal binding capacity (biosorption) and EPS-bubbling monomers (Glc, Man, Gal, and GlcN) within the tested metals (Cd, Pb, and Zn); (C) metal binding capacity (biosorption), EPS building components (protein, total sugar, and phenolic compounds) within the tested strains (DEMFc2, DEMFc5, and DEMFc37); and (D) metal binding capacity (biosorption), growth components, and obtained EPS (protein, total sugar, and phenolic compounds) in the range of the tested metals (Cd, Pb, and Zn).
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Figure 5. Biplot of the Principal Component Analysis (PCA) describing correlations between the (A) metal binding capacity (biosorption) and EPS-bubbling monomers (Glc, Man, Gal, and GlcN) within the types of EPS tested (C and P); and (B) metal binding capacity (biosorption) and bulking components of the resulting EPS (protein, total sugar, and phenolic compounds) within the types of EPS (C and P) tested.
Figure 5. Biplot of the Principal Component Analysis (PCA) describing correlations between the (A) metal binding capacity (biosorption) and EPS-bubbling monomers (Glc, Man, Gal, and GlcN) within the types of EPS tested (C and P); and (B) metal binding capacity (biosorption) and bulking components of the resulting EPS (protein, total sugar, and phenolic compounds) within the types of EPS (C and P) tested.
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Figure 6. Biplot of the Principal Component Analysis (PCA) describing correlations between (A) the amount of synthesised (EPS), the biomass obtained from the mycelium (Biomass), and the final pH value (pH) of the culture within the tested strains (DEMFc2, DEMFc5, and DEMFc37); (B) the amount of synthesised (EPS), the biomass obtained from the mycelium (Biomass), and the final pH value (pH) of the culture for the DEMFc2 strain within the tested metals (Cd, Pb, and Zn); (C) the amount of synthesised (EPS), the biomass obtained from the mycelium (Biomass), and the final pH value (pH) of the culture for the DEMFc5 strain within the tested metals (Cd, Pb, and Zn); and (D) the amount of synthesised (EPS), the biomass obtained from the mycelium (Biomass), and the final pH value (pH) of the culture for the DEMFc37 strain within the tested metals (Cd, Pb, and Zn).
Figure 6. Biplot of the Principal Component Analysis (PCA) describing correlations between (A) the amount of synthesised (EPS), the biomass obtained from the mycelium (Biomass), and the final pH value (pH) of the culture within the tested strains (DEMFc2, DEMFc5, and DEMFc37); (B) the amount of synthesised (EPS), the biomass obtained from the mycelium (Biomass), and the final pH value (pH) of the culture for the DEMFc2 strain within the tested metals (Cd, Pb, and Zn); (C) the amount of synthesised (EPS), the biomass obtained from the mycelium (Biomass), and the final pH value (pH) of the culture for the DEMFc5 strain within the tested metals (Cd, Pb, and Zn); and (D) the amount of synthesised (EPS), the biomass obtained from the mycelium (Biomass), and the final pH value (pH) of the culture for the DEMFc37 strain within the tested metals (Cd, Pb, and Zn).
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Figure 7. Biplot of the Principal Component Analysis (PCA) describing correlations between (A) the amount of synthesised EPS, the biomass obtained (Biomass), and the final pH value (pH) of the culture within the individual metals (Cd, Pb, and Zn); (B) the amount of synthesized EPS, the biomass obtained (Biomass), and the final pH value (pH) of the culture or the metal (Cd) within the tested strains (DEMFc2, DEMFc5, and DEMFc37); (C) the amount of synthesised EPS, the biomass obtained (Biomass), and the final culture pH value (pH) or the metal (Pb) within the tested strains (DEMFc2, DEMFc5, and DEMFc37); and (D) the amount of synthesised EPS, the biomass obtained (Biomass), and the final pH value (pH) of the culture or the metal (Zn) within the tested strains (DEMFc2, DEMFc5, and DEMFc37).
Figure 7. Biplot of the Principal Component Analysis (PCA) describing correlations between (A) the amount of synthesised EPS, the biomass obtained (Biomass), and the final pH value (pH) of the culture within the individual metals (Cd, Pb, and Zn); (B) the amount of synthesized EPS, the biomass obtained (Biomass), and the final pH value (pH) of the culture or the metal (Cd) within the tested strains (DEMFc2, DEMFc5, and DEMFc37); (C) the amount of synthesised EPS, the biomass obtained (Biomass), and the final culture pH value (pH) or the metal (Pb) within the tested strains (DEMFc2, DEMFc5, and DEMFc37); and (D) the amount of synthesised EPS, the biomass obtained (Biomass), and the final pH value (pH) of the culture or the metal (Zn) within the tested strains (DEMFc2, DEMFc5, and DEMFc37).
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Table 1. Compositions of EPS preparations (crude (C) and proteolysed (P)) obtained from cultures of three F. culmorum strains: DEMFc2, DEMFc5, and DEMFc37.
Table 1. Compositions of EPS preparations (crude (C) and proteolysed (P)) obtained from cultures of three F. culmorum strains: DEMFc2, DEMFc5, and DEMFc37.
StrainDEMFc2DEMFc5DEMFc37
Component Composition of water fraction of EPS (µg/mg)
CPCPCP
Proteins2.301.200.440
Phenolic compounds31.614.426.920.727.720.5
Total sugars143.752.4103.9200.8147.1260.2
Table 2. Monosugar composition, molecular weights, and linkage analysis of crude (C) and proteolysed (P) EPS preparations obtained from cultures of three F. culmorum strains: DEMFc2, DEMFc5, and DEMFc37.
Table 2. Monosugar composition, molecular weights, and linkage analysis of crude (C) and proteolysed (P) EPS preparations obtained from cultures of three F. culmorum strains: DEMFc2, DEMFc5, and DEMFc37.
StrainDEMFc2DEMFc5DEMFc37
Component Sugar composition [μg/mg] ± SD *
CPCPCP
Mannose (Man)6.97 (SD = 1.670)12.40 (SD = 0.552)29.22 (SD = 3.466)58.39 (SD = 3.828)37.39 (SD = 4.957)128.99 (SD = 19.94)
Glucose (Glc)36.14 (SD = 4.230)12.69 (SD = 1.419)18.18 (SD = 3.200)75.48 (SD = 9.038)15.18 (SD = 3.760)34.04 (SD = 5.726)
Galactose (Gal)3.12 (SD = 1.290)8.01 (SD = 0.329)2.80 (SD = 0.129)6.09 (SD = 0.531)9.41 (SD = 1.299)13.59 (SD = 1.574)
Glucosamine (GlcN)-1.09 (SD = 0.155)----
Fraction **Molecular weights [kDa]
PPP
HMW1000–735-1000–735
MMW15.873.5–34.118.5
LMW -5.4
Type of linkageGlycosidic bonds [%]
PPP
tHex I (Man (1→)18.331.646.9
tHex III (Gal (1→)13.4Tr.Tr.
→2) Hex (1→24.520.322.8
→3) Hex (1→Tr.1.8Tr.
→4) Hex (1→20.915.65.1
→6) Hex (1→-Tr.6.7
→3,4) Hex (1→13.8-Tr.
→3,6) Hex (1→9.1-11.6
→2,3,4,6) Hex (1→-30.76.9
* SD values were calculated based on the results from three independent repetitions of sugar analysis; Tr.-traces; ** HMW—high molecular weight; MMW—medium molecular weight; LMW—low molecular weight polymers.
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Jaroszuk-Ściseł, J.; Nowak, A.; Pac-Sosińska, M.; Kołodyńska, D.; Komaniecka, I. Heavy Metal Biosorption Ability of EPS Obtained from Cultures of Fusarium culmorum Strains with Different Effects on Cereals. Sustainability 2025, 17, 3744. https://doi.org/10.3390/su17083744

AMA Style

Jaroszuk-Ściseł J, Nowak A, Pac-Sosińska M, Kołodyńska D, Komaniecka I. Heavy Metal Biosorption Ability of EPS Obtained from Cultures of Fusarium culmorum Strains with Different Effects on Cereals. Sustainability. 2025; 17(8):3744. https://doi.org/10.3390/su17083744

Chicago/Turabian Style

Jaroszuk-Ściseł, Jolanta, Artur Nowak, Małgorzata Pac-Sosińska, Dorota Kołodyńska, and Iwona Komaniecka. 2025. "Heavy Metal Biosorption Ability of EPS Obtained from Cultures of Fusarium culmorum Strains with Different Effects on Cereals" Sustainability 17, no. 8: 3744. https://doi.org/10.3390/su17083744

APA Style

Jaroszuk-Ściseł, J., Nowak, A., Pac-Sosińska, M., Kołodyńska, D., & Komaniecka, I. (2025). Heavy Metal Biosorption Ability of EPS Obtained from Cultures of Fusarium culmorum Strains with Different Effects on Cereals. Sustainability, 17(8), 3744. https://doi.org/10.3390/su17083744

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