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Article

Reduction of Ferric Chloride in Yeast Growth Media, by Sugars and Aluminum

by
Kęstutis Mažeika
1,*,
Vytautas Melvydas
2 and
Dovilė Čepukoit
3
1
Center for Physical Sciences and Technology, Savanoriu 231, LT-02300 Vilnius, Lithuania
2
Independent Researcher, LT-05128 Vilnius, Lithuania
3
Nature Research Center, Akademijos 2, LT-08412 Vilnius, Lithuania
*
Author to whom correspondence should be addressed.
Inorganics 2024, 12(5), 137; https://doi.org/10.3390/inorganics12050137
Submission received: 9 April 2024 / Revised: 6 May 2024 / Accepted: 7 May 2024 / Published: 10 May 2024
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Abstract

:
Iron compounds can be used in antimicrobial applications by exploiting the toxicity of divalent iron to living organisms due to the Fenton reaction. In this study, the growth inhibitory effects of ferrous sulfate FeSO4·7H2O and ferric chloride FeCl3·6H2O were observed on Metschnikowia clade and Saccharomyces cerevisiae yeast cells. The relatively high amount of reduced Fe3+ to Fe2+ in the growth medium determined by Mössbauer spectroscopy may contribute to the antimicrobial activity of ferric chloride. In order to test the reducing ability of sugars in the growth media of yeasts, the reaction of ferric chloride FeCl3·6H2O with sugars was investigated. In mixtures of FeCl3·6H2O and fructose, approximately two thirds of Fe3+ can be reduced to Fe2+. When the mixture of FeCl3·6H2O and fructose is placed on the surface of aluminum foil, an iron film is formed on the surface of the aluminum due to the reduction by both fructose and aluminum. The relative amount of Fe3+ which was reduced to Fe0 reached 68%.

1. Introduction

Metal oxidation and reduction take place in biological cells, in their medium, in the environment and elsewhere. In industry, obtaining pure metals from metal compounds with non-metals requires metal reduction, which is achieved using carbon, carbon monoxide, hydrogen and metallothermic reduction applying reactive metals, aqueous or molten salt electrolysis [1,2]. Many compounds can be used as reducing agents depending on the purpose. When investigating the interaction of Metschnikowia clade yeasts with iron in their environment [3,4], it was found that the reduced iron form, Fe2+, appears both in the growth media and in yeast biomass. Since divalent iron can have toxic or inhibitory effects on yeast cells [5,6], it is important to determine the ability of compounds in growth media to reduce Fe3+. Although many nutrients, including glucose or fructose, are added to the growth media, sugars have previously been reported to be mild reductants of iron [7,8].
Yeasts of the Metschnikowia clade are used in winemaking (together with Saccharomyces cerevisiae) [9] and in other biotechnological processes such as oil production [10], and their promising biocidal properties are also widely studied [11,12,13,14,15,16]. Metschnikowia spp. are characterized by the production of pulcherriminic acid, which binds with iron in the environment to form the red pigment pulcherrimin [17]. Some bacteria such as Bacillus suptilis are also characterized by pulcherrimin synthesis [18]. It was found that the amount of iron in the environment of pulcherriminic-acid-producing yeast and bacteria is regulated with the help of pulcherrimin, thus avoiding oxidative stress [5,6,16,18].
Ferric chloride is widely used as a mild oxidant or etchant, in organic polymerization reactions, in water treatment as a flocculant and coagulant and in many other reactions as a precursor and catalyst [19,20,21,22,23,24]. Ferric chloride can also be used as an oxidizer of glucose [25].
Due to the growing resistance of pathogenic microorganisms to antibiotics, the biocidal properties of various inorganic materials, including iron-containing ones such as ferritic nanoparticles and iron salts (ferrous sulfate and ferric chloride), are being studied [26,27,28,29]. In experiments with Metschnikowia yeast, ferric chloride was used mainly as an iron source in growth media [3,4]. In this study, the growth inhibition effects of ferric chloride FeCl3·6H2O and ferrous sulfate FeSO4·7H2O on Metschnikowia clade yeasts which produce pulcherrimin-immobilizing excess iron were observed. Saccharomyces cerevisiae, which has many domesticated strains and has a well-explored genome and is a widely used model yeast [30,31], was applied in this study to compare the antimicrobial effects of iron compounds as pulcherrimin non-synthesizing yeast. The interaction of ferric chloride (FeCl3·6H2O) with sugars was investigated to determine the ability of sugars to reduce iron in ambient conditions. It was also found that when placing the mixture of fructose with FeCl3·6H2O on the aluminum foil, Fe3+ is reduced to the metallic state. Aluminum was used as an additional reducing agent. It can be noted that iron reduction to the metallic state has previously been observed in iron–aluminum chloride melts, but at significantly higher temperatures than ambient [32]. In another study [33], the growth inhibition of Metschnikowia yeast was also observed during the decomposition of metallic iron in the growth medium.

2. Results

Ferrous sulfate FeSO4·7H2O applied to the growth medium significantly inhibited the growth of Metschnikowia yeasts inoculated as streaks (Figure 1a–c). Yeast inhibition is also visible in the center of the area with brown precipitates formed at the sites of the application of ferric chloride FeCl3·6H2O. Over time, the yeast biomass became redder, indicating the formation of a red pigment—pulcherrimin—inside the yeast biomass. Growth inhibition and red pigmentation were also observed when FeSO4·7H2O and FeCl3·6H2O were applied to the Metschnikowia shanxiensis M10 strain yeast lawn (Figure 1d). The inhibitory effects of ferrous sulfate FeSO4·7H2O and ferric chloride FeCl3·6H2O on the Saccharomyces cerevisiae lawn are shown in Figure 2a. Ferric chloride FeCl3·6H2O also produces brown precipitates (Figure 1 and Figure 2a), but at the edges the inhibitory effects are similar to those of FeSO4·7H2O. The results for four Metschnikowia clade yeast strains, M. sinensis M4 and M. pulcherrima MP strains exposed to FeCl3·6H2O and M. sinensis M6, M. shanxiensis M10 exposed to FeSO4·7H2O, and repeated experiments with substitution of strains show little strain dependence for the inhibitory effect. With ferrous sulfate FeSO4·7H2O, the effect is more widely distributed, which is probably due to an easier diffusion of Fe2+, as in the case of FeCl3·6H2O, where insoluble precipitates fall out.
The inhibition zone in Figure 2b is delimited by a red pigment rim around the inoculated Metschnikowia sinensis M4 spots and streaks indicating the active formation of insoluble pulcherrimin that binds incoming iron from the surrounding growth medium containing 5 mg/L of elemental iron. No inhibition effect in the lawn is produced by S. cerevisiae streaks, which do not produce pulcherriminic acid. In response to the higher iron concentration when additional ferric chloride (in solution) is applied (Figure 2b), increased red pigmentation reflects increased secretion of pulcherriminic acid. In this way, the inhibition of the S. cerevisiae lawn observed here is not due to the effect of Fe compounds, but due to competition with Metschnikowia yeasts for iron [12] when the amounts of applied iron compounds are much lower than in Figure 1.
The presence of Fe2+ in the growth medium after supplementing the medium with ferric chloride FeCl3·6H2O was observed previously [3]. In this study, in order to exclude the influence of yeasts, experiments with growth media without inoculated yeasts were performed. Upon supplementing the yeast growth media with ferrous sulfate FeSO4·7H2O and ferric chloride FeCl3·6H2O, changes in the valency of iron were observed, with both Fe3+ and Fe2+ detected in the Mössbauer spectra (Figure 3). In the case of FeSO4·7H2O, one-third of the iron ions in the dried growth medium were found in the oxidized Fe3+ state, while the remainder remained Fe2+ (Table 1). With FeCl3·6H2O, about a quarter of the iron in the growth medium was reduced to Fe2+.
In order to exclude the influence of other compounds which are present in growth media, only ferric chloride FeCl3·6H2O and sugars were mixed. In the experiments with FeCl3·6H2O and sugars, immediately after mixing FeCl3·6H2O (hexahydrate ferric chloride) and fructose (or sucrose), the mixture became wet. After that, sugar dissolved in the released water and a viscous mass was formed. Accordingly, the Mössbauer spectrum of FeCl3·6H2O, which is a characteristic asymmetric doublet with its isomer shift δ = 0.41 ± 0.01 mm/s and quadrupole splitting Δ = 0.94 ± 0.01 mm/s (Figure 4a) [34], transformed to a symmetric low-intensity doublet (Figure 4b). However, the valence state according to the isomer shift δ = 0.40 ± 0.03 mm/s and quadrupole splitting Δ = 0.60 ± 0.06 mm/s of doublet still remained Fe3+.
After drying slightly above ambient temperature (≈30 °C) for about a day or more, the mixture of FeCl3·6H2O and fructose turned more or less black (Figure 5a,b). In the case of the mixture on plastic tape a doublet attributed to Fe2+ (26–68% of the total spectral area depending on the conditions, Table 2) appeared in the Mössbauer spectrum (Figure 4c). The isomer shift δ = 1.12–1.3 mm/s and quadrupole splitting of the doublet Δ = 2.2–2.8 mm/s indicate ferrous chloride FeCl2·nH2O. For comparison, the contribution of Fe2+ was only 12% of the total spectral area when mixing ferric sulfate Fe2(SO4)3·H2O and fructose, even after 6 days (Table 2).
When the mixture of FeCl3·6H2O and fructose was placed on aluminum foil gas bubbles formed inside the viscous black mass (Figure 5b). In the case when the mixture was on Al foil, in addition to Fe3+ and Fe2+ doublets, a sextet with parameters characteristic of α-Fe appeared (Figure 4d). The dependence on the surface on which the mixture was placed indicates that the mixture reacts with the aluminum foil. The reaction of pure FeCl3·6H2O with aluminum is characterized by strong corroding of aluminum foil, which damages its integrity. In this case, Fe2+ chloride is the dominant reaction product, while the α-Fe sextet is barely noticeable in the Mössbauer spectrum (Figure 4e).
In the Mössbauer spectrum of a mixture of FeCl3·6H2O and fructose on aluminum foil (Figure 4d), the sextet attributed to metallic iron is the most intense subspectrum (47% of the total area), while the Fe3+ and Fe2+ doublets account for 24 and 29% of the total area, respectively. After washing off the mixture from the aluminum surface, most of the metallic iron remains as a film on the Al foil, as shown in Figure 5c, and only the α-Fe sextet is visible in the spectrum (Figure 4f).
Metallic iron can also form on the surface of aluminum powder. Aluminum powder was added to the mixture of FeCl3·6H2O and fructose (mass ratio 1:1:1) (Figure 5d). In this case, the contribution of the α-Fe sextet increased to 68% of the total spectral area (Figure 4g). The magnetization data (Figure 6) confirm the formation of ferromagnetic α-Fe. The saturation magnetization ms ≈ 7.5 emu/g is obtained when extrapolating magnetization dependence of the mixture, m = ms − const/H, with H→∞. The coercivity of ≈110 Oe was determined for this sample.

3. Discussion

The inhibitory activity of two iron compounds shown in Figure 1 and Figure 2 can be explained on the basis of detailed antimicrobial studies of ferric chloride and ferrous sulfate against pathogenic bacteria. The high bactericidal efficacy of ferric chloride against drug-resistant Pseudomonas aeruginosa has been linked to an increase in intracellular Fe2+ and the induction of ferroptosis via the Fenton reaction [28]. Cell lysis was observed at high concentrations. The antimicrobial mechanism of ferrous sulfate against Staphylococcus aureus was similar [29]. Ferric chloride was more effective than ferrous sulfate against Pseudomonas aeruginosa [28]. This can be explained by the existence of different Fe2+ and Fe3+ assimilation systems in P. aeruginosa cells. Since the growth inhibition of S. cerevisiae and Metschnikowia yeasts is greater in the case of ferrous sulfate FeSO4·7H2O than ferric chloride FeCl3·6H2O, the mechanism of inhibition may be related more to a higher concentration of Fe2+ in the growth medium than to total excess of iron [5,6].
In the cases observed in Figure 1 and Figure 2, iron in much larger quantities (of Fe2+ and Fe3+) than the yeast cells need comes to yeast biomass from the growth medium. However, due to the production of pulcherrimin, the species of yeast belonging to the Metschnikowia clade can lower the concentration of free iron, binding it either outside the biomass or accumulating it in the form of red pigment—pulcherrimin—in the specialized cells—chlamydospores [3]. In this way, the initial toxic effect of iron on the yeast cells can be neutralized.
As the use of Metschnikowia yeast for fruit and berry protection is under investigation [11], the application of ferric chloride in combination with pulcherrimin-synthesizing yeast may be beneficial for stronger initial and long-lasting subsequent antimicrobial effects. That is, iron compounds can perform an initial biocidal function, after which the effects of iron compounds disappear as they dissipate, turn into insoluble hydroxides or pulcherrimin in the case of application of Metschnikowia spp., without having negative effects on plants or other living tissues. As antimicrobial substances, such iron compounds are suitable because they are biocompatible [28,29], and their effects disappear after performing the antimicrobial function.
The natural Metschnikowia spp. yeast environment, fruits and berries [12], is characterized by a high fructose content. The reducing properties of the sugar-containing medium (Figure 3b) may increase the antimicrobial effect of ferric chloride. This suggests that the reducing properties of the medium should be taken into account when studying the antimicrobial effects of iron compounds.
Much more brown precipitates were observed in the places where ferric chloride FeCl3·6H2O was applied compared to ferrous sulphate FeSO4·7H2O (Figure 1 and Figure 2a). The precipitation of Fe3+ can occur through hydrolysis and complexation reactions [35,36,37,38]. For example, due to Fe2+ oxidation and the formation of hydroxides, the concentration of free iron is very low in mildly acidic or alkaline natural waters [35]. Fe3+ hydrolysis and the formation of complexes will depend on the pH of the medium, its composition and other properties. A non-buffered growth medium with 5–6 pH was used in the study. It should be noted that hydroxide precipitates of Fe3+ can form at pH > 4 [35,36]. The amounts of ferric chloride FeCl3·6H2O and ferrous sulfate FeSO4·7H2O (1–2 mg) applied can change pH only locally and for a short time since there was no change in the characteristics of the agar growth medium (liquefaction of agar medium would be visible at pH < 3–4). The precipitates here are probably visible because of the high local concentration of Fe3+.
Because of the low concentrations of free iron at biological pH, microorganisms have developed the ability to secrete special compounds of high affinity to iron—siderophores—to acquire iron from an insoluble form [39]. Metschnikowia spp. yeast secretes pulcherriminic acid, which is similar to siderophores but has another function—to reduce the availability of iron—which may be useful in preventing oxidative stress and restricting the Fenton reaction [18]. At the relatively high elemental iron concentration of 80 mg/L in the growth medium (when ferric chloride is distributed over the entire volume of the medium before its solidification and yeast inoculation), the accumulation of more than half of the total iron content in the yeast biomass is observed, mainly in the red pigment—pulcherrimin [3]. At the same time, such accumulation indicates the relative mobility of iron in the agar growth media. The movement of iron can be facilitated by iron reduction to Fe2+, the formation of unstable complexes [40] and the secretion of compounds of the siderophore type.
A high ability of sugars to reduce ferric chloride was observed in the yeast growth medium and in the mixtures of ferric chloride (FeCl3·6H2O) with sugars (Figure 3b and Figure 4c, Table 1 and Table 2). The occurrence of Fe2+ in the growth medium after supplementing the medium with ferric chloride is consistent with previous observations [3]. About 30% of iron in the form of Fe2+ was observed in M. shanxiensis M10 strain yeast biomass after 22 h of yeast growth with ≈5 mg/L of Fe in the growth medium. However, after another 22 h, the relative amount of Fe2+ decreased several times. The time dependence of the Fe2+ concentration can probably be attributed to the depletion of nutrients, including sugars, when yeast is present, so divalent iron may be more abundant in the initial yeast growth phase.
Due to the specificity of Mössbauer spectroscopy, a good spectral recording efficiency is achieved only for solids, so the samples were dried. The decrease in absorption area (Table 2) shortly after mixing ferric chloride (FeCl3·6H2O) with fructose can be explained by the wetness of the mixture. It can be concluded that, first of all, mixing ferric chloride hexahydrate FeCl3·6H2O with fructose (the same as sugar) breaks down the crystalline structure and partially releases crystalline water.
In the mixture of FeCl3·6H2O and fructose, the isomeric shift δ = 1.12–1.3 mm/s and quadrupole splitting of the doublet Δ = 2.2–2.8 mm/s indicate the chemical state of Fe2+ corresponding to ferrous chloride FeCl2·nH2O. The variation in the quadrupole splitting could be due to the different amount of water in the FeCl2·nH2O formula, where n < 4 [41]. In the case of yeast growth media, there are more opportunities for the formation of various iron compounds, so it is difficult to draw the same conclusion, even though the Mössbauer parameters are similar to those obtained for the mixtures.
It can also be assumed that iron complexes with fructose are not formed in the mixtures of fructose and FeCl3·6H2O because the quadrupole splitting of the ferric fructose complexes, Δ = 0.895 mm/s [8], is much larger than that of the observed Fe3+ doublet: Δ = 0.37–0.41 mm/s (Table 2). It should be noted that iron complexes with fructose have been observed in distilled water or methanol solutions [8,42]. In this study, water in mixtures is released from FeCl3·6H2O when it is mixed with fructose. However, the Fe3+ doublet in the Mössbauer spectra of the growth medium with ferrous sulfate FeSO4·7H2O and ferric chloride FeCl3·6H2O is broader and its lines are wider (Figure 3, Table 1), so the formation of Fe3+ complexes with fructose in the growth medium cannot be ruled out.
Although the reaction in mixtures of ferric chloride FeCl3·6H2O and fructose occurs near ambient temperature, the oxidizing power of ferric chloride increases with increasing ferric chloride concentration when oxidizing glucose with ferric chloride to obtain gluconic acid [25]. The release of gas (gas bubbles in the viscous mass of the mixture) is observed only when the mixture is placed on aluminum or the mixture contains aluminum powder (Figure 5b,d). As ferric chloride can remove the surface oxide layer on aluminum, the reaction of water with aluminum is possible [43]:
3H2O + Al = Al(OH)3 + 1.5H2.
When placing pure ferric chloride FeCl3·6H2O on aluminum foil, the Mössbauer results (20% Fe3+, 70% Fe2+ and 10% Fe0 according to Table 1) generally correspond to the reaction of etching [20]
3FeCl3 + Al = 3FeCl2 + AlCl3.
However, this reaction does not explain the formation of metallic iron when placing the mixture of FeCl3·6H2O and fructose on aluminum. The hydrogen formed in reaction (1) can participate in the reduction of ferrous chloride to metallic iron [1]:
FeCl2 + H2 = Fe + 2HCl.
Moreover, the following reactions were considered to occur in iron–aluminum chloride melts [32]:
FeCl3 + Al = AlCl3 + Fe,
3FeCl2 + 2Al = 2AlCl3 + 3Fe,
2FeCl3 + Fe = 3FeCl2,
of which the first two were responsible for the formation of metallic iron. However, reactions (4) and (5) occurred efficiently at much higher than room temperature. In the mixtures of FeCl3·6H2O with fructose and aluminum, the reduction to metallic iron should occur mainly due to reactions (3) and (5), while the efficiency of the reverse reaction, (6), should decrease at the Al and Fe surfaces because the amount of ferric chloride (FeCl3) decreases as it reacts with fructose and aluminum. This is shown by the experimental data, where the amount of Fe3+ is less than that of Fe2+ (Table 2). Furthermore, the interaction of Fe3+ with Fe0 is hindered by the viscous medium which also traps hydrogen.
In the case of a mixture with Al, more Fe3+ is converted to metallic iron due to the larger surface area of the Al powder. Magnetization of 7.5 emu/g (Figure 6) corresponds to a 3.4% weight fraction of metallic iron in the sample when Fe3+ initially makes up about 6.6% of the sample mass. This is more or less consistent with the Mössbauer data (68% of Fe0, Table 2). It is assumed that metallic iron is deposited as a thin film on the surface of the Al powder, so the surface plane of the film is, on average, oriented equally in all directions. The saturation of the magnetization of the sample with increasing magnetic field strength is achieved relatively slowly, because it is more difficult to magnetize the film when the direction of magnetization deviates from the plane of the film. The coercivity of ≈110 Oe is probably due to shape anisotropy, which depends on the thickness and structure of the Fe film [44].

4. Materials and Methods

Ferric chloride hexahydrate FeCl3·6H2O (Reachem Slovakia, Bratislava, Slovakia, 99% or Fluka Chemie GmbH, Buchs, Switzerland, 97%), ferrous sulfate heptahydrate FeSO4·7H2O (Carl Roth GmbH, Karlsruhe, Germany, 99.5%), ferric sulfate hydrate Fe2(SO4)3·H2O (Reachem Slovakia, Bratislava, Slovakia, 99%) and aluminum powder (Sigma-Aldrich, Steinheim, Germany, 99%, <75 μm) were used in this study. Fructose, sucrose and aluminum foil were bought at a regular store.
For yeast growth, non-buffered MR growth medium (pH 5–6) was applied. MR growth medium was prepared from 1% peptone (mycological, Liofilchem, Roseto, Italy) or 2% peptone M66 universal (Merck, Darmstadt, Germany or Sigma-Aldrich, Steinheim, Germany), 1% yeast extract (Liofilchem, Roseto, Italy), 2% glucose (Oriola, Espoo, Finland or Liofilchem, Roseto, Italy) and 2% agar (Difco Laboratories, Detroit, MI, USA). According to previous studies, the growth media contain 1.1 mg/L or 5 mg/L of elemental Fe. Small crystals (≈1–2 mg) of FeSO4·7H2O and FeCl3·6H2O or droplets (5 μL) of a 10 mg/L ferric chloride solution were applied directly to yeast streaks, spots or lawn grown in Petri dishes 2 h after yeast inoculation. Metschnikowia clade yeasts—Metschnikowia sinensis (strains—M4 and M6), M. shanxiensis (M10), M. pulcherrima (MP) [3,4], Saccharomyces cerevisiae haploid strain α′1 and diploid strain Rom 100—were used to show the antimicrobial effects of iron compounds. Metschnikowia strains were isolated from spontaneous fermentations and identified as described before [3,12]. Saccharomyces cerevisiae Rom 100, the strain used in winemaking, and α′1, the strain widely used in genetic research, were taken from the collection of the Institute of Botany (Nature Research Center, Vilnius, Lithuania). Yeast was inoculated after growth medium solidification and grown in the incubator at 20 °C. Experiments were repeated several times.
In the experiments with ferric chloride and sugars, FeCl3·6H2O and fructose or sucrose were mixed in a 1:1 (200 mg:200 mg) or 2:1 mass ratio (400 mg:200 mg) using a mortar and pestle. For comparison one sample of the mixture of ferric sulfate and fructose was made. The mixtures were placed on plastic tape or aluminum foil covering ≈ 4 cm2 of area. Before recording the Mössbauer spectra, the mixtures were dried at approximately 30 °C for one or more days. In a further experiment, aluminum powder was added to the mixture of FeCl3·6H2O and fructose and placed on paper. Mixing with sucrose gives quite similar results to mixing with fructose; therefore, the results of experiments with sucrose are not presented here. Table 2 uses abbreviated sample names: FeClFP1d (1:1), FeClSAl2d (1:1), etc., where FeCl is ferric chloride; FeS is ferric sulfate Fe2(SO4)3·H2O; F is fructose; P is plastic tape; Al is aluminum foil; Alp—aluminum powder; nd—dried for n days; and (1:1)—components ratio.
Mössbauer spectra were measured using a 57Co(Rh) source and Mössbauer spectrometer (Wissenschaftliche Elektronik GmbH, Starnberg, Germany). The quadrupole distributions or separate doublets and the sextet were used to fit to the Mössbauer spectra applying WinNormos Site and Dist, version 3.0 software. Isomer shifts are given relative to α-Fe. The changes in the valence state of iron Fe3+→Fe2+→Fe0 were evaluated according to the parameters of the Mössbauer spectra. Since Fe3+, Fe2+ and Fe0 (metallic iron) states have a sufficiently different isomer shift δ, quadrupole splitting Δ and hyperfine field B, these states can be easily distinguished as shown in the Mössbauer spectra by different colors.
A vibrating sample magnetometer consisting of a lock-in amplifier SR510 (Stanford Research Systems, Sunnyvale, CA, USA), a Gauss-/Teslameter FH-54 (Magnet Physics, Cologne, Germany) and a laboratory magnet supplied by a power source SM 330-AR-22 (Delta Elektronika, Zierikzee, The Netherlands) were applied for magnetization measurements.

5. Conclusions

A significant reduction of Fe3+, 26% in the yeast growth media with ferric chloride FeCl3·6H2O, and iron, up to 68% in the FeCl3·6H2O and fructose mixtures, is observed. If the mixture of ferric chloride and fructose is placed on aluminum foil, a thin film of metallic iron is formed on its surface. In this case, fructose reduces ferric chloride to Fe2+, traps hydrogen resulting from the reaction of aluminum with water and also protects the aluminum surface from significant corrosion. Ferrous sulfate FeSO4·7H2O significantly inhibited the growth of Metschnikowia and S. cerevisiae yeasts. In the case of ferric chloride, FeCl3·6H2O, the zone of growth inhibition was smaller. Since the effect of reduced Fe3+ to Fe2+ in growth media cannot be ruled out, it is generally concluded that the reducing ability of the medium may be important for the antimicrobial applications of iron compounds.

Author Contributions

Conceptualization, K.M. and V.M.; methodology, K.M. and V.M.; investigation, K.M., V.M. and D.Č.; writing, K.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Inorganics 12 00137 g001
Figure 1. Effects of ferric chloride FeCl3·6H2O and ferrous sulfate FeSO4·7H2O on Metschnikowia spp. yeast after (a) 1.5 days, (b) 3 days and (c,d) 5 days. Yeast grown at 20 °C: (a–c) M. sinensis M4, M6, M. shanxiensis M10 and M. pulcherrima MP yeast biomass streaks; (d) M10 lawn on MR growth medium containing 1.1 mg/L of elemental Fe. At indicated places: 1—1–2 mg of FeCl3·6H2O, 2—1–2 mg of FeSO4·7H2O applied 2 h after yeast inoculation.
Figure 1. Effects of ferric chloride FeCl3·6H2O and ferrous sulfate FeSO4·7H2O on Metschnikowia spp. yeast after (a) 1.5 days, (b) 3 days and (c,d) 5 days. Yeast grown at 20 °C: (a–c) M. sinensis M4, M6, M. shanxiensis M10 and M. pulcherrima MP yeast biomass streaks; (d) M10 lawn on MR growth medium containing 1.1 mg/L of elemental Fe. At indicated places: 1—1–2 mg of FeCl3·6H2O, 2—1–2 mg of FeSO4·7H2O applied 2 h after yeast inoculation.
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Figure 2. Effects of ferric chloride FeCl3·6H2O and ferrous sulfate FeSO4·7H2O after 5 days (a) and M. sinensis M4 and S. cerevisiae Rom 100 spots after 3 days (b) on S. cerevisiae lawn. MR growth medium contains (a) 1.1 mg/L and (b) 5 mg/L of elemental Fe. Yeast grown at 20 °C. 1—1–2 mg of FeCl3·6H2O, 2—1–2 mg of FeSO4·7H2O, 3—5 μL of 10 mg/L FeCl3·6H2O solution applied 2 h after inoculation.
Figure 2. Effects of ferric chloride FeCl3·6H2O and ferrous sulfate FeSO4·7H2O after 5 days (a) and M. sinensis M4 and S. cerevisiae Rom 100 spots after 3 days (b) on S. cerevisiae lawn. MR growth medium contains (a) 1.1 mg/L and (b) 5 mg/L of elemental Fe. Yeast grown at 20 °C. 1—1–2 mg of FeCl3·6H2O, 2—1–2 mg of FeSO4·7H2O, 3—5 μL of 10 mg/L FeCl3·6H2O solution applied 2 h after inoculation.
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Figure 3. Mössbauer spectra of dried yeast growth media with applied FeSO4·7H2O (a) and FeCl3·6H2O (b) kept for 5 days. Right: quadrupole splitting distribution. Yellow is Fe3+ and green is Fe2+.
Figure 3. Mössbauer spectra of dried yeast growth media with applied FeSO4·7H2O (a) and FeCl3·6H2O (b) kept for 5 days. Right: quadrupole splitting distribution. Yellow is Fe3+ and green is Fe2+.
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Inorganics 12 00137 g004
Figure 4. Mössbauer spectra of FeCl3·6H2O (a), mixture of FeCl3·6H2O with fructose before (b) and after drying on plastic tape for 4 days (c), dried mixture of FeCl3·6H2O with fructose on Al foil (d), FeCl3·6H2O on Al foil (e), Al foil when mixture of FeCl3·6H2O with fructose was washed out (f), and dried mixture of FeCl3·6H2O with fructose and Al powder on paper (g). Grey subspectrum is for α-Fe, yellow is Fe3+ and green is Fe2+.
Figure 4. Mössbauer spectra of FeCl3·6H2O (a), mixture of FeCl3·6H2O with fructose before (b) and after drying on plastic tape for 4 days (c), dried mixture of FeCl3·6H2O with fructose on Al foil (d), FeCl3·6H2O on Al foil (e), Al foil when mixture of FeCl3·6H2O with fructose was washed out (f), and dried mixture of FeCl3·6H2O with fructose and Al powder on paper (g). Grey subspectrum is for α-Fe, yellow is Fe3+ and green is Fe2+.
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Inorganics 12 00137 g005
Figure 5. Mixture of FeCl3·6H2O with fructose on plastic tape (a) and Al foil (b), dried for 1 day at ≈30 °C, the aluminum foil with mixture washed out (c), mixture of FeCl3·6H2O with fructose and Al powder (d).
Figure 5. Mixture of FeCl3·6H2O with fructose on plastic tape (a) and Al foil (b), dried for 1 day at ≈30 °C, the aluminum foil with mixture washed out (c), mixture of FeCl3·6H2O with fructose and Al powder (d).
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Figure 6. Magnetization dependence of mixture of FeCl3·6H2O, fructose and aluminum powder on applied magnetic field.
Figure 6. Magnetization dependence of mixture of FeCl3·6H2O, fructose and aluminum powder on applied magnetic field.
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Table 1. The parameters of Mössbauer spectra of ferric chloride FeCl3·6H2O and ferrous sulfate FeSO4·7H2O applied to growth media: I—relative intensity; δ—isomer shift; Δ—mean quadrupole splitting of distribution or quadrupole splitting of separate doublets.
Table 1. The parameters of Mössbauer spectra of ferric chloride FeCl3·6H2O and ferrous sulfate FeSO4·7H2O applied to growth media: I—relative intensity; δ—isomer shift; Δ—mean quadrupole splitting of distribution or quadrupole splitting of separate doublets.
SampleI, %δ, mm/sΔ, mm/s
FeSO4·7H2O35 (34 ± 1) *0.427 ± 0.0040.695 (0.66 ± 0.01)Fe3+
65 (35 ± 1; 31 ± 1)1.245 ± 0.0042.36 (1.97 ± 0.02; 2.75 ± 0.01)Fe2+
FeCl3·6H2O74 (73 ± 1)0.423 ± 0.0020.656 (0.63 ± 0.01)Fe3+
26 (27 ± 1)1.215 ± 0.0102.33 (2.44 ± 0.02)Fe2+
* in parenthesis: data for separate doublets.
Table 2. The parameters of Mössbauer spectra of experiments with mixtures of ferric chloride FeCl3·6H2O and sugars: I—relative intensity; Γ—linewidth; δ—isomer shift; Δ—quadrupole splitting; B—hyperfine field.
Table 2. The parameters of Mössbauer spectra of experiments with mixtures of ferric chloride FeCl3·6H2O and sugars: I—relative intensity; Γ—linewidth; δ—isomer shift; Δ—quadrupole splitting; B—hyperfine field.
SampleI, %Γ, mm/sδ, mm/sΔ, mm/sB, T
FeCl3·6H2O1001.07 ± 0.05 *0.41 ± 0.010.94 ± 0.01 Fe3+
FeClFP0d (1:1)1000.83 ± 0.050.40 ± 0.030.60 ± 0.06 Fe3+
FeClFP1–2d (1:1)74 ± 20.55 ± 0.020.37 ± 0.130.40 ± 0.01 Fe3+
26 ± 20.91 ± 0.091.14 ± 0.042.39 ± 0.07 Fe2+
FeClFP1d (2:1)32 ± 10.50 ± 0.040.36 ± 0.010.41 ± 0.02 Fe3+
68 ± 10.28 ± 0.011.17 ± 0.012.35 ± 0.01 Fe2+
FeClFP4d (1:1)41 ± 20.57 ± 0.090.37 **0.40 ± 0.04 Fe3+
59 ± 30.67 ± 0.091.11 ± 0.022.28 ± 0.04 Fe2+
FeClFAl1d (1:1)24 ± 10.53 ± 0.040.37 **0.37 ± 0.02 Fe3+
29 ± 10.76 ± 0.041.16 ± 0.012.49 ± 0.03 Fe2+
47 ± 10.33 ± 0.010.00 ± 0.010.00 ± 0.0133.17 ± 0.02Fe0
After washing FeClFAl1d 1000.33 ± 0.020.00 ± 0.01−0.01 ± 0.0133.23 ± 0.03Fe0
FeCl3·6H2O on Al20 ± 10.33 ± 0.030.36 ± 0.050.71 ± 0.11 Fe3+
70 ± 20.36 ± 0.011.14 ± 0.022.28 ± 0.04 Fe2+
10 ± 20.34 ± 0.020.00 ± 0.060.00 **33.7 ± 0.4Fe0
FeClFAlp1d 2 ± 10.20 ± 0.090.46 ± 0.040.22 ± 0.05 Fe3+
(1:1:1)30 ± 10.45 ± 0.011.15 ± 0.012.28 ± 0.01 Fe2+
68 ± 10.30 ± 0.010.01 ± 0.010.01 ± 0.0133.2 ± 0.01Fe0
Washed 19 ± 20.6 ± 0.20.29 ± 0.050.69 ± 0.09 Fe3+
FeClFAlp1d81 ± 20.35 ± 0.010.00 ± 0.010.00 ± 0.0133.18 ± 0.04Fe0
FeSFP6d (1:1)88 ± 10.33 ± 0.010.42 ± 0.010.15 ± 0.01 Fe3+
12 ± 10.78 ± 0.091.24 ± 0.062.57 ± 0.08 Fe2+
* lines area ratio 0.79 ± 0.05; linewidth ratio 0.36 ± 0.02; ** fixed.
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Mažeika, K.; Melvydas, V.; Čepukoit, D. Reduction of Ferric Chloride in Yeast Growth Media, by Sugars and Aluminum. Inorganics 2024, 12, 137. https://doi.org/10.3390/inorganics12050137

AMA Style

Mažeika K, Melvydas V, Čepukoit D. Reduction of Ferric Chloride in Yeast Growth Media, by Sugars and Aluminum. Inorganics. 2024; 12(5):137. https://doi.org/10.3390/inorganics12050137

Chicago/Turabian Style

Mažeika, Kęstutis, Vytautas Melvydas, and Dovilė Čepukoit. 2024. "Reduction of Ferric Chloride in Yeast Growth Media, by Sugars and Aluminum" Inorganics 12, no. 5: 137. https://doi.org/10.3390/inorganics12050137

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