The effect of heavy metal ions (Me⁺) on the process of anaerobic fermentation is associated with the uncontrolled content of metal-containing compounds in the raw material entering the fermentation (for example, organic municipal waste [1] ), which can lead to inhibition of anaerobic association of microorganisms by the following mechanisms:

· Metal ions can interact with electron donor (hydroxyl, carboxyl, amino and sulfhydryl) groups of organic compounds, changing their structure and influencing their biological action [2];

· Metal ions can irreversibly replace divalent cations of the active centers of enzyme systems and other components of cells, which causes growth inhibition and death of microorganisms [3];

· Oxidizing metals—have a detrimental effect on microorganisms due to irreversible oxidation of enzymes and structural components of cells [4];

· Metal ions can act as antimetabolites, inactivating their catabolism or accelerating it [5].

In an anaerobic process, with the participation of a complex anaerobic association of microorganisms, metal ions can be involved in many physicochemical processes, including precipitation in the form of sulfides (except Cr³⁺), carbonates and hydroxides; sorption on solids of the anaerobic system; formation of complexes in solution with intermediate metabolites, substrates and products formed during fermentation; binding to the cell wall of bacteria, etc. [6].

The role of iron in the vital activity of the anaerobic association of microorganisms is manifested in biological action in the composition of enzymes: electron transfer chains, hydrogenases, CO-dehydrogenase, methane monooxygenase, NO-reductase, superoxide dismutase, nitrite and nitrate reductase, etc. [7].

The most accepted mechanism of bioavailability of iron metal is related to corrosion: H₂, which is formed, can be used to reduce CO₂, which is an intermediate product in the stages of anaerobic fermentation, to methane by hydrogenotrophic methanogens [8].

During the accumulation of iron in the environment, microorganisms encapsulate the metal, which can damage the cell structure.

Iron (III) ions affect the granulation of anaerobic sludge due to the fact that they act as centers of granule formation, increase the diameter of the granules and improve its granularity [9].

Inhibition of the activity of microorganisms by iron (II) oxide can be caused by its high concentrations and as a subsequent pH value, which provides conditions that are incompatible with the development of bacteria [10].

Cu²⁺ is a high-potential oxidant (Eo) (Cu²⁺/Cu⁰ = +338 mV), which is able to replace divalent metals in the active centers of enzymes [11]. Microorganisms can reduce Cu (II) ions to Cu (I) and Cu (0) (for redox systems formed by Cu (II), Cu (I) are characterized by lower values of standard potentials—+160 mV Cu²⁺/Cu⁺, and + 518 mV for Cu⁺/Cu⁰) [12].

Microorganisms tightly regulate the concentration of cytoplasmic Cu²⁺ to minimize its toxic effects, while providing a sufficient amount of proteins containing copper ion (Cu-proteins). In turn, Cu proteins in bacteria are periplasmic or extracellular rather than cytoplasmic [11]. If the Cu²⁺ content becomes too high for the vital activity of microorganisms, the inclusion of cell defense mechanisms are activated: Cue (Cu efflux) system [13], CsoR (copper sensitive operon) system [14], oxidation of Cu (I) to obtain less toxic ion Cu (II) [12].

The process of methanogenesis at high concentrations of copper ions is influenced by factors such as the presence in the environment of chelating agents (inhibitory effect is observed at higher concentrations), density of microorganisms (with increasing concentration of microorganisms—the maximum allowable salt content increases), pH (at neutral value or slight leaching—permissible concentration of copper ions increases), the ratio of iron and copper ions (the higher the content of iron ions, the higher the value of the inhibitory concentration of copper ions).

Zn (II) is part of enzymes that affect phosphate, carbohydrate, protein metabolism, RNA and ribosome synthesis and regulation of redox potential of cells (hydrogenase, dehydrogenase formate, superoxide dismutase, etc.), it is a stabilizer of membrane components, determining their reactivity [7] [15] [16]. The minimum concentration of zinc ions that is required for different types of microorganisms varies widely.

Inhibitory content of zinc ions in the environment—3 - 400 mg/dm³, toxic— 250 - 600 mg/dm³ depending on the species of microorganisms and living conditions [17].

Cr (III) is an important trace element required for glucose, lipid and amino acid metabolism, play a regulatory role in the processes of replication and transcription in microorganisms [18] [19] [20], but in high concentrations causes a negative effect on cellular structures. Cr (VI) is 1000 times more toxic and mutagenic than Cr (III) due to the oxidative capacity of compounds [21]. When chromium (III) methionine is added to the medium at a concentration of 0.5 - 1.5 mmol/dm³, activation of anabolic processes, cellulose and amylolytic activity, cell growth of microorganisms is observed, as a result of which their mass increases [22]. Chromium (III) chloride in concentrations up to 1.0 mmol/dm³ increases cell biomass growth. Increasing the concentration of chromium (III) salts to 2.5 mmol/dm³ leads to inhibition of hydrolytic processes and cell growth.

Under anaerobic conditions, Cr (VI) can serve as the ultimate acceptor of electrons in the respiratory chain for a wide range of electron donors, including carbohydrates, proteins, fats, hydrogen, NAD(F)N [23], both cytoplasmic and membrane enzymes are involved in the reduction of Cr (VI) under anaerobic conditions [24].

For different types of anaerobic microorganisms and their associations, there is a range of heavy metal ions in the environment, which does not adversely affect biogas production: for Fe³⁺—4 - 150 mg/dm³, for Cr³⁺—28 - 50 mg/dm³; for Cu²⁺—0.3 - 5 mg/dm³, for Zn²⁺—5 - 20 mg/dm³.

The goal of the research is to establish the dependence of the yield of biogas and methane on the concentration of Me⁺ ions in the anaerobic environment.

As shown in Figure 1(a), Fe³⁺ has a positive effect on the activity of the anaerobic association of microorganisms in the anaerobic fermentation of cattle manure. Compared to the sample without the addition of iron salts, the samples under study have a higher biogas yield. In samples containing 40 and 60 mg/dm³ Fe³⁺, biogas formation began on the day of fermentation. At a content of 20 mg/dm³ Fe³⁺, biogas formation occurred starting from 2 days. At a content of 80 mg/dm³ Fe³⁺, the yield of biogas slows down.

As shown in Figure 1(b), the highest yield of biogas 4300 dm³ for the entire fermentation period was obtained at a content of 20 mg/dm³ Fe³⁺, which is 76.79 ± 3.84 dm³/g DOM (Figure 1(c)), and 35% more compared to biogas yield in the control sample. When the concentration of iron ions increases to 60 mg/dm³, the yield of biogas and methane content in it decreases. At the same time with increasing content of iron ions decreases the methane content in biogas (Figure 1(b)). At such concentrations of iron ions, the yield of biogas exceeds the yield of the control sample by 19.3% and reaches the values of 67.86 ± 3.39 dm³/g DOM (Figure 1(c)). Based on the fact that gas formation decreases after 9 days of fermentation,

it can be assumed that Fe³⁺ has a more positive effect on acetogenic and acidogenic microorganisms of the association, due to participation in a number of enzymes: hydrogenase (absorption or release of H₂), monoxide dehydrogenase (CODH)—an enzyme for the formation of acetic acid by anaerobic bacteria [7]. A slight increase in biogas yield after 16 and 20 days for samples containing iron ions of 20 and 40 mg/dm³, respectively, may be associated with hydrogenotrophic methanogens that use hydrogen and CO₂ to form methane, because the concentration of iron ions decreases in the process of fermentation due to the increase in biomass.

As shown in Figure 2, a, the content of 40 mg/dm³ Cu²⁺ in the anaerobic system has a positive effect on the biogas yield. At the same time, the methane yield is the same for Cu²⁺ 40 and 60 mg/dm³ concentrations. That is, when the content of copper ions increases to 60 mg/dm³, the concentration of methane in biogas increases and reaches 83%. While at a content of 40 mg/dm³ of copper ions, the concentration of methane is 60%, as for other concentrations. Based on the fact that the methane content in biogas increases while reducing its yield, it can be assumed that this concentration of copper ions has a negative effect on microorganisms—hydrogen consumers, which are contained in the association, thereby increasing methane production from CO₂ and hydrogen. This leads to increased methane yield and reduced CO₂ content in biogas.

Copper ions (Figure 2(b)) affect methanogenic microorganisms at a concentration of 80 mg/dm³, the concentration of methane in biogas begins to reduce, also reduces the yield of biogas. The concentration of Cu²⁺ 10 mg/dm³ slightly improves biogas yield (Figure 2(b)), but has a positive effect on methane yield in biogas (Figure 2(b)). Instead of the expected inhibitory effect, we obtained a stimulating effect on the yield of biogas and biomethane, which can be explained by the formation of complex copper compounds with environmental components contained in raw materials and high concentrations of microorganisms in the system and sufficient Cu proteins [11].

Another mechanism of general resistance to copper is the diversity of anaerobic association, as evidenced by the lack of inhibition of Methanobacteriumthermoautotrophicum in cocultivation with sulfate-reducing bacteria Desulfotomaculumspp. at a concentration of Cu²⁺ in a medium of 63.5 mg/dm³, which causes complete inhibition of Methanobacteriumthermoautotrophicumt [28]. In this case, part of the hydrogen sulfide reacts with metal ions in the environment with the formation of metal sulfides, so the inhibitory effect on microorganisms is reduced [29]. Desulfovibrio strain R2 is resistant to 800 mg/dm³ Cu²⁺ because it contains the plasmid gene for resistance to Cu²⁺ [30]. The content of copper ions in superoxide dismutase (SODM) and hydrogenase in methanogens [31] may also affect the lack of inhibition.

Given the data obtained, it can be taken into account that the concentration range of Cu²⁺ 20… 80 mg/dm³ increases the biogas yield from 56.88 dm³/g DOM (control) to 114.29 dm³/g DOM (Figure 2(c)), that can be used in the production of micronutrients for biogas plants.

Figure 3(a) shows that the addition of Zn²⁺ does not stimulate biogas production in the anaerobic association of microorganisms, and even at the lowest concentration (10 mg/dm³) biogas yield decreases from 80.36 dm³/g DOM (control sample) to 74.11 dm³/g DOM, (Figure 3(c)). This confirms the fact that the inhibitory effect of zinc ions on microorganisms at concentrations of 3 mg/dm³ in the environment [17]. In this case, the methane content in biogas increases from 56% in the control to 72% at a zinc concentration of 10 mg/dm³ (Figure 3(b)). Concentrations of 20 and 50 mg/dm³ have the same biogas yield for 13 days of fermentation (Figure 3(b)), but at 20 mg/dm³ the methane yield is slightly higher than at 50 mg/dm³ (Figure 3(b)). It is also worth noting that the action of zinc ions increases the content of H₂ in biogas and reaches 7% - 10%, which indicates a positive effect of zinc ions on hydrogen-producing microorganisms.

As shown in Figure 4(a), Cr³⁺ has a stimulating effect on the activity of the association of microorganisms, including methanogens, which is manifested by peak biogas yields for 8 - 10 days for all test samples containing chromium salts. At the same time, the peak value of biogas yield is almost twice the value of the control sample. This confirms the fact that Cr (III) is involved in the metabolism of organic compounds, which can promote the growth of cells of microorganisms, thereby increasing their productivity [22].

Methane production also increases (Figure 4(b)) with the addition of chromium salts: if for the control sample the methane content in biogas was 56%, then for the tested samples—not less than 68%. Figure 4(b) and Figure 4(c) show that the biogas yield at a concentration of 10 mg/dm³ is 37.8% higher than the control values and 5% higher than the biogas yield in the concentration range of 20 - 50 mg/dm³. But increasing the concentration of chromium ions, despite the same biogas yield at this concentration range, leads to a decrease in methane content from 70% for 20 mg/dm³ to 68% for 50 mg/dm³.

It is shown that ions of such metals as Fe³⁺, Cu²⁺, Cr³⁺ have a positive effect on the yield of biogas and methane content in it. All experimental concentrations of Fe³⁺, Cu²⁺, Cr³⁺ and Zn²⁺ ions lead to increased methane production by the anaerobic association of microorganisms due to the effect on the activity of enzyme systems.

Rational concentrations to increase methane yield in biogas are: Fe³⁺—20 - 40 mg/dm³, Cu²⁺—40 - 60 mg/dm³, Cr³⁺—10 mg/dm³. Zinc ions have a positive effect on methane production, but reduce the overall yield of biogas and, accordingly, the degree of conversion of organic raw materials. Therefore, the rational concentration of Zn²⁺ is 10 mg/dm³.

All authors contributed to the concept and design of the study. Experiment planning performed by N. B. Golub, M. V. Kozlovets. Preparation of material, construction of laboratory installation performed by A. V. Shynkarchuk, O. A. Kozlovets. The experiment, data collection was performed by A. V. Shynkarchuk, O. A. Kozlovets. Data analysis was performed by M. V. Kozlovets, N. B. Golub. The first draft of the manuscript was written by A. V. Shynkarchuk, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

The authors thank Igor Levtun for the technical assistance provided in writing up this manuscript.

The authors declare no conflicts of interest regarding the publication of this paper.

Golub, N.B., Shynkarchuk, A.V., Kozlovets, O.A. and Kozlovets, M.V. (2022) Effects of Heavy Metal Ions (Fe³⁺, Cu²⁺, Zn²⁺ and Cr³⁺) on the Productivity of Biogas and Biomethane Production. Advances in Bioscience and Biotechnology, 13, 1-14. https://doi.org/10.4236/abb.2022.131001