Methanogenesis—General Principles and Application in Wastewater Remediation

Abstract

The first discovery of methanogens led to the formation of a new domain of life known as Archaea. The Archaea domain exhibits properties vastly different from previously known Bacteria and Eucarya domains. However, for a certain multi-step process, a syntrophic relationship between organisms from all domains is needed. This process is called methanogenesis and is defined as the biological production of methane. Different methanogenic pathways prevail depending on substrate availability and the employed order of methanogenic Archaea. Most methanogens reduce carbon dioxide to methane with hydrogen through a hydrogenotrophic pathway. For hydrogen activation, a group of enzymes called hydrogenases is required. Regardless of the methanogenic pathway, electrons are carried between microorganisms by hydrogen. Naturally occurring processes, such as methanogenesis, can be engineered for industrial use. With the growth and emergence of new industries, the amount of produced industrial waste is an ever-growing environmental problem. For successful wastewater remediation, a syntrophic correlation between various microorganisms is needed. The composition of microorganisms depends on wastewater type, organic loading rates, anaerobic reactor design, pH, and temperature. The last step of anaerobic wastewater treatment is production of biomethane by methanogenesis, which is thought to be a cost-effective means of energy production for this renewable biogas.

1. Introduction

In nature, methane can be produced in anaerobic environments by methanogenic microorganisms. The starting point of methane production is the degradation of biomass [1,2]. Sources of renewable biomass vary from food industry waste to agricultural waste, animal manure, and forest industry waste [3]. Carbon dioxide acquired from biomass degradation is reduced with hydrogen in a process called methanogenesis. As methanogenesis is a multi-step process, a syntrophic association between two or more organisms is needed [4]. Methanogenesis plays a crucial role in the global carbon cycle. Moreover, processes for waste conversion to biomethane by methanogenesis are being implemented in industry, and more than 25% of all bioenergy is predicted to originate from waste-produced biogas [1,3].

2. Hydrogen for Methane Formation

In Earth’s lower atmosphere, molecular hydrogen is present in trace concentrations. Nonetheless, H₂ plays a key role in the biogeochemical cycles of numerous elements, thus connecting different parts of ecosystems, with the most prominent function in anoxic environments. In anaerobic environments, hydrogen occurs in very low concentrations and exhibits a fast turnover rate (turnover times are in a matter of minutes) [5,6]. Despite low concentrations, annual formation and consumption of hydrogen from biomass add up to 0.3 Gt. Half of this quantity is used for methanogenesis from carbon dioxide. In environments where the reduction of carbon dioxide to methane is the predominant reaction, H₂ steady-state partial pressure is around 10 Pa. Because of the ability to diffuse through cytoplasmic membranes, H₂ is the best electron carrier between microorganisms [7,8]. Consumption of hydrogen in methanogenic environments (where no inorganic electron acceptors other than carbon dioxide are available) is possible by methanogenic Archaea. More than 80 species from the Archaea domain are capable of conducting methanogenesis. Almost all of the hydrogen utilized by methanogenic microorganisms is of biological origin. However, a fraction of the hydrogen for sustaining methanogen growth is of geochemical origin [6,8,9].

3. Global Methane Cycle

Methane gas (CH₄) is one of the main greenhouse gases, with a global warming potential greater than that of carbon dioxide. Alongside CO₂ and N₂O, CH₄ plays a key role in degradation of the ozone layer. Human-related CH₄ emissions make up two-thirds of the total global methane emissions. The concentration of methane in the atmosphere has doubled over the past century [1,4].

Biological production of methane is mediated by methanogenic Archaea (methanogens). This process is called methanogenesis or biomethanation, and it plays a key role in the carbon cycle [1]. In total, 1 Gt of methane is produced annually from biomass in anoxic environments from acetate, carbon dioxide, and hydrogen through methanogen activity. Another 1 Gt is released into the environment by the melting of methane hydrates. From the 2 Gt of CH₄ produced annually, 1 Gt is oxidized by anaerobic Archaea and 0.6 Gt by aerobic bacteria, and 0.4 Gt diffuses to the atmosphere, with an additional 0.2 Gt released from other sources [4]. Biomass consists mostly of carbohydrates (~50%), proteins (around 30 to 40%), and lipids (~10%). Because of the complex biomass composition, different metabolic groups are required for degradation [2]. Methanogens function in a syntrophic manner, meaning that biomass degradation is catalyzed by a combination of multiple organisms that, on their own, cannot catabolize biomass. A combination of bacteria, protozoa, and fungi is needed to decompose biomass. The products are then transformed into acetic acid, carbon dioxide, and hydrogen by syntrophic bacteria [4]. In the last step of the process, methanogenic Archaea use hydrogenases for the activation of hydrogen, resulting in the formation of methane [8]. Methanogenesis is beneficial for treatment of organic waste in industry. Methane harnessed from renewable carbon feedstock by anaerobic digestion with methanogens is a useful tool for biogas production. Biogas is comprised of 30% to 90% CH₄ and can be used as a fuel to generate electricity or to power transportation [1,10]. It is estimated that 1 m³ of CH₄ has a calorific value around 9.17 kWh [11], meaning that biogas containing the aforementioned ranges of CH₄ can have calorific values up to 9 kWh. When produced by anaerobic digestion, biomethane is a non-poisonous, colorless, and odorless gas. In comparison to other liquid biofuels, the separation of biomethane from the liquid phase is relatively easy and therefore has reduced production costs [3].

4. Methanogens

By studying prokaryotic relationships, Woese and Fox [12] discovered that microbes producing methane (methanogens) are Archaea. The discovery of this domain allowed better understanding of different properties methanogens exhibit in comparison to members of the Bacteria and Eukarya domains [13]. Nowadays, in the Archaea domain, there are seven methanogenic orders: Methanobacteriales, Methanocellales, Methanococcales, Methanomicrobiales, Methanoplasmatales, Methanopyrales, and, lastly, Methanosarcinales [14]. Methanogens populate anaerobic environments, especially low-sulfate habitats, and can be isolated from a broad range of thermochemical gradients. It is important to note that the presence of oxygen inhibits methanogen growth [1]. Methanogenic microbes can originate from acidophilic to alkaliphilic environments (pH 3.0 to 10.2) and psychrophilic to hyperthermophilic temperatures (−2 °C to 122 °C) [10]. In recent years, many methanogens have been discovered to be hyperthermophiles with optimum growth temperatures such as 98 °C (e.g., Methanopyrus kandleri) and 85 °C (e.g., M. jannaschii) [4]. Freshwater and marine sediments, marshes, geothermal systems, rice paddies, and human and animal gastrointestinal tracts are some of the typical methanogenic habitats [1,15]. In many environments, methanogens are consumers of hydrogen (hydrogenotrophs). They are the most numerous and the fastest-growing microbes [13]. Generation times can range from minutes (e.g., 25 min for Methanocaldococcus jannaschii) to a few hours (e.g., 6 h for Methanosarcina barkeri) [10]. The highest growth rates are observed in the Methanococcales order [16]. Methanogens grow by reducing coal, acetate, or one-carbon compounds, such as carbon monoxide, carbon dioxide, and methanol to methane. Generally, methanogen growth can be described by Equation (1). By producing CH₄, methanogenic microorganisms can also conserve energy [10].

e⁻ donor + e⁻ acceptor → CH₄ + byproduct

(1)

Methanogenic Archaea required for biological CH₄ production can be with or without cytochromes. An overview of the differences between these methanogens is presented in

Table 1. Differences between methanogens.

Methanogens with Cytochromes	Methanogens Without Cytochromes	Ref.
contain methanophenazine	do not contain methanophenazine	[4]
only some grow on CO2 and H2; most grow on methylamines and acetate; cannot grow on formate	grow on CO2 and H2; cannot grow on methylamines and acetate; most can grow on formate
growth yields on CO2 and H2 ≤ 7 g/mol CH4	growth yields on CO2 and H2 ≤ 3 g/mol CH4
H2 partial pressure threshold is >10 Pa	H2 partial pressure threshold is <10 Pa
not hyperthermophilic	hyperthermophilic

[4].

Hydrogenases from Methanogenic Origin and Their Function

Hydrogenases are widely distributed in microbes. These enzymes serve different physiological functions in various metabolic pathways, e.g., in remediation of toxic heavy metals, parasites, and pathogenic bacteria, as well as in methane formation [17]. Organisms from three domains of life (Archaea, Bacteria, and Eucarya) utilize hydrogenase activity to produce or consume hydrogen [16]. For example, hydrogen gas for methanogenesis is formed in anaerobic environments with the help of hydrogenases [2].

Hydrogenases were first discovered by Stephenson and Stickland [18,19] and are classified based on their active site structure as [NiFe]-hydrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenases. Besides the main classification based on active site cofactors, hydrogenases can be further categorized by location within the harboring cell (cytoplasmic or membrane-bound), ability to conserve energy, and their electron-carrying redox partners [16]. From hydrogenase classes, the most studied are [NiFe]-hydrogenases [17].

As Stephenson and Stickland [20] studied methane-forming microorganisms from river sediments, the correlation between hydrogen-forming bacteria and hydrogen-consuming methanogens became evident. Additionally, the nickel requirement for hydrogenase activity was first discovered in methanogens. In methanogenic Archaea, only [NiFe]-hydrogenases and [Fe]-hydrogenases were found. [FeFe]-hydrogenases, present in Eucarya and Bacteria domains, have not yet been discovered in Archaea. Genetic and biochemical studies have been conducted on a limited number of methanogen species, namely Methanococcus maripaludis, Methanosarcina barkeri, and Methanosarcina mazei [8].

Functional hydrogenases are not expressed in all methanogens [10]. However, in methanogens expressing hydrogenases, mostly [NiFe]-hydrogenases are found [8]. M. maripaludis is a typical representative of methanogens without cytochromes [4]. In methanogens without cytochromes, four types of hydrogenases produced are as follows: (a) membrane-bound, ferredoxin-dependent energy-converting hydrogenase (Ech), (b) cytoplasmic, F₄₂₀-dependent hydrogenase–F₄₂₀-reducing hydrogenase (Frh), (c) cytoplasmic, electron-bifurcating Mvh hydrogenase, and (d) cytoplasmic, [Fe]-hydrogenase [21].

On the other hand, all Methanosarcinales organisms, with representatives such as M. barkeri and M. mazei, belong to methanogens with cytochromes [4]. Methanogens with cytochromes encode three types of hydrogenases: (a) membrane-bound, ferredoxin-dependent energy-converting hydrogenase (Ech), (b) cytoplasmic, F₄₂₀-dependent hydrogenase–F₄₂₀-reducing hydrogenase (Frh), and (c) membrane-bound, methanophenazine-dependent hydrogenase (Vht) [21].

Even though methanogens with and without cytochromes have different mechanisms for energy conservation, both contain [NiFe] ferredoxin-dependent energy-converting hydrogenases and F₄₂₀-reducing hydrogenases. With the exception of the bimetallic [NiFe] active site and three FeS clusters found in all [NiFe]-hydrogenases, membrane-bound energy-converting hydrogenases (Ech) have few sequence similarities to other hydrogenases of the [NiFe] family [16,22]. Ech contains six subunits, EchA–F. The EchE subunit harbors the bimetallic active center for the formation and oxidation of H₂. The remaining subunits are in charge of transferring electrons between ferredoxin (Fd) and H₂/H⁺ [16].

Furthermore, the coenzyme F₄₂₀-reducing hydrogenase (Frh) is a heterotrimeric enzyme with three subunits. The FrhA subunit harbors the [NiFe] active site responsible for hydrogen oxidation. Flavin adenine dinucleotide (FAD) containing subunit FrhB functions as an active site for oxidation and reduction of F₄₂₀ coenzyme. Electrons travel from the FrhA subunit through the FrhG subunit to the active site in the FrhB subunit. This hydrogenase is a requirement in all methanogens possessing the ability of CO₂ reduction with H₂-derived electrons [16]. The methanophenazine-dependent hydrogenase was initially identified as viologen-reducing hydrogenase (two) and thus has the Vht abbreviation. The VhtA subunit contains a [NiFe] active center for hydrogen oxidation. Electrons from H₂ oxidation reduce methanophenazine (MP), which is later used for the reduction of the terminal electron acceptor CoM-CoB, a disulfide of coenzyme M (CoM) and coenzyme B (CoB), common in all methane-forming pathways. This type of hydrogenase is found only in methanogens from the Methanosarcinales order. One cytoplasmic [Fe]-hydrogenase, limited to methanogens without cytochromes, is a substitute for F₄₂₀-reducing [NiFe]-hydrogenase in nickel-limiting conditions [8,16].

5. Methanogenic Pathways—Dependence on Substrates and Mechanism

Production of methane is a multi-step process classified based on the substrate utilized in the production pathway on the hydrogenotrophic Equation (2) and acetoclastic Equation (3) pathways. The hydrogenotrophic pathway utilizes inorganic carbon dioxide as a substrate, while acetoclastic involves the use of acetic acid [1]. Another less common pathway is the methylotrophic production of methane, where the substrates are methanol or methylamines [1,14]. Equation (4) describes the methylotrophic production pathway [11].

4H₂ + CO₂ → CH₄ + H₂O

(2)

CH₃COOH → CH₄ + CO₂

(3)

4CH₃OH → 3CH₄ + CO₂ + H₂O

(4)

The determination of the substrate used for successful methanogenesis is mostly guided by pH and temperature [1]. The substrate abundance affects total methanogenesis yield but does not play a role in controlling the rate of reactions [9]. Depending on the methanogenic order, different methanogenesis pathways are predominant. With the exception of Methanoplasmatales (also referred to as Methanomassiliioccales), which are only methylotrophic methanogens, all orders support the hydrogenotrophic methanogenesis pathway [10]. This is because the hydrogenotrophic pathway is more energetically favorable than acetoclastic and methylotrophic production pathways [14]. Nevertheless, around two-thirds of biologically produced methane comes from acetoclastic methanogenesis [16]. Alongside hydrogenotrophic, Methanosarcinales are capable of acetoclastic and methylotrophic methanogenesis [10].

To understand existing and predicting new methanogenic pathways, thermodynamic studies are the key. As shown in Equation (1), methanogen metabolism can be simply described as electron flow from electron donor to electron acceptor. Thermodynamic studies focus on determining whether the reaction is feasible or not, i.e., whether an organism can conserve energy for growth or not. This estimation is done by calculating Gibbs free energy. The value of Gibbs free energy is used to predict the amount of ATP synthesized depending on the amount of substrate consumed and should be negative for a reaction to be favorable [10]. By comparing Gibbs free energy values, general conclusions about microbial growth and feasibility of reactions can be drawn. These studies have introduced a spectrum of new substrates available for methanogenesis beyond the three previously mentioned and commonly described in literature. A case study conducted by Cozannet et al. based on thermodynamic calculations elucidated new substrates for methanogenesis, bringing the number of known substrates from 10 (until 1979) up to 152 (in 2023). Alongside 152 proven substrates, 41 putative substrates were predicted on account of thermodynamic calculations [23]. To tie in the notion of Gibbs free energy with the metabolic productivity of methanogen, it is worth mentioning that methanogenic reactions with these (152 + 41) substrates have value ranges of >0 kJ mol⁻¹ CH₄ (for 6.4% of substrates), 0 to −30 kJ mol⁻¹ CH₄ (62.8% of substrates), −30 to −100 kJ mol⁻¹ CH₄ (5.2% of substrates), −100 to −200 kJ mol⁻¹ CH₄ (62.8% of substrates), and <−200 kJ mol⁻¹ CH₄ (2.9% of substrates). A few examples of substrates promoting methane production, alongside the methanogenic order of a methanogen responsible for conversion of these substrates to methane, are shown in

Table 2. Methanogenesis substrates excluding previously mentioned carbon dioxide, acetic acid, and methanol.

Substrate	Methanogen Order	Ref.
pyruvate	Methanococcales, Methanosarcinales	[23]
carbon monoxide	Methanosarcinales
primary and secondary alcohols (ethanol, 2-propanol and 2-butanol)	Methanomicrobiales
methylated amine compounds (monomethylamine, dimethylamine, trimethylamine, choline and glycine betaine)	Methanosarcinales
organosulfur compounds (methanethiol and dimethylsulfide)	Methanosarcinales
metoxylated aromatic compounds	Methanosarcinales, Methanomicrobiales

[23]. Methanosarcinales have been an outstanding order of organisms for studies on methanogenic pathways [24]. Here, this statement is supported with Table 2, seeing that most of methanogens derive from the Methanosarcinales order. Methanogenic pathways utilize several cofactors and coenzymes shown in

Table 3. Coenzymes and their role in methanogenesis.

Coenzymes	Functional Role	Ref.
methanofuran (MF)	carriers of carbon moiety for generation of methane	[1]
tetrahydromethanopterin (H4MPT)
tetrahydrosarcinapterin (H4SPT)
coenzyme M (HS-CoM)
coenzyme B (HS-CoB)	transferring of electrons for carbon reduction
coenzyme F420
coenzyme F430
methanophenazine

[1].

Hydrogenotrophic pathway is described as a stepwise reduction of CO₂ to CH₄ with electrons acquired from hydrogen oxidation by hydrogenases. Even though methanogenic pathways have been greatly described, the involvement of hydrogenases in electron flow and different aspects of energy conservation need further characterization [16]. In the case of methanogens with cytochromes, energy conservation and production of methane from CO₂ relies on 13 reactions. The energy conservation relies on, among other proteins, energy-converting [NiFe]-hydrogenase and methanophenazine-reducing [NiFe]-hydrogenase, in a manner briefly explained previously. The methanogenic pathway in methanogens without cytochromes generally proceeds in the same manner as methanogenesis with cytochromes, with most of the enzymes and coenzymes being the same [4]. The final step for methane production in all methanogenic pathways is catalyzed by methyl-coenzyme M reductase [14]. All anaerobic methanogenic pathways produce CoM-CoB disulfide in the final step of methane production. Methanogens with cytochromes utilize multiple mechanisms for electron transfer depending on the electron source (that being H_2, F₄₂₀, or Fd) and whether the microorganism is capable of utilizing hydrogen as a substrate. In contrast, methanogens without cytochromes utilize only one mechanism called flavin-based electron bifurcation [16]. Due to the dependence on hydrogenase enzymes and thiol cofactors (coenzyme M and coenzyme B), methanogenesis is a seemingly reversible reaction [10].

6. Factors Effecting Methanogenesis

In view of the close interdependence between hydrogen-producing microorganisms and hydrogen-utilizing methanogens, a change in any environmental factor influencing one of the syntrophic microorganism results in a change in the overall extent and rate of methanogenesis [25].

6.1. Hydrogen Concentration

Firstly, methanogenesis is under the control of the environmental hydrogen concentrations. A steady-state H₂ concentration is reached when the production and utilization rates are equal. When the production rate is greater than the rate of utilization, H₂ concentration increases, resulting in higher utilization of H₂. A higher steady-state hydrogen concentration does not persist for long, as H₂ utilizers grow and increase their biomass. From this, a general conclusion that the concentration of hydrogen is controlled by the kinetic characteristics of hydrogen utilizers can be made. Maximum growth rate (µ_max), specific maximum hydrogen utilization rate (u_max), hydrogen concentration at half-maximum growth rate (K_s), and Michaelis constant (K_m) are specific for each methanogenic microorganism [6]. Hydrogen concentration, i.e., H₂ accumulation, is also one of the factors that can limit hydrogenase activity. H₂ concentration can reverse the metabolism of a microorganism from H₂-oxidising to H₂-reducing [26]. This characteristic explains the ‘reversible’ metabolism of methanogens [10].

6.2. Oxygen and Sulfate Presence

Methane production from biomass degradation is feasible only in anoxic environments with low sulfate concentrations [4]. Oxygen inhibition is apparent in methanogen growth, so much so that methanogens will not grow or produce CH₄ even if trace levels of O₂ are present. Most of the hydrogenases present in methanogens are rapidly inactivated by oxygen [8,25]. Only membrane-bound [NiFe]-hydrogenases are known to retain catalytic activity in the presence of oxygen. O₂ tolerance may be the key in allowing methanogenic microorganisms to persist in environments with fluctuating anoxic and oxic conditions [25,27].

Furthermore, in the presence of sulfate as an electron acceptor, methanogen activity is out-competed by sulfate reducers. Because of the lower K_s value, sulfate reducers grow faster as opposed to methanogens, resulting in faster utilization of hydrogen. After sulfate reducers outgrow the methanogenic population, a complete reaction inhibition is achieved. For this reason, in environments where sulfate concentrations are high, methanogens are replaced by sulfate reducers. Analogously, the addition of Fe (III) or nitrate inhibits methanogen activity, with iron and nitrate reducers becoming more predominant in the methane-forming pathway [6].

6.3. Environmental Salinity

Higher environmental salinity may have an inhibitory effect on methanogen activity [9]. In hypersaline conditions, characterized by high concentrations of magnesium chloride, sodium chloride, magnesium sulfate, and other salts, halophilic and extremely halophilic methanogens can be found. The prevailing methanogenesis pathway in these environments is methylotrophic [25]. The mechanism of the inhibitory effect salts have on hydrogenase activity is not fully understood [26]. Given that conditions in hypersaline environments are a result of higher temperatures, methanogenic microorganisms inhabiting these environments may face multiple extremes [25].

6.4. Temperature

A key factor in controlling methanogenic reactions, alongside nutrient availability, is the temperature. Methanogens exhibit the highest activity at temperatures between 35 and 45 °C. In contrast to mesophilic methanogens, psychrophiles and (hyper)thermophiles are not as important in the methane formation pathways. Only methanogens without cytochromes exhibit hyperthermophily [4,9]. Interestingly, M. kandleri is the only known methanogen with activity above 100 °C. Methanogenesis reaction rates increase more steeply with the increase in temperature than those of other biological processes, such as photosynthesis or heterotrophic respiration [16,25].

6.5. pH Values

Optimal pH values for methanogenic archaea are in a range from 6.8 to 7.5 [11]. Some methanogen activity was recorded in environments with pH values as low as 3. However, for optimal activity, higher values are required. Low temperature ranges frequently accompany acidic habitats. For acidophilic methanogens, pH values can be 4.3, but optimally are above 5. Like acidic environments, alkaline environments are characterized by some extremes for methanogen activity. Alkalinity is commonly associated with higher temperatures and high salt concentrations; therefore, some alkaliphilic methanogen representatives are also halophilic. Alkaliphilic methanogens have an optimum pH of 9.0 to 9.5 [25].

6.6. Pressure

Pressures of 10 to 20 MPa hinder cellular processes, such as nutrient uptake and mobility in mesophilic methanogens. By contrast, elevated pressures enhance solubility of gaseous substrates and hydrogen transport between microorganisms, allowing for greater substrate conversion [25]. Although it is generally considered a denaturant for mesophilic methanogens, high pressure helps with stabilization of proteins at high temperatures [26]. An example of piezophilic methanogen is M. jannaschii, which displays optimum activity at a pressure of 75 MPa [25]. Moreover, activity assays of hydrogenases isolated from this piezophilic methanogen at 90 °C show a 4-fold increase in the half-life when the pressure was raised to 50.7 MPa, compared to the half-life at 1 MPa and the same temperature [26].

7. Application of Methanogenesis in Wastewater Remediation

The amount of industrial wastewater and organic waste is an ever-growing problem [3]. Since the end of the 19th century, systems for anaerobic wastewater treatment have been in use. Recently, extensive research has been conducted to gather a deeper understanding of anaerobic processes employed in wastewater treatment. Methanogenic pathways are the primary pathways for most organic matter disposal [28]. Methanogens reduce wastewater oxygen demand and, as a result, produce renewable biogas in the form of biomethane [10]. However, this remediation method can be utilized only when a high level of readily biodegradable soluble organic matter is present. Anaerobic remediation techniques are usually used for high-loaded wastewaters with COD (chemical oxygen demand) greater than 1.5–2 g L⁻¹. Besides substantial results in the treatment of heavily polluted wastewaters with a COD between 3 and 4 g L⁻¹, anaerobic treatment has some success in the remediation of wastewaters with a COD lower than 1.5 g L⁻¹ [28]. In wastewater, organic matter is decomposed by a variety of microorganisms. Primary producers decompose raw waste into organic fatty acids or other substrates, which are then consumed by methanogens to produce biomethane. A simplified representation of these reactions is shown in Figure 1. The composition of syntrophic microorganisms utilized in wastewater remediation is guided by wastewater type, pH, temperature, reactor design, and organic loading rates [3,28]. In anaerobic reactors, methanogens take up 5 to 6% of the total microbial population, while the rest is occupied by the bacterial population [28].

7.1. Classification of Anaerobic Reactors

Anaerobic wastewater treatments have, in recent years, evolved from lab-scale trials to successful application in various industries. Numerous anaerobic reactors are implemented in wastewater management. The classification of anaerobic reactors is based on the retention characteristics of components involved in biomethane production [3,28]. Type A is the simplest reactor design, with equal retention times of microorganisms (RT_m), solid (RT_s), and liquid (RT_l). This type of reactor allows uniform substrate and temperature distribution. However, it suffers from potential microorganism washout and incomplete homogenization at larger scales. The type B anaerobic reactor has higher RT_m and RT_s than RT_l. Because of this, higher process efficiencies can be achieved. One of the drawbacks is the loss of microorganisms at high loading rates. Lastly, the type C reactor has a microorganism retention time that is higher than the retention times of solid and liquid. The highest biogas performance and overall reaction efficiency are achieved by the type C reactor. Adaptability to a variety of loading rates is a key advantage of type C reactors. Like type B reactors, type C reactors do not need extensive mixing systems. In contrast to the large volume requirements of type A reactors, type C reactors function with smaller tank volumes. Wastewaters with high solid and grease contents are not suitable for treatment in type C reactors, but for diluted wastewaters this reactor is the best choice. A few examples of reactors representing each type are shown in

Table 4. Reactor examples by type. Adapted from Ref. [3].

Reactor Type	Retention Times	Examples	Ref.
Type A	RTm = RTs = RTl	closed digester tank, continuously stirred tank reactor	[3]
Type B	RTm, RTs > RTl	upflow anaerobic sludge bed reactor, closed digester tank with solid recycle
Type C	RTm > RTs, RTl	membrane bioreactor, upflow anaerobic sludge fixed film reactor

and Figure 2 [3].

7.2. Microbial Interactions Within Reactors

If the steps of anaerobic wastewater treatment, illustrated in Figure 1, are viewed in more depth, then a question about microbial interactions within the reactor arises. Conversion of organic waste is carried out in four consecutive steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. Hydrolysis is a process controlled by the presence of hydrolytic bacteria, such as Bacteroidetes and Firmicutes. The ratio of Firmicutes to Bacteroidetes serves as indication of process performance, with higher values indicating a balanced function. Products from hydrolysis are converted by acidogenesis, followed by the step of acetogenesis. These steps rely on bacterial microorganisms. The last step of the anaerobic process highlights the importance of methanogens belonging to the Archaea domain [29]. One of the most important microbial interactions is granule formation, especially in type B reactors, such as the upflow anaerobic sludge bed reactor. Mechanisms of granule formation differ depending on reactor type. The outer layer of microbial granule is formed by methanogens, such as Methanosatcinales and Methanococcales, while internal cavities contain non-methanogenic microorganisms. Even though the mechanism of microbial granule formation is not fully elucidated, their role in stabilizing anaerobic systems and improving wastewater treatment efficiency has been observed. Acetate-forming bacteria, such as Acetobacterium and Clostridium, form a considerable syntrophic relationship with methanogens by providing acetate and H₂ for methane formation [30]. Depending on the substrates present, the co-existence of fungi is needed. For example, if waste contains lignocellulosic materials, a syntrophic relationship between anaerobic fungi and methanogens is needed for efficient methane production [31]. Additionally, to prevent accumulation of hydrogen above a certain threshold that would disrupt the system balance, removal of hydrogen is conducted by methanogens, sulfate-reducing bacteria, and homoacetogens. This hydrogen interspecies transfer is considered a key factor in the syntrophic relationship between microorganisms in anaerobic reactors. Moreover, interspecies formate transfer has been observed between acetogens and methanogens [30].

7.3. Wastewater Types and the Presence of Heavy Metals

Wastewater compositions vary depending on the industry branch and greatly affect microbiome diversity in the anaerobic reactor [28]. A few examples of wastewater types, their main characteristics, the dominant methanogens used for remediation, and the most used reactor types can be found in

Table 5. Main characteristics, methanogens, and reactors used for wastewater remediation.

Industry Branch	Amount of Waste Produced	Characteristics of Wastewater	Methanogens	Reactor Type	Ref.
Beer industry	3–10 L of wastewater per 1 L of beer	high level of oxygen demand pH from 4.5 to 12 temperatures from 18–40 °C	Methanosaeta concilii Methanosarcina mazei	B	[3,32]
Oil extraction	0.5–0.75 t of palm oil mill effluent per 1 t of oil palm branch	high level of oxygen demand pH from 4 to 5 temperatures from 80 to 90 °C	Methanosaeta concilii	A, B, C	[3]
	1200–1800 L of olive mill wastewater per 1 t of olives	high level of oxygen demand pH around 5 high temperatures	Methanosaeta concilii Methanobacterium formicicum	B	[3]
Paper industry	at least 30,000 L of wastewater per 1 t of paper pulp	high level of oxygen demand fluctuating pH temperatures from 50 to 60 °C	Methanosarcina barkeri	B	[3]
Dairy industry	500–2000 L of wastewater per 1 L of milk	high level of oxygen demand pH from 5.7 to 7.8 temperatures from 30 to 40 °C	Methanosaeta spp.	B	[3,11,28]
Fruit and vegetable processing industry	n.a.	low level of oxygen demand low pH temperature range—n.a.	Methanothrix spp.	B	[28]
Slaughterhouse wastewater	90–140 L of wastewater per 1 slaughtered pig; although variable with type of animal	high level of oxygen demand fluctuating pH temperature range—n.a.	Methanobacterium Methanosarcinales	n.a.	[28]
Chemical industry	variable with the type of chemical produced	variable with the type of chemical produced	Methanothrix spp.	B, C	[28]

[3,11,28,32]. With the emergence of industries such as mining, ceramic and glass, metal plating, an increase in heavy metal concentrations in wastewaters has been observed. Heavy metals are not biodegradable and tend to accumulate in high concentrations, causing toxicity and carcinogenicity in living organisms. Because of this toxicity, the removal of heavy metals from industrial wastewaters has gained major attention. Even though heavy metals have an inhibitory effect, the interactions between syntrophic microorganisms utilized in wastewater treatments may offer a protective effect. Because of this, heavy metal removal from wastewater is possible [33]. Methanogenic archaea have the ability to methylate almost all heavy metals from Groups IV, V, and VI in the periodic system of elements [13].

7.4. Effect of Methanogenesis on Wastewater Treatments

Methanogenesis implemented in waste management stops methane leakages to the atmosphere. Waste conversion to biomethane is thought to be a cost-effective means of energy production [3]. Anaerobic wastewater treatment is considerably cheaper than an aerobic process. The lower economic expenses stem from the fact that the wastewater does not need additional aeration [11]. Moreover, excess sludge formed by anaerobic treatment does not need further stabilization, unlike in aerobic treatments where this step is a necessity. It is estimated that up to 90% savings in operational costs can be achieved, as well as 40 to 60% savings in investment costs, by applying anaerobic wastewater treatments [28].

Methanogenic microorganisms are a key group in anaerobic wastewater treatments. If methanogen activity is inhibited, the process stops at acidogenesis, ultimately leading to incomplete organic waste degradation [33]. By controlling methanogenic activity during remedial action and maintaining lower levels of methanogenesis, some benefits were observed. One of these is the maintenance of anoxic conditions in treatment zones, and another is the regulation of co-metabolic activity between all microorganisms involved in wastewater treatment. On the contrary, excessive methanogenesis during wastewater remediation results in efficiency and cost issues, regulatory issues, and, most importantly, potential health and safety issues [13].

Anaerobic microorganisms, especially methanogens, have high sensitivity to environmental conditions and are inhibited by toxic compounds present in wastewater. Some microorganisms have long generation times, which result in long start-up times of the remediation process. These properties hinder the optimization of methanogenesis and its application [28,33].

To conclude, methane production is an indisputable indication that hydrogen generated in previous steps is used by methanogens instead of other microorganisms present in the reactor [13]. The assessment of the methane formation potential from industrial wastewaters relies on concentrations of organic substrates in any given type of wastewater. High CH₄ production potential is identified in numerous industrial wastewaters [28].

8. Methanogenesis for Renewable Energy Production

8.1. Methane for Hydrogen Storage

Specific activities of methanogenic Archaea are high enough for methanogens to be viewed as useful catalysts in industrial energy transformation. An attractive means of storing hydrogen, via the formation of methane from hydrogen and carbon dioxide catalyzed by methanogenic archaea (Equation (2)), is being considered. Methanogens catalyze this reduction process at hydrogen pressures well under 1 bar and at room temperatures. Methane is easily stored/transported, and almost all of the hydrogen combustion energy is conserved in methane. However, the feasibility of this hydrogen storage application is hindered by the difficulty of employing methanogenic hydrogenases in large-scale processes, the economic costs, and hydrogenase deactivation in oxic conditions [8].

8.2. Power-to-Gas

Organic waste in the form of biomass contains stored energy that can be converted into more useful forms, which could potentially replace fossil fuels. This energy is recovered by anaerobic digestion. Microbial populations in anaerobic reactors support high conversion efficiencies for methane production [34]. Hydrogenotrophic methanogens have gained attention for their ability to produce CH₄ from hydrogen and carbon dioxide, in a process called power-to-gas, by mediating the biomethanation step [35]. The foundation of the power-to-gas concept is the transformation of electricity into different products, with methane and hydrogen being the most common products [36]. If methane is the desired product, electric power is utilized for hydrogen production by water electrolysis. This conversion is followed by a biomethanation step, where H₂ and CO₂ are converted to methane by methanogenic Archaea. An example of the successful implementation of the power-to-gas process is a pilot plant in Denmark, where methane gas is produced with a purity grade of more than 98 vol%. Biomethane production from the power-to-gas process is in development phases and further research for commercial-size usage is needed [35].

8.3. Bioelectrochemical Methane Production

As methanogens are electroactive microorganisms, they can be utilized in bio-electrochemical methane production. Bio-electrochemical methanogenic systems rely on exploiting the capability of methanogens to interact with electrodes in order to utilize or produce electric power [35]. Electrochemistry can be implemented in the methanogenic process by integrating electrodes in reactors for wastewater treatments or treatments of other biodegradable wastes, such as solid wastes found in the agricultural sector. Furthermore, the methanogenic bio-electrochemical process can be a separate process integrated in biogas production plants. The conversion mediated by methanogenic Archaea takes place at the cathode. The second option of separating processes reduces inhibitory effects from anodic reactions, such as oxygen generation. Electromethanogenesis allows for increased methane production and biogas purity. When implemented into wastewater treatment, the CH₄ content of produced biogas reaches up to 98.1% [37].

8.4. Micro Biogas Plants

Micro biogas plants are an interesting methanogen application that has recently gained attention in developing countries. These plants convert household organic wastes to biogas, that can later be used as a direct source of energy needed for heating or electrical power. These systems have only a small positive impact on solving the energy problem. However, in some countries, micro biogas production is welcomed and supported by the government. This results in further optimization and reactor engineering of the micro plants in the hopes of producing higher electricity volumes [37].

8.5. Methanogens in the Agricultural Sector

The agricultural sector is the most abundant source of biodegradable organic waste for biogas production. Methanogenic treatments of agricultural wastes, such as production slurry and animal manure, are advantageous not only for biogas production. These treatments help in reducing pathogens and odors present in fertilizers, thus increasing the quality of the fertilizer. Biogas production by methanogenic Archaea does not have to be linked to waste treatments. The source of biodegradable material can also be maize or sugar beet silage, as well as grass and microalgae. Biogas produced by methanogens in the agricultural sector has a methane content ranging from 50 to 70%, with food waste being the most abundant biogas source [37].

9. Conclusions

Methanogenic habitats are anaerobic and range from acidophilic to alkaliphilic environments with psychrophilic to hyperthermophilic temperatures. Even though methanogens are a diverse group of microorganisms, they are capable of utilizing only inorganic carbon dioxide, acetic acid, methanol, or methylamines as a substrate for methane production. Recently, new substrates were shown to benefit methanogenic activity, with the number of available substrates increasing up to 193. As an electron source for reactions, methanogenic Archaea use hydrogen, which is activated by hydrogenase enzymes.

More anaerobic processes are being employed for wastewater remediation. These processes are more economically and environmentally friendly, due to reduction in operational costs and high conversion of waste materials to biogas. Anaerobic wastewater treatments consist of four steps, three of which mediated by bacterial populations. For the last step, that is, biogas formation, anaerobic wastewater treatments use methanogens in a process called methanogenesis. The dominant methanogen used in anaerobic treatment depends on the wastewater type. Methanogenesis, as a complex multi-step process, greatly depends on environmental factors such as pH, temperature, composition of wastewater, and the syntrophic interactions between Bacteria and Archaea populations present. Depending on the wastewater type, different reactor configurations are needed. Each type has benefits and disadvantages, although generally the biggest problem is potential microorganism washout, which would prevent biomass conversion to the final product, i.e., methane. Furthermore, methanogenic Archaea require anaerobic conditions. This is because hydrogenases found in methanogens are rapidly inactivated by oxygen, thus stopping the wastewater treatment process. Additionally, remediation by methanogenesis can only be used in the purification of wastewaters with high levels of soluble organic content. Despite these challenges, methanogenesis is of significant use in wastewater remediation. While most efforts of implementing methanogens in industrial processes are applied in wastewater remediation, methanogenesis can be implemented in other biogas production processes. Examples of such processes are power-to-gas or bio-electrochemical methane production.