Anaerobic digestion, a process that ultimately generates methane and carbon dioxide, is common in natural anoxic ecosystems where concentrations of electron acceptors such as nitrate, the oxidized forms of metals and sulphate are low. It also occurs in landfill sites and wastewater treatment plants. The general scheme of anaerobic digestion is well known and comprises four major steps: (i) hydrolysis of complex organic polymers to monomers; (ii) acidogenesis that results in the formation of hydrogen and carbon dioxide as well as non-gaseous fermentation products that are further oxidized to hydrogen, carbon dioxide and acetate in (iii) acetogenesis based on syntrophic metabolism and (iv) methanogenesis. Approaches to the analysis of methane-yielding microbial communities and data acquisition are described. There is currently great interest in the development of new technologies for the production of biogas (primarily methane) from anaerobic digestion as a source of renewable energy. This includes the modernization of landfill sites and wastewater treatment plants and the construction of biogas plants. Moreover, research effort is being devoted to the idea of separating hydrolysis and acidogenesis from acetogenesis and methanogenesis under controlled conditions to favour biohydrogen and biomethane production, respectively. These two stages occur under different conditions and are carried out in separate bioreactors.
- anaerobic digestion
- renewable energy
Anaerobic digestion of biomass under mesophilic conditions (anaerobic microbial decomposition/degradation of organic matter), whose final products are methane and carbon dioxide, contributes to the energy flow and circulation of matter in ecosystems. It is a key process in the global carbon cycle that is promoted by the activity of many different groups of microorganisms. Anaerobic digestion commonly occurs in natural anoxic ecosystems such as freshwater sediments, wetlands, marshlands, paddy fields and deeper zones of marine sediments. The digestive tracts of animals, especially ruminants and termites, are also sites of methane production by this process. It is estimated that biological methanogenesis is responsible for more than 70% of total global methane emissions [1, 2].
Anaerobic decomposition of biomass to carbon dioxide and methane only occurs in anoxic environments with a low redox potential, i.e., where concentrations of other electron acceptors including nitrate, oxidized forms of metals such as Mn(IV) and Fe(III) or sulphate are low. The inhibition of anaerobic digestion by nitrate, oxidized metal ions and sulphate is determined by the redox potential. As shown in Figure 1, a decrease in redox potential leads to changes in the dominant type of anaerobic respiration towards low energy-yielding processes. The nature of the final electron acceptors present in an environment is a key factor in determining the ecological niches for particular microorganisms.
The general scheme of anaerobic digestion is well known (Figure 2). It is a complex process promoted by the interaction of many groups of microorganisms and has four major steps. The first is hydrolysis of complex organic polymers to monomers. The second step is acidogenesis that results in the formation of hydrogen and carbon dioxide as well as non-gaseous fermentation products, i.e., low-molecular weight organic acids (short-chain fatty acids) and alcohols. In the third step, known as acetogenesis, these non-gaseous products are further oxidized to hydrogen, carbon dioxide and acetate, mainly by syntrophic degradation processes. The fourth step is methanogenesis. The final two steps, acetogenesis and methanogenesis, are closely linked and involve syntrophic associations between hydrogen-producing acetogenic bacteria and hydrogenotrophic methanogens. These associations keep the hydrogen partial pressure sufficiently low to allow acetogenesis to become thermodynamically favourable. This process, referred to as interspecies electron transfer, is in fact a hydrogen/formate transfer. Acetate is a direct substrate for methanogenesis and can also be syntrophically oxidized to hydrogen and carbon dioxide [3–8].
Anaerobic digestion is common in landfill sites and anaerobic wastewater treatment plants. The process of anaerobic decomposition of biomass, such as energy crops or organic agro-waste, is commonly used to produce biogas as an alternative energy source. There is currently great interest in the development of new technologies for the modernization of landfills and wastewater treatment plants to control the release of biogas and collect methane to use as fuel. Moreover, for the purpose of innovative technologies based on microbial processes, it is desirable to build modern biogas plants where the hydrogen-yielding (hydrolysis and acidogenesis) and methane-yielding (acetogenesis and methanogenesis) stages of anaerobic digestion are separated to, respectively, favour the production of hydrogen and methane under controlled conditions. Optimization of methane or hydrogen and methane production from organic matter requires a good understanding of anaerobic digestion at the molecular level, namely the structure and diversity of microbial communities and metabolic pathways, leading to transformation of the organic substrate to the desired gaseous products.
2. Meta-omics approaches for exploring microbial communities
Current knowledge of microbial ecology and physiology, derived from culture-dependent techniques, is limited and incomplete because the majority of microorganisms have not been cultivated. It has been predicted that only 1% or less of all microorganisms present in natural ecosystems may be cultivated as a pure culture using standard methods . Moreover, syntrophy is believed to be common in microbial communities, and syntrophic bacteria cannot be grown as a monoculture. However, culture-dependent techniques have permitted the isolation and characterization of some species involved in specific metabolic processes during anaerobic digestion, and numerous genomes have been sequenced. Data from genome sequence analyses supported by the results of physiological and biochemical studies on cultivated bacteria have provided hints as to which physiological groups of microorganisms are responsible for the key steps of anaerobic digestion. Information on methane-yielding microbial communities is now being obtained using culture-independent analytical techniques (Figure 3).
The recent increase in the number of culture-independent molecular biology techniques and bioinformatic tools for exploring microbial communities has helped to develop the field of meta-omics. Meta-omics encompasses metagenomics, metatranscriptomics, metaproteomics and metabolomics, based on analyses of, respectively, total DNA, mRNA, total proteins and metabolites isolated from the microbial communities [10–14]. Metagenomics shows microbial potential by describing the genes present in a microbial community or ecosystem. Metatranscriptomics analyses gene expression and thus represents potential microbial function. Messenger RNA (mRNA) can be sequenced directly or used to generate cDNA (by reverse transcription) that is subsequently sequenced using metagenomics platforms. Metaproteomics is focused on microbial function—it investigates proteins expressed within a microbiome. Metabolomics analyses the intermediates and end-products of metabolism and then shows microbial activity.
The data generated by these novel methodologies have provided significant insights into the structure and function of microbial communities in both natural environments and man-made systems. However, meta-omics-based approaches do suffer from certain limitations: the variable extraction efficiency of DNA/RNA/protein/metabolites may affect the results, and reference databases used for comparative analyses often contain false or missing assignments of DNA and protein sequences or chromatography/mass spectrometry data. For example, metagenomic analyses always generate large numbers of sequences that are of low complexity, unclassified, not assigned or show no hits. Such unidentified reads usually constitute a significant proportion of the total reads, as discussed by Chojnacka et al. . It is noteworthy that the limited number of microorganisms that can be propagated as pure cultures determines the number of sequenced reference genomes available for genomic studies. So far, only five genomes of syntrophic bacteria involved in acetogenesis have been sequenced:
Using metagenomic sequence data and genomic assembly procedures, it is possible to reconstruct genomes of bacteria that have not been cultivated. One example is a reconstruction of the genome of
In the case of anaerobic digestion, the combined use of meta-omic approaches and isotope labelling techniques in both natural anoxic environments and bioreactors plus the analysis of reactor performance data will allow us to develop a fundamental understanding of the processes leading to methane production. Meta-omic data can also be used to validate commonly accepted theses.
Other cultivation-independent techniques include isolation of total DNA from microbial communities, amplification, cloning and sequencing of marker genes, most frequently 16S rRNA or others such as
3. Hydrolysis and acidogenesis: the anaerobic digestion steps yielding short-chain fatty acids and hydrogen
Hydrolysis is the first step in the anaerobic decomposition of organic matter. It involves the conversion of polymeric organic matter (e.g., polysaccharides, lipids, proteins) to monomers (e.g., sugars, fatty acids, amino acids) by hydrolases secreted to the environment by microorganisms. Three key groups of hydrolases are involved in the process of anaerobic digestion: esterases, glycosidases and peptidases, which catalyse the cleavage of ester bonds, glycoside bonds and peptide bonds, respectively . The bacteria most commonly associated with hydrolysis include representatives of the
Metaproteomic analysis of microbial communities mediating the decomposition of dead plant material in forest leaf litter revealed fungi to be the main producers of extracellular hydrolytic enzymes, the most prominent of which are cellulolytic enzymes: exo- and endo-glucanases as well as β-glucosidases. Other hydrolases involved include phosphatases, pectinases, xylanases, lipases, amylases, chitinases and oxidoreductases. Moreover, the species of fungi – the main cellulase producers – changed depending on the season. In a sample collected in February,
3.2.1. Fermentation of sugars
During acidogenesis, the products of hydrolysis are converted to non-gaseous short-chain fatty acids, alcohols, aldehydes and the gases, such as carbon dioxide and hydrogen . The dominant end-products of the fermentation process determine the type of fermentation (Figure 4A).
The main hydrogen-yielding fermentations under mesophilic conditions are butyric acid fermentation (
In the mixed-acid fermentation (also known as formic acid fermentation), pyruvate is converted to acetyl-CoA and formic acid by pyruvate formate-lyase (PFL). The formic acid can then be degraded into hydrogen and carbon dioxide by formate hydrogen-lyase (FHL) complex. One of the FHL subunits is the [FeNi] hydrogenase Hyd-3. There are two types of mixed-acid fermentation: the
Besides glycolysis, other pathways of pyruvate formation exist, e.g., the 2-keto-3-deoxy-6-phosphogluconate (Entner-Doudoroff) pathway. Two intermediates of glycolysis, glyceraldehyde-3-phosphate and fructose-6-phosphate, are also formed in the pentose phosphate pathway. Monosaccharides other than glucose can enter glycolysis or other pathways leading to pyruvate formation. Pyruvate can also be formed from glycerol .
In addition to the hydrogen-yielding fermentations, other fermentations occur during acidogenesis, including lactic, propionic and ethanol fermentations. Two types of lactic acid fermentation are distinguished: homolactic and heterolactic, whose products are, respectively, lactate only or lactate, ethanol, acetate and carbon dioxide.
3.2.2. Fermentation of amino acids
Members of the
Notably, glutamate may be fermented through five different pathways by various bacterial species: Pathway 1—through 3-methylaspartate; Pathway 2—through 3-methylaspartate to acetate, propionate, carbon dioxide and ammonium; Pathway 3—through 2-hydroxyglutarate to acetate, butyrate, hydrogen, carbon dioxide and ammonium; Pathway 4—through 4-aminobutyrate to acetate, butyrate and ammonium and Pathway 5—through 5-aminovalerate to acetate, propionate, valeriate and ammonium .
3.2.3. Transformation of lipids during acidogenesis
The products of lipid hydrolysis are glycerol and long-chain fatty acids (Figure 4A). Glycerol can enter (i) a reductive pathway and be converted to 1,3-propanediol or (ii) an oxidative pathway and be transformed to phosphoenolopyruvate in a four-step process. Phosphoenolopyruvate can then be converted to succinate and propionate and/or to pyruvate. In the latter case, further transformations of pyruvate occur through glycolytic fermentations as described for sugars [33, 35]. Significant hydrogen production was observed when
Long-chain fatty acids are transformed to acetate and hydrogen through the beta-oxidation pathway, requiring syntrophic cooperation between acetogens and methanogens (described in Section 2.3). However, long-chain fatty acids have an inhibitory effect on anaerobic digestion due to their adherence to microbial cell walls, which can block the passage of nutrients through the cell membrane and/or cause flotation of the cells.
Symbiotic interactions between lactic acid bacteria and butyrate-producing bacteria involving clostridia, called “cross-feeding”, have been detected in the gastrointestinal tract (Figure 4A). Numerous observations in different animal models have described lactate and acetate conversion to butyrate by butyrate-producing intestinal bacteria, stimulated by lactic acid bacteria (for review, see Ref. ). The incubation of human microflora in media containing 13C-labelled lactate revealed that butyrate was the major net product of lactate conversion . Other studies performed using 2H-labelled acetate and 13C-labelled lactate showed that acetic and lactic acids are important precursors of butyrate production in human faecal samples . The metabolic pathway of lactate and acetate utilization to produce butyrate proposed for
It is commonly accepted that anaerobic digestion requires symbiotic interactions between specific groups of microorganisms. Some studies have indicated that lactic acid bacteria (LAB), often detected within mesophilic hydrogen-producing microbial communities, may support hydrogen production during acidogenesis. Based on our own research and the findings of other groups, we have considered the true role of LAB in bioreactors and their influence on hydrogen producers . Our metagenomic survey of microbial communities in anaerobic bioreactors, performed using 454-pyrosequencing, revealed that
An analysis of the hydrogen-yielding granular sludge using the FISH technique  revealed that
Others researchers have examined the effects of lactic acid on hydrogen production by communities of fermentative bacteria. In one study, the complete consumption of lactic acid increasing hydrogen production and butyric acid formation was observed . Subsequently, another group demonstrated that lactic acid increased the efficiency of hydrogen production . FISH analysis revealed that
Many studies have examined the conversion of lactate and acetate to butyrate and hydrogen by clostridial species, and all point to pH as a critical factor for this process. It is noteworthy that the results of studies on gastrointestinal microflora indicate that acidity is a key regulatory factor in lactate metabolism. The pH may influence both bacterial growth and the development of specific groups of bacteria, as well as fermentation processes affecting the relative proportions of short-chain fatty acids (for review, see Ref. ).
A phenomenon analogous to cross-feeding observed in the gastrointestinal tract may occur in hydrogen-producing bioreactors and natural environments [38, 43] (Figure 4A).
4.1. The essence of acetogenesis
The two final steps of anaerobic digestion, acetogenesis (Stage III) and methane formation (Stage IV), are tightly connected. Acetogenesis supplies substrates for methanogens. Three groups of substrates for methane production and three types of methanogenic pathways have been recognized: (i) splitting of acetate (aceticlastic/acetotrophic methanogenesis); (ii) reduction in CO2 with H2 or formate and rarely ethanol or secondary alcohols as electron donors (hydrogenotrophic methanogenesis) and (iii) reduction in methyl groups of methylated compounds such as methanol, methylated amines or methylated sulphides (hydrogen-dependent and hydrogen-independent methylotrophic methanogenesis) [2, 48–51].
Due to the limited number of substrates for methanogenesis, methanogens are strictly dependent on partner microbes with which they form syntrophic systems. Syntrophy is a special type of symbiotic cooperation between two metabolically different types of microorganisms, which depend on one another for the degradation of a certain substrate, typically through the transfer of one or more metabolic intermediate. In this case, the partner microbes oxidize non-gaseous products of acidogenesis to acetate, carbon dioxide, hydrogen and formate that are directly utilized by the methanogens, making the entire syntrophic metabolism efficient and thermodynamically favourable. This is the essence of acetogenesis. The process of hydrogen or formate transfer (interspecies hydrogen/formate transfer) between acetogenic bacteria and methanogenic
Under standard conditions, the oxidation of butyrate, propionate, acetate, ethanol and other non-gaseous products of acidogenesis, coupled to hydrogen or formate production, is endergonic, as demonstrated by the positive change in Gibbs free energy. However, when the oxidation processes are coupled to methane production, the conversion is energetically feasible (exergonic) due to the very low hydrogen partial pressure ensured by hydrogen-consuming methanogens (Figure 5). Oxidation of non-gaseous products of acidogenesis during acetogenesis is based on reverse electron transfer: the energetically unfavourable movement of electrons that requires the input of energy to drive the oxidation/reduction reaction (Figure 5). This involves multiple systems, most of which are membrane-located, comprising formate dehydrogenases (FDHs), ferredoxin:NAD+ oxidoreductase, hydrogenases,
The second known mechanism of interspecies electron transfer in methanogen-yielding communities is direct transfer. This was described between
Our current understanding of the microbial ecology and physiology associated with anaerobic digestion is restricted to culture-dependent techniques and thus is incomplete. The majority of microorganisms involved in the process of anaerobic digestion have yet to be cultivated. It is noteworthy that acetogenic bacteria are unable to grow without their syntrophic partners and cannot be cultivated as a monoculture. Thus, the mechanisms of acetogenesis are poorly characterized at the molecular level. Data derived using recently developed meta-omics approaches are likely to give a deeper insight into syntrophic metabolic pathways of anaerobic digestion.
4.2. Biochemistry of syntrophic oxidation of non-gaseous products of acidogenesis
The metabolic pathways utilized for syntrophic oxidation of common non-gaseous products of acidogenesis include beta-oxidation for butyrate, the methylmalonyl-CoA pathway for propionate, the Wood-Ljungdahl pathway for acetate, the pathway of lactate oxidation recognized in
In the first reaction of butyrate oxidation, butyrate is activated with acetyl-CoA to butyryl-CoA by butyrate-CoA transferase. This is followed by the conversion of butyryl-CoA to crotonyl-CoA catalysed by butyryl-CoA dehydrogenase, to release electrons as hydrogen or formate, which requires ATP. This process is only possible by a reverse electron transport through electron transfer flavoprotein EtfAB and a membrane-anchored DUF224 protein to the menaquinone pool in the membrane, cytochromes and other electron transfer complexes, terminating at the formate dehydrogenase and hydrogenase/formate dehydrogenase complexes. Crotonyl-CoA is transformed to 3-hydroxy-butyryl-CoA by crotonase and then to aceto-acetyl-CoA by 3-hydroxybutyryl-CoA dehydrogenase. The latter reaction also yields electrons as hydrogen or formate due to reverse electron transfer and the activity of the NADH:hydrogenase/formate dehydrogenase complex. Aceto-acetyl-CoA is split into two moieties of acetyl-CoA by acetyl-CoA acetyltransferase: one is used for butyrate activation and the second is transformed to acetate by phosphotransacetylase and acetate kinase activity, accompanied by the release of ATP [52, 56, 57].
In the first reaction of propionate oxidation, propionate is activated with acetyl-CoA to propionyl-CoA by propionate-CoA transferase. This is then transformed to (S) methylmalonyl-CoA, (M) methylmalonyl-CoA, succinyl-CoA and succinate by, respectively, methylmalonyl-CoA decarboxylase, methylmalonyl-CoA epimerase, methylmalonyl-CoA mutase and succinyl-CoA synthetase. The final step generates ATP. The next reaction is the conversion of succinate to fumarate by fumarate reductase, which releases electrons. This is the first key reaction that requires reverse electron transport. Fumarate is transformed to malate by fumarate hydratase. Malate is then converted to oxaloacetate by malate dehydrogenase in the second key reaction coupled to reverse electron transport. Pyruvate formed from oxaloacetate by pyruvate carboxylase is then transformed to acetyl-CoA by pyruvate:ferredoxin oxidoreductase. Finally, acetyl-CoA is converted to acetate in the third step generating electrons during propionate oxidation. The oxidation of oxaloacetate to fumarate involves coupling menaquinone reduction, proteins encoded by cytochrome c gene homologues, cytochrome b:quinone oxidoreductases, formate dehydrogenases, and hydrogenases including confurcating [FeFe]-hydrogenases [7, 52, 55, 57].
Acetogens that synthesize acetate from hydrogen and carbon dioxide use the reductive carbon monoxide dehydrogenase/acetyl-CoA synthase pathway (reductive CODH/ACS) known as the Wood-Ljungdahl pathway. Acetate-oxidizing syntrophs use the same pathway in reverse (oxidative CODH/ACS). Electrons as hydrogen or formate are released in the reactions catalysed by the carbon monoxide dehydrogenase/acetyl-CoA synthase, methylene-tetrahydrofolate (methylene-THF) reductase and methylene-THF dehydrogenase formate dehydrogenase. Reverse electron transfer during acetate oxidation has yet to be confirmed. It is likely that the same electron transfer mechanism is used in both pathways (reductive and oxidative) [52, 58].
It is believed that ethanol is oxidized to acetaldehyde coupled to NADH formation. Subsequently, acetaldehyde is oxidized to acetate and reduced ferredoxin is formed. Ethanol-oxidizing
The key reaction of syntrophic lactate oxidation in
Worm and co-workers analysed the genomes of the butyrate- or propionate-oxidizing syntrophs
In the same study, functional domains involved in electron transfer were also identified . These were found in the following proteins: cytoplasmic FDH, extra-cytoplasmic FDH, formate transporter, Fe-Fe hydrogenase, NiFe hydrogenase, Rnf complex, Ech complex, Etf alpha, Etf beta, Bcd, cytochromes c, cIII, b561 and b5 and the DUF224 protein complex.
Notably, the genomes of sulphate-reducing non-syntrophs were found to lack the syntrophy-specific domains. However, these domains are present in other sulphate reducers that have never been tested for syntrophy:
4.3. A model of methane-yielding granules
According to the model of methane-yielding granules proposed more than 25 years ago acetotrophic methanogens constitute a central core of the granule surrounded by acetogenic bacteria and hydrogenotrophic methanogens, and the external layer is composed of microorganisms responsible for acidogenesis. The physical distances (proximities) necessary for energetically favourable hydrogen transfer between acetogenic bacteria and hydrogenotrophic methanogens have been estimated from studies on the propionate-, propanol-, ethanol-oxidizing syntroph
4.4. Syntrophic relationships between acetogenic bacteria and methanogens during anaerobic digestion
The most well-studied examples of syntrophic metabolism in methanogenic communities are described below. The
The most frequently recognized butyrate oxidizers are representatives of the
The propionate-oxidizing bacteria are members of the
Li and co-workers  developed specific PCR assays for propionate-CoA transferase genes (
Acetate is the major intermediate product during anaerobic digestion of organic matter to methane and carbon dioxide. It can be directly transformed to methane and carbon dioxide by acetoclastic methanogens (Section 2.4) or syntrophically oxidized to hydrogen and carbon dioxide. The latter reaction requires the participation of two microbial partners: an acetate-oxidizing bacterium and a hydrogenotrophic methanogen. Recognized acetate-oxidizing bacteria include members of the
Ito and co-workers  used MAR-FISH combined with phylogenetic analysis of 13C-labelled bacterial 16S rRNA and tracing of [2-14C]-labelled acetate degradation to study metabolic pathways of acetate transformation in methanogenic sludge from an anaerobic digester fed with mineral medium containing powdered whole milk. These analyses identified
Lee and co-workers  presented evidence that in anaerobic digesters fed with a medium containing acetate as the sole carbon source,
Interestingly, current knowledge concerning the oxidation of lactate in methanogenic consortia is limited to members of the
Recent studies on anaerobic digestion of molasses wastewater in an upflow anaerobic sludge blanket (UASB) reactor revealed the significant contribution of
Chojnacka and co-workers  hypothesized that a symbiotic interaction between lactic acid bacteria and clostridia, known as lactate cross-feeding (described in Section 2.2.3.), may also occur in methanogenic communities. Butyrate and hydrogen are the products of lactate transformation. The hydrogen and the products of further syntrophic butyrate oxidation constitute substrates for methanogenesis.
Ethanol is also effectively utilized by the methane-yielding microbial communities [15, 66]. Apart from
Members of the orders
We have examined the microbial community processing an acidic effluent from molasses fermentation to methane in a UASB bioreactor . Total DNA isolated from the methanogenic community formed in the reactor was sequenced by 454-pyrosequencing. The results revealed that the biodiversity of methanogenic sludge is significantly higher than that of the hydrogen-producing community. The ratio of
Some of the aforementioned bacterial phyla are capable of oxidizing other compounds, including 1-propanol, benzoate, hydroxybenzoate, phenol and phthalates.
5. Methane formation
Methane formation, Stage IV of anaerobic digestion, is a complex process requiring specific enzymes and cofactors not found in other microorganisms. The course of the reaction depends on the substrates utilized by the methanogens. Three groups of substrates are recognized: (i) acetate, (ii) CO2 and H2 or formate, and rarely ethanol or secondary alcohols and (iii) methylated compounds including methanol, methylated amines and methylated sulphides. These substrates are, respectively, processed through three recognized pathways of methanogenesis: aceticlastic/acetotrophic, hydrogenotrophic and methylotrophic (hydrogen-dependent and hydrogen-independent) (Figure 6) . Irrespective of the substrate, the final step in each methanogenic pathway is the reaction of methyl-coenzyme M (CH3-S-CoM) and coenzyme B to produce heterodisulphide CoM-S-S-CoB and methane:
This reaction is catalysed by methylcoenzyme M reductase (Mcr), the key enzymatic complex of the methanogenic process. It possesses a unique prosthetic group, coenzyme F430, containing nickel. CoM-S-S-CoB acts as the final electron acceptor during anaerobic respiration and is the key compound for energy gain by methanogens. Methane is a by-product of methanogen metabolism. The pathways of methanogenesis are in fact pathways of CoM-S-S-CoB synthesis.
Splitting of acetate (acetotrophic methanogenic pathway) involves the formation of acetyl-CoA, the transfer of methyl groups to tetrahydrosarcinopterin (H4SPT) and the formation of methyl tetrahydrosarcinopterin CH3-H4STP. CH3-S-CoM is formed in the reaction of CoM with CH3-H4STP. The electrons required to reduce CH3-S-CoM to methane come from oxidation of the carboxyl group of acetate.
The formation of CH3-S-CoM by the reduction in CO2 with H2, formate or alcohols constitutes the hydrogenotrophic methanogenic pathway. This pathway is comprised of the following steps: (i) the formation of formylmethanofuran (formyl-MFR) from methanofuran (MFR) and CO2, (ii) the reaction of formyl-MFR and tetrahydromethanopterin (H4MPT) to produce formyl tetrahydromethanopterin (formyl H4MPT), (iii) the formation of methylene H4MPT that in reaction with F420, a derivative of 5′ dezaflavin, produces methyl H4MPT and (iv) the reaction of methyl H4MPT with CoM to generate CH3-S-CoM. The electrons required to reduce CH3-S-CoM to methane come from hydrogen, formate or alcohols.
In the methylotrophic pathway of methanogenesis, CH3-S-CoM is formed by the direct transfer of methyl groups from methylated compounds to CoM. One methyl group bound to CoM is oxidized to CO2 and hydrogen (in the form of F420H2 and reduced ferredoxin) to reverse the hydrogenotrophic pathway. The reducing equivalents are used to reduce CH3-S-CoM to methane.
In the recently discovered process of hydrogen-dependent methylotrophic methanogenesis, CH3-S-CoM is also formed through the direct transfer of methyl groups from methylated compounds to CoM. However, the electrons required to reduce CH3-S-CoM to methane come from externally supplied hydrogen. Genomic analysis revealed that organisms generating methane by this process lack genes encoding the enzymes of hydrogenotrophic methanogenesis [50, 51].
The known cultured methanogens are strict anaerobes and comprise seven orders in the class
It has been estimated that 70% of methane is produced from acetate. When biomass is transformed into methane under mesophilic conditions in anaerobic digesters or natural environments, it is first fermented to acetate, carbon dioxide and hydrogen and formate, as well as short-chain fatty acids during acidogenesis. The theoretical maximum hydrogen yield during dark fermentation occurs with the conversion of one-third of the substrate to hydrogen and carbon dioxide and two-thirds of the substrate to acetate. Therefore, it follows that two-thirds of methane originates from acetate and one-third is from hydrogen, formate and carbon dioxide .
Culture-independent analyses of methanogenic communities (mainly from anaerobic digesters) based on cloning and sequencing of 16S rRNA and
It should be noted that the acetoclastic pathway provides only a small amount of energy available for growth:
In comparison, the hydrogenotrophic pathway produces fourfold more energy:
4HCOO- + 4H+ → CH4 + 3CO2 + H2O (ΔG0´ = - 144.5 kJ / mol) 
Thus, the hydrogenotrophic pathway is much more energetically effective, and this may be one of the reasons for the dominance of the
An analysis of the substrate preferences of the recognized methanogenic
In all methanogenic microbial communities examined by high-throughput DNA sequencing, the contribution of unidentified sequences is usually high. As phylogenetic analyses are dependent on comparison with DNA sequences present in databases and the majority of the recognized genera of methanogens produce methane through the hydrogenotrophic pathway, it is possible that acetoclastic methanogens are hidden among the unidentified sequences. Therefore, the apparent dominance of hydrogenotrophic methanogens such as
Recently, Dziewit and co-workers  described four novel molecular markers—other than 16S rRNA and
It is commonly recognized that methanogenic granular sludge is rich in minerals, mainly ferric sulphide and Ca-, Mg-, Na-, K- or Al-containing compounds. They constitute between 10 and 90% of the dry mass, depending on the composition of the wastes and nature of the methanogenic process . The inorganic components of the extracellular matrix of methanogenic granules may inhibit some metabolic pathways and thus determine the processes leading to methane production by the microbial community. Both Al and K are undesirable elements in the methanogenic sludge due to their competition with other essential metals, inhibiting microbial growth and consequently their adverse effect on the methanogenic process. In contrast, Ca and Mg have a positive effect due to their promotion of the granulation process. Sodium plays a role in the formation of ATP and oxidation of NADH and then is essential for the growth of methanogens. However, high concentrations of Ca2+, Mg2+ and Na+ ions cause inhibitory effects on methanogen activity. The optimum concentration of Ca2+ and Na+ ions for methane synthesis from acetate was found to be 200 and 230 mg/L, respectively, whereas a concentration of 8000 mg/L of either ion inhibited the process . Interestingly, the combination of various elements can mitigate the toxicity of others, e.g., magnesium, sodium and ammonium counteract potassium toxicity. It is noteworthy that the acetoclastic pathway of methanogenesis and the oxidation of propionate are particularly sensitive to raised levels of certain minerals . Moreover, it has been observed that inhibition of the acetotrophic pathway of methane formation is usually accompanied by inhibition of propionate oxidation .
6. Hydrogen and methane production in a two-stage anaerobic digestion
There is currently great interest in the development of new technologies for the production of energy from renewable sources, of which fermentation processes generating methane and hydrogen show great promise. Hydrogen-yielding fermentation is considered to be one of the most attractive alternative biological methods of hydrogen production. However, there are two major drawbacks: low productivity of the process and the formation of large amounts of environmentally unfriendly non-gaseous fermentation products [29, 72]. The theoretical maximum hydrogen yield during
This gives the highest possible yield of hydrogen during dark fermentation. The complete oxidation of glucose provides 12 moles of hydrogen per mole of glucose:
Theoretically, only one-third of the biomass can be converted to hydrogen by the process of hydrogen-yielding fermentation. In practice, this value is lower due to the formation of non-gaseous products such as organic acids and alcohols. For example, when the glucose is converted to butyrate, the hydrogen yield drops to two moles. It is estimated that the efficiency of hydrogen production must reach 60–80% to be economically attractive [73, 74]. This level of efficiency may be attained by using two-stage systems to achieve the transformation of substrates into hydrogen and methane. In such systems, the hydrogenic (hydrolysis and acidogenesis) and methanogenic (acetogenesis and methanogenesis) steps are performed separately under controlled conditions to favour biohydrogen and biomethane production, respectively. In the first stage, hydrogen-rich fermentation gas is produced, while in the second, the non-gaseous products of hydrogen fermentation act as substrates for methanogenic consortia. These two processes are carried out in separate bioreactors that differ in design and have different pH conditions and hydraulic retention times.
A growing number of reports describe the use of two-stage systems for hydrogen and methane production. Such systems have shown promise at the laboratory and pilot scales using various substrates including organic wastes, plant biomass, by-products of the food industry and pure hydrocarbons [66, 75–90]. Increases in energy recovery of up to 20–30% have been achieved using these systems compared to one-stage biogas-producing bioreactors [76, 78, 85, 90]. Effective biomethane production from non-gaseous fermentation products could make biological production of hydrogen through fermentation economically attractive. It has been estimated that by 2040, biohydrogen may be produced on an industrial scale .
The idea of two-phase anaerobic digestion as a method for the effective degradation of biomass to methane and carbon dioxide is not new . The novel aspect is the co-production of hydrogen and methane. Many studies on the production of both hydrogen and methane by the anaerobic digestion of biomass have focused on the performance and efficiency of the entire process, but they have lacked any in-depth analysis of the microbial communities in the bioreactors where the two steps are performed. Recognition of the structure and diversity of the microbial communities capable of syntrophic cooperation in the transformation of substrate to the desired gaseous products should facilitate the optimization of hydrogen and methane co-production from organic matter in two-stage systems.
Research on two-stage anaerobic digestion has been conducted in our laboratory for several years. We have developed and described a laboratory-scale two-stage anaerobic digestion system that produces hydrogen (in Stage 1) and methane (in Stage 2) from sucrose-rich by products of the sugar beet refining industry as the primary energy substrate under mesophilic conditions [15, 43]. Initially, hydrogen is generated through processes of acidogenesis in a three-litre packed bed reactor (PBR) by a hydrogen-yielding microbial community fermenting molasses. Subsequently, non-gaseous organic products from this first stage feed a 3.5-litre UASB reactor in which methane (biogas) is produced by a methane-yielding microbial community. A detailed molecular characterization of this two-stage anaerobic digestion system producing hydrogen and methane from sugar beet molasses was achieved using optimized DNA extraction protocols and high-throughput pyrosequencing (454 Roche) [15, 43].
Recently, the two-stage system for hydrogen and methane production described above has been successfully scaled up 10 times and is currently being trialled in a Polish sugar factory.
We acknowledge the support of the National Centre for Research and Development, Poland, through grant PBS1/B9/9/2012 awarded for Years 2012–2016.
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