Open access peer-reviewed chapter

Searching for Metabolic Pathways of Anaerobic Digestion: A Useful List of the Key Enzymes

By Anna Sikora, Anna Detman, Damian Mielecki, Aleksandra Chojnacka and Mieczysław Błaszczyk

Submitted: May 29th 2018Reviewed: September 1st 2018Published: November 5th 2018

DOI: 10.5772/intechopen.81256

Downloaded: 1022


The general scheme of anaerobic digestion is well known. It is a complex process promoted by the interaction of many groups of microorganisms and has four major steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. The aim of the study was to prepare a systematized list of the selected enzymes responsible for the key pathways of anaerobic digestion based on the Kyoto Encyclopedia of Genes and Genomes database resource. The list contains (i) key groups of hydrolases involved in the process of degradation of organic matter; (ii) the enzymes catalyzing reactions leading to pyruvate formation; (iii) the enzymes of metabolic pathways of further pyruvate transformations; (iv) the enzymes of glycerol transformations; (v) the enzymes involved in transformation of gaseous or nongaseous products of acidic fermentations resulting from nonsyntrophic nutritional interactions between microbes; (vi) the enzymes of amino acid fermentations; (vii) the enzymes involved in acetogenesis; and (viii) the enzymes of the recognized pathways of methanogenesis. Searching for the presence and activity of the enzymes as well as linking structure and function of microbial communities allows to develop a fundamental understanding of the processes, leading to methane production. In this contribution, the present study is believed to be a piece to the enzymatic road map of anaerobic digestion research.


  • anaerobic digestion
  • enzymes
  • hydrolysis
  • acidogenesis
  • acetogenesis
  • methanogenesis
  • syntrophy
  • metabolic pathways

1. Introduction

Anaerobic digestion (AD), whose final products are methane and carbon dioxide, is a common process in natural anoxic environments such as water sediments, wetlands, or marshlands. The environments have to be rich in organic matter and poor with other electron acceptors such as nitrate, compounds containing oxidized forms of metals, and sulfate. AD is also common in landfills and wastewater treatment plants and was used by man to produce biogas from waste biomass as an alternative energy source.

AD is a complex process that requires the metabolic interaction of many groups of microorganisms responsible for four closely related major steps. The first one is hydrolysis of complex organic polymers (e.g., polysaccharides, lipids, proteins) to monomers (sugars, fatty acids, amino acids). The second step is acidogenesis that results in formation of hydrogen and carbon dioxide as well as nongaseous fermentation products, that is, low-molecular-weight organic acids and alcohols. These products are further oxidized to hydrogen, carbon dioxide, and acetate in acetogenic step that involves mainly syntrophic degradation of nongaseous fermentation products. The fourth step is methanogenesis. Three groups of substrates for methane production and three types of methanogenic pathways are known: splitting of acetate (aceticlastic/acetotrophic methanogenesis); reduction of CO2 with H2 or formate and rarely ethanol or secondary alcohols as electron donors (hydrogenotrophic methanogenesis); and reduction of methyl groups of methylated compounds such as methanol, methylated amines, or methylated sulfides (hydrogen-dependent and hydrogen-independent methylotrophic methanogenesis). The two last steps, acetogenesis and methanogenesis, are closely related and involve syntrophic associations between hydrogen-producing acetogenic bacteria and hydrogenotrophic methanogens (Figure 1) [1, 2, 3, 4, 5].

Figure 1.

A scheme of anaerobic digestion of organic matter. Enzymes catalysing specific reactions of AD are presented inTables 14. Thus in Figure 1 there are the links toTables 14. Furthermore, background colours in the Figure correspond to the background colours of the title rows in theTables 14: hydrolysis is indicated in green, acidogenesis in orange, acetogenesis in blue and methanogenesis in yellow. A, B, C, D, E refer to the title rows inTable 2; F, G refer to the title rows inTable 3.

Recently, there has been a rapid development in culture-independent techniques (meta-omics approaches such as metagenomics, metatranscriptomics, metaproteomics, metabolomics) for exploring microbial communities, which have led to a new insight into their structure and function in both natural environments and anaerobic digesters. The current trends involve the combined use of meta-omic approaches and detailed reactor performance data as well as isotope labeling techniques that allow us to develop a fundamental understanding of the processes occurring in AD. Those activities are aimed to improve biogas production and increase the share of renewable energy in total energy consumption [6, 7, 8, 9].

Analysis of many studies on metagenomes of microbial communities from anaerobic digesters shows that (i) contribution of methanogens in the methane-yielding microbial communities is relatively small, below 20%; (ii) the most abundant phyla of bacteria are usually Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria; (iii) methanogenic archaea are dominated by acetotrophs or hydrogenotrophs with a certain contribution of methylotrophs; (iv) substrate, operational conditions such as temperature, pH, ammonia concentration, etc. shape the structure, percentage distribution of specific taxons, and functioning of the community of microorganisms; (v) it is important to describe interactions within microbial communities and assign functions in AD steps to specific groups of microbes; and (vi) the majority of sequences are not classified at the genus level confirming that most of the microorganisms are still unrecognized [6, 10, 11, 12, 13, 14, 15].

In this contribution, the purpose of the study was to prepare a list of the selected enzymes and their catalyzed reactions, being a specific enzymatic road map of AD metabolic pathways, useful in molecular studies. The available metabolic pathway databases such as KEGG PATHWAY Database [16, 17, 18], MetaCyc Metabolic Pathway Database, BioCyc Database Collection [19], and BRENDA—The Comprehensive Enzyme Information System [20] were used to select metabolic pathways dedicated only to AD from hydrolysis to methanogenic steps exerted by microbes.

Hydrolytic enzymeReaction/processEC number
EsterasesActing on ester bondsEC 3.1
GlycosidasesActing on glycoside bondsEC 3.2
Acting on cellulose
Cellulase; endo-1,4-beta-d-glucanaseEndohydrolysis of (1 → 4)-beta-d-glucosidic linkages in cellulose, lichenin, and cereal beta-d-glucansEC
Cellulose 1,4-beta-cellobiosidase (nonreducing end)Hydrolysis of (1 → 4)-beta-d-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose from the nonreducing ends of the chainsEC
Beta-glucosidaseHydrolysis of terminal, nonreducing beta-d-glucosyl residues with release of beta-d-glucoseEC
Acting on hemicellulose
Endo-1,4-beta-xylanaseEndohydrolysis of (1 → 4)-beta-d-xylosidic linkages in xylansEC
Xylan 1,4-beta-xylosidaseHydrolysis of (1 → 4)-beta-d-xylans, to remove successive D-xylose residues from the nonreducing terminiEC
Mannan endo-1,4-beta-mannosidaseRandom hydrolysis of (1 → 4)-beta-d-mannosidic linkages in mannans, galactomannans, and glucomannansEC
Beta-mannosidaseHydrolysis of terminal, nonreducing beta-d-mannose residues in beta-d-mannosidesEC
Alpha-galactosidaseHydrolysis of terminal, nonreducing alpha-d-galactose residues in alpha-d-galactosides, including galactose oligosaccharides, galactomannans,and galactolipidsEC
Alpha-glucuronidaseAn alpha-d-glucuronoside + H2O → an alcohol + d-glucuronateEC
PeptidasesActing on peptide bondsEC 3.4
Other hydrolases
Hydrolases acting on carbon-nitrogen bonds, other than peptide bondsEC 3.5
Hydrolases acting on ether bondsEC 3.3
Hydrolases acting on carbon-carbon bondsEC 3.7
Hydrolases acting on halide bondsEC 3.8
Hydrolases acting on phosphorus-nitrogen bondsEC 3.9
Hydrolases acting on sulfur-nitrogen bondsEC 3.10
Hydrolases acting on carbon-phosphorus bondsEC 3.11
Hydrolases acting on sulfur-sulfur bondsEC 3.12
Hydrolases acting on carbon-sulfur bondsEC 3.13
Hydrolases acting on acid anhydridesEC 3.6

Table 1.

The selected enzymes of hydrolytic step of anaerobic digestion [21, 22].

EnzymeReaction/processEC number
A. Pyruvate formation from carbohydrates [23]
Glycolysis (the Embden-Meyerhof-Parnas pathway)
Hexose kinaseD-Glucose + ATP ↔ D-glucose-6-phosphate + ADPEC
Phosphoglucose isomeraseD-Glucose 6-phosphate ↔ D-fructose 6-phosphateEC
Phosphofructose kinaseATP + D-fructose 6-phosphate ↔ ADP + D-fructose 1,6-bisphosphateEC
Fructose-bisphosphate aldolaseFructose-1,6-bisphosphate ↔ dihydroxyacetone phosphate + glyceraldehyde-3-phosphateEC
Triose phosphate isomeraseGlyceraldehyde 3-phosphate ↔ dihydroxyacetone phosphateEC
Glyceraldehyde-3-phosphate dehydrogenaseD-Glyceraldehyde 3-phosphate + phosphate + NAD+ ↔ 1,3-bisphosphoglycerate + NADH + H+EC
Phosphoglycerate kinase1,3-Bisphosphoglycerate + ADP ↔ 3-phosphoglycerate + ATPEC
Phosphoglycerate mutase3-Phosphoglycerate ↔ 2-phosphoglycerateEC
Enolase2-Phospho-D-glycerate ↔ phosphoenolpyruvate + H2OEC
Pyruvate kinasePhosphoenolpyruvate + ADP ↔ pyruvate + ATPEC
2-Keto-3-deoxy-6-phosphogluconate (the Entner-Doudoroff pathway)
Glucose-6-phosphate dehydrogenaseD-glucose 6-phosphate + NADP+ ↔ 6-phospho-D-glucono-1,5-lactone + NADPH + H+EC
Phosphogluconate dehydrogenase6-Phospho-D-gluconate + NAD(P)+ ↔ 6-phospho-2-dehydro-D-gluconate + NAD(P)H + H+EC
2-Keto-3-deoxy-6-phosphogluconate aldolase2-Dehydro-3-deoxy-6-phospho-D-gluconate ↔ pyruvate + D-glyceraldehyde 3-phosphateEC
B. Further transformations of pyruvate—glycolytic fermentations [2327]
Lactate dehydrogenasePyruvate + NADH ↔ lactate + NAD+EC
Pyruvate:ferredoxin oxidoreductase, PFORPyruvate + CoA + oxidized Fd ↔ acetyl-CoA + reduced Fd + CO2 + H+EC
NADH:ferredoxin oxidoreductase, NFOROxidized Fd + NADH ↔ reduced Fd + NAD+ + H+EC
Ferredoxin hydrogenase2 reduced ferredoxin + 2 H+ ↔ H2 + 2 oxidized ferredoxinEC
PhosphotransacetylaseCoA + acetyl phosphate ↔ acetyl-CoA + phosphateEC
Acetate kinaseATP + acetate ↔ ADP + acetyl phosphateEC
NAD+-dependent ethanol dehydrogenaseAcetaldehyde + NADH + H+ ↔ ethanol + NAD+
An aldehyde + NADH + H+ ↔ a primary alcohol + NAD+
Acetaldehyde dehydrogenaseAcetaldehyde + CoA + NAD+ ↔ acetyl-CoA + NADH + H+EC
Acetyl-CoA acetyltransferase2-acetyl-CoA ↔ CoA + acetoacetyl-CoAEC
3-Hydroxybutyryl-CoA dehydrogenase3-Acetoacetyl-CoA + NADPH + H+ ↔ 3-hydroxybutanoyl-CoA + NADP+EC
3-OH-butyryl-CoA dehydratase
3-Hydroxybutanoyl-CoA ↔ crotonoyl-CoA + H2OEC
2NADH+ oxidized Fd + crotonyl-CoA → 2 NAD+ reduced Fd + butyryl-CoA catalyzed by butyryl CoA dehydrogenase/electron-transfer flavoprotein complex
Butyryl-CoA dehydrogenaseA short-chain acyl-CoA + electron-transfer flavoprotein ↔ a short-chain trans-2,3-dehydroacyl-CoA + reduced electron-transfer flavoproteinEC
Butyryl-CoA dehydrogenase/Etf complexButanoyl-CoA + 2 NAD+ + 2 reduced Fd ↔ Crotonoyl-CoA + 2 NADH + 2 oxidized FdEC
PhosphotransbutyrylaseButanoyl-CoA + phosphate ↔ CoA + butanoyl phosphateEC
Butyrate kinaseButanoyl phosphate + ADP ↔ butanoate + ATPEC
PFL—pyruvate formate lyasePyruvate + CoA ↔ acetyl-CoA + formateEC
FHL—formate hydrogen lyaseFormate → H2 + CO2EC
Pyruvate carboxylaseATP + pyruvate + HCO3 ↔ ADP + phosphate + oxaloacetateEC
Malate dehydrogenaseMalate + NAD+ ↔ oxaloacetate + NADH + H+EC
Fumarate hydrataseMalate ↔ fumarate + H2OEC
Fumarate reductaseFumarate + a quinol ↔ succinate + a quinoneEC
Fumarate + NADH ↔ succinate + NAD+EC
Succinyl-CoA synthetaseGTP + succinate + CoA = GDP + phosphate + succinyl-CoAEC
Methylmalonyl CoA mutaseSuccinyl-CoA ↔ (R)-methylmalonyl-CoAEC
Methylmalonyl CoA epimerase(R)-methylmalonyl-CoA ↔ (S)-methylmalonyl-CoAEC
Methylmalonyl-CoA decarboxylase(S)-methylmalonyl-CoA ↔ propanoyl-CoA + CO2EC
Propionate-CoA transferaseAcetate + propanoyl-CoA ↔ acetyl-CoA + propanoateEC
C. Transformation of gaseous and nongaseous products of acidic fermentations (the selected examples)
Transformation of lactate and acetate to butyrate, hydrogen, and carbon dioxide([28] and cited therein)
Lactate dehydrogenases(S)-lactate + NAD+ ↔ pyruvate + NADH + H+EC
(R)-lactate + NAD+ ↔ pyruvate + NADH + H+EC
Lactate + 2 NAD+ + 2 reduced Fd ↔ pyruvate + 2 NADH + 2 oxidized Fd
See Table 3
Pyruvate is oxidized to acetyl coenzyme A, which is further routed to acetate and butyrate with hydrogen release. See Part B: Further transformations of pyruvate—glycolytic fermentations
Transformation of ethanol and acetate to butyrate and hydrogen in Clostridium kluyveri[29]
Acetate kinaseSee Part B. Further transformations of pyruvate—glycolytic fermentationsEC
Acetyl-CoA acetyltransferaseEC
3-Hydroxybutyryl-CoA dehydrogenaseEC
3-Hydroxyacyl-CoA dehydrataseEC
Butyryl-CoA dehydrogenase/Etf complexEC
Acetate CoA-transferaseAcyl-CoA + acetate ↔ a fatty acid anion + acetyl-CoAEC
Reductive carbon monoxide dehydrogenase/acetyl-CoA synthase pathway (reductive CODH/ACS)[30]
NADP-dependent formate dehydrogenaseCO2 + NADPH ↔ formate + NADP+EC
Formyltetrahydrofolate synthetaseATP + formate + tetrahydrofolate ↔ ADP + phosphate + 10-formyltetrahydrofolateEC
Methenyltetrahydrofolate cyclohydrolase10-Formyltetrahydrofolate ↔ 5,10-methenyltetrahydrofolate + H2OEC
NADP-dependent methylenetetrahydrofolate dehydrogenase5,10-Methenyltetrahydrofolate + NADPH + H+ ↔ 5,10-Methylenetetrahydrofolate + NADP+EC
Ferredoxin-dependent methylenetetrahydrofolate reductase5,10-Methylenetetrahydrofolate + 2 reduced Fd + 2 H+ ↔ 5-methyltetrahydrofolate + 2 oxidized FdEC
5,10-Methylenetetrahydrofolate reductase5,10-Methylenetetrahydrofolate + NAD(P)H + H+ ↔ 5-methyltetrahydrofolate + NAD(P)+EC
5-Methyltetrahydrofolate:corrinoid/iron–sulfur protein Co-methyltransferase[Co(I) corrinoid Fe-S protein] + 5-methyltetrahydrofolate ↔ [methyl-Co(III) corrinoid Fe-S protein] + tetrahydrofolateEC
Carbon monoxide dehydrogenaseCO2 + 2 reduced Fd + 2 H+ ↔ CO + H2O + 2 oxidized FdEC
CO-methylating acetyl-CoA synthaseCO + CoA + [methyl-Co(III) corrinoid Fe-S protein] ↔ acetyl-CoA + [Co(I) corrinoid Fe-S protein]EC
D. Glycerol transformations [31, 32]
Oxidative pathway
Glycerol dehydrogenaseGlycerol + NAD+ ↔ glycerone (dihydroxyacetone) + NADH + H+EC
Dihydroxyacetone kinaseATP + glycerone ↔ ADP + glycerone phosphateEC
For further reactions, see Part A: Pyruvate formation
Reductive pathway
Glycerol dehydrataseGlycerol ↔ 3-hydroxypropionaldehyde + H2OEC
1,3-Propanediol dehydrogenase3-Hydroxypropionaldehyde + NADH + H+ ↔ 1,3-propanediol + NAD+EC
E. Amino acids fermentations [3337]
Syntrophy with H2-scavenging microorganism: amino acid degradation involves NAD(P)- or FAD-dependent deamination of amino acids to the corresponding α-keto acids by amino acid dehydrogenases (EC 1.4.1.X): RCH(NH4+)COO + H2O → RCOCOO + NH4+ + H2 and further conversion of α-keto acids via oxidative decarboxylation to fatty acids: RCOCOO + H2O → RCOO + CO2 + H2 [33]
Without syntrophy with H2-scavenging microorganism: Stickland Reaction—coupled oxidation-reduction reactions between suitable amino acids (coupled deamination of amino acids); one member of the pair is oxidized (dehydrogenated) and the other is reduced (hydrogenated) [34], for example,
Alanine and glycine: alanine + 2 glycine + 3H2O → 3 acetate + 3NH4+ + HCO3 + H+
Valine and glycine: valine + 2 glycine + 3H2O → isobutyrate + 2 acetate + 3NH4+ + HCO3 + H+
Leucine and glycine: leucine + 2 glycine + 3H2O → isovalerate + 2 acetate + 3NH4+ + HCO3 + H+
Examples of amino acid dehydrogenases catalyzing deamination of amino acids to the corresponding α-keto acids [33]
Aspartate dehydrogenaseL-aspartate + H2O + NAD(P)+ ↔ oxaloacetate + NH3 + NAD(P)H + H+EC
Valine dehydrogenaseL-valine + H2O + NADP+ ↔ 3-methyl-2-oxobutanoate + NH3 + NADPH + H+EC
Alanine dehydrogenaseL-alanine + H2O + NAD+ ↔ pyruvate + NH3 + NADH + H+EC
Leucine dehydrogenaseL-leucine + H2O + NAD+ ↔ 4-methyl-2-oxopentanoate + NH3 + NADH + H+EC
Key enzymes of Stickland reaction [3436]
Glycine reductase GR pathway (grdoperon)
Glycine reductaseGlycine + phosphate + reduced thioredoxin + H+ ↔ acetyl phosphate + NH3 + oxidized thioredoxin + H2OEC
Acetate kinaseAcetyl phosphate + ADP ↔ acetate + ATPEC
Proline reductase PR pathway (prdoperon)
D-proline reductase (dithiol)D-proline + dihydrolipoate ↔5-aminopentanoate (5-aminovalerate) + lipoateEC
Others examples [33]
Serine dehydrataseL-serine ↔ pyruvate + NH3 (overall reaction)
(1a) L-serine ↔ 2-aminoprop-2-enoate + H2O
(1b) 2-Aminoprop-2-enoate ↔ 2-iminopropanoate (spontaneous)
(1c) 2-Iminopropanoate + H2O ↔ pyruvate + NH3 (spontaneous)
Threonine dehydrataseL-threonine ↔ 2-oxobutanoate + NH3 (overall reaction)
(1a) L-threonine ↔ 2-aminobut-2-enoate + H2O;
(1b) 2-Aminobut-2-enoate ↔ 2-iminobutanoate (spontaneous)
(1c) 2-Iminobutanoate + H2O ↔ 2-oxobutanoate + NH3 (spontaneous)
Detailed pathways of glutamate fermentation via 3-methylaspartate [37]
Glutamate mutase (methylaspartate mutase)L-glutamate ↔ L-threo-3-methylaspartateEC
Methyl aspartaseL-threo-3-methylaspartate mesaconate (2-methylfumarate) + NH3EC
Mesaconase (2-methylmalate dehydratase)2-Methylfumarate + H2O (S)-2-methylmalate4.2.1.34
Citramalate lyase(2S)-2-hydroxy-2-methylbutanedioate acetate + pyruvate
(S)-2-methylmalate = 2-hydroxy-2-methylbutanedioate
For further transformations of pyruvate to acetate and butyrate, see Part B.
For further transformations of pyruvate to propionate, see Part B.
Detailed pathway of glutamate fermentation via 2-hydroxyglutarate [37]
Glutamate dehydrogenaseL-glutamate + H2O + NAD+ ↔ 2-oxoglutarate + NH3 + NADH + H+
2-Hydroxyglutarate dehydrogenase(S)-2-hydroxyglutarate + acceptor ↔ 2-oxoglutarate + reduced acceptor1.1.99.2
Glutaconate (2-hydroxyglutarate) CoA-transferaseAcetyl-CoA + (E)-glutaconate ↔ acetate + glutaconyl-1-CoA2.8.3.12
2-Hydroxyglutaryl-CoA dehydratase(R)-2-hydroxyglutaryl-CoA ↔ (E)-glutaconyl-CoA + H2OEC
Glutaconyl-CoA decarboxylase4-Carboxybut-2-enoyl-CoA ↔ but-2-enoyl-CoA + CO24.1.1.70

Table 2.

The selected enzymes of acidogenic step of anaerobic digestion. A, B, C, D, and E refer to the processes indicated in Figure 1.

EnzymeReaction/processEC number
F. Acetogenesis dependent on syntrophic relations between microorganisms
Acetate oxidation by, for example,Clostridium ultunense—oxidative carbon monoxide dehydrogenase/acetyl-CoA synthase pathway (oxidative CODH/ACS):
Acetate + 4H2O → 2 HCO3 + 4H2 + H+, ΔG0’ = + 104.6 kJ/mol, with the H2 consuming methanogen, ΔG0’ = −31.0 kJ/mol [38]
NADP-dependent formate dehydrogenaseSee Table 2, Part C
Formyltetrahydrofolate synthetase
Methenyltetrahydrofolate cyclohydrolase
NADP-dependent methylenetetrahydrofolate dehydrogenase
Ferredoxin-dependent methylenetetrahydrofolate reductase
5,10-Methylenetetrahydrofolate reductase
5-Methyltetrahydrofolate:corrinoid/iron-sulfur protein Co-methyltransferase
Carbon monoxide dehydrogenase
CO-methylating acetyl-CoA synthase
Reverse electron transfer during acetate oxidation has yet to be confirmed. Direct interspecies electron transfer (DIET) is not excluded (Westerholm et al., 2016)
Acetate oxidation byGeobacter sulfurreducens:
Acetate oxidation coupled to reduction of fumarate to succinate (∆G°′ = −249 kJ per mol acetate), acetate metabolism proceeds via reactions of the citric acid cycle [39]
Acetate kinaseSee Table 2, Part B
Citric acid cycle
Citrate synthaseAcetyl-CoA + H2O + oxaloacetate ↔ citrate + CoAEC
AconitaseCitrate ↔ isocitrate (overall reaction)EC
Isocitrate dehydrogenase (NADP+-dependent)Isocitrate + NADP+ ↔ 2-oxoglutarate + CO2 + NADPH + H+EC1.1.1.42
2-Oxoglutarate:ferredoxin oxidoreductase2-Oxoglutarate + CoA + 2 oxidized Fd = succinyl-CoA + CO2 + 2 reduced Fd + 2 H+EC
Succinyl-CoA:acetate CoA-transferaseSuccinyl-CoA + acetate ↔ acetyl-CoA + succinateEC
Succinate dehydrogenasesuccinate + a quinone ↔ fumarate + a quinolEC
Fumarate hydratase(S)-malate ↔ fumarate + H2OEC
Malate dehydrogenase(S)-malate + NAD+ ↔ oxaloacetate + NADH + H+EC
Butyrate oxidation bySyntrophomonas wolfei:
Butyrate + 2H2O → 2 acetate + 2H+ + 2H2, ΔG0’ = + 48.3 kJ/mol, with the H2 consuming methanogen, ΔG0’ = −17.3 kJ/mol [4]
CoA transferaseButyrate + acetyl-CoA ↔ butyryl-CoA + acetateEC
Butyryl-CoA dehydrogenaseSee Table 2, Part B
Crotonase-3-OH-butyryl-CoA dehydratase
3-Acetyl-CoA acetyltransferase
Hydroxybutyryl-CoA dehydrogenase
Acetate kinase
Butyrate oxidation coupled with a reverse electron transfer that involves electron transfer flavoprotein EtfAB, membrane-anchored electron carrier DUF224 protein, the menaquinone pool in the membrane, a membrane-bound cytochrome, NADH:hydrogenase/formate-dehydrogenase complex (NDH/HYD1/FDH-1 complex), Rnf (proton-translocating ferredoxin:NAD+ oxidoreductase) [40]
Propionate oxidation bySyntrophobacter wolinii:
Propionate + 3H2O → acetate + HCO3 + H+ + 3H2, ΔG0’ = + 76.0 kJ/mol, with the H2 consuming methanogen, ΔG0’ = −22.4 kJ/mol [4]
Pyruvate carboxylaseSee Table 2, Part B
Malate dehydrogenase
Fumarate hydratase
Fumarate reductase
Succinate dehydrogenaseSuccinate + a quinone ↔ fumarate + a quinolEC
Succinyl-CoA synthetaseSee Table 2, Part B
Methylmalonyl CoA mutase
Methylmalonyl CoA epimerase
Methylmalonyl-CoA decarboxylase
Propionate-CoA transferase
Propionate oxidation coupled with a reverse electron transfer that involves menaquinone, proteins encoded by cytochrome c homologous genes, cytochrome b:quinone oxidoreductases, formate dehydrogenases, hydrogenases including confurcating [FeFe]-hydrogenases [41]
Six syntrophy-specific functional domains found in the genomes of the butyrate- or propionate-oxidizing syntrophs [42]InterPro number
Extra-cytoplasmic formate dehydrogenase (FDH) alpha subunit, EC
FdhE-like protein—tightly connected with FDHIPR024064
FDH accessory protein—tightly connected with FDHIPR006452
CapA—a membrane-bound complex, a protein involved in capsule or biofilm formation that may facilitate syntrophic growth (also present in acetate-oxidizers)IPR019079
FtsW, RodA, SpoVE—membrane-integrated proteins involved in membrane integration, cell division, sporulation, and shape determinationIPR018365
Ribonuclease P involved in tRNA maturationIPR020539
Functional domains involved in electron transfer identified by [42]InterPro number
Cytoplasmic FDHIPR027467, IPR006655, IPR006478, IPR019575, IPR001949
Extracytoplasmic FDHIPR006443
Formate transporterIPR000292, IPR024002
Fe-Fe hydrogenaseIPR004108, IPR009016, IPR003149, IPR013352
NiFe hydrogenaseIPR001501, IPR018194
Rnf complex: 2 reduced Fd + NAD+ + H+ + Na+ ↔ 2 oxidized Fd + NADH + Na+ (EC, IPR010207, IPR026902, IPR010208, IPR004338, IPR011303, IPR007329
Ech complex: 2 reduced Fd + NADP+ + H+ ↔ 2 oxidized Fd + NADPH (EC, IPR001516, IPR001694, IPR006137, IPR001268, IPR012179, IPR001135
Etf alpha, Etf beta, Bcd (Butyryl-CoA dehydrogenase): see Table 2, Part B (EC, IPR012255, IPR006089, IPR009075, IPR006092, IPR006091, IPR013786, IPR009100
IPR023155, IPR024673
IPR020942, IPR002322
IPR016174, IPR000516
DUF224 protein complexIPR003816, IPR004017, IPR023234
Lactate oxidation byDesulfovibrio vulgaris:
Lactate + H2O → acetate + CO2 + 4 H2, ΔG0’ = −8.8 kJ/mol with the H2 consuming methanogen, ΔG0’ = −74.2 kJ/mol [43]
Lactate dehydrogenaseSee Table 2, Part B
Pyruvate:ferredoxin oxidoreductase
Phosphate acetyltransferase
Acetate kinase
Alcohol dehydrogenase
Lactate oxidation coupled with a reverse electron transfer that involves the membrane-bound Qmo complex, cytochromes, hydrogenases (Coo, Hyn, Hyd, Hys), formate dehydrogenases, menaquinone, membrane-bound Qrc complex [43, 44]
Ethanol oxidation byPelobacter carbinolicus
Ethanol + H2O → acetate + H+ + 2H2, ΔG0’ = + 9.6 kJ/mol with the H2 consuming methanogen, ΔG0’ = − 56 kJ/mol [4]
NAD+-dependent ethanol dehydrogenaseSee Table 2, Part B
Acetaldehyde dehydrogenase (acetylating)
Nonacetylating acetaldehyde dehydrogenaseAn aldehyde + NAD+ + H2O ↔ a carboxylate + NADH + H+EC
PhosphotransacetylaseSee Table 2, Part B
Acetate kinase
Ethanol oxidation coupled with a reverse electron transfer that involves membrane-bound ion-translocating ferredoxin:NAD+ oxidoreductase, formate dehydrogenases, and confurcating hydrogenases [1, 45]
G. Acetogenesis independent on syntrophic relations between microorganisms
Ethanol oxidation byAcetobacterium woodii: 2 ethanol + 2 CO2 → 3 acetate—75.4 kJ/mol [46]
Bifunctional acetaldehyde-CoA/alcohol dehydrogenaseEthanol + NAD+ → acetaldehyde + NADH + H+
acetaldehyde + NAD+ + CoA → acetyl-CoA + 2 NADH + H+
Ethanol is oxidized to acetyl-CoA in a two-step reaction by a bifunctional acetylating ethanol/aldehyde dehydrogenase
Acetyl-CoA is transformed to acetate with the release of ATPSee Table 2, Part B
Reduction of ferredoxin by NADH by reverse electron flow in a reaction catalyzed by Rnf complexSee Part F
Carbon dioxide is reduced to acetate via the Wood-Ljungdahl pathwaySee Table 2, Part C
Lactate oxidation byAcetobacterium woodii: 2 lactate → 3 acetate—61 kJ/mol [47]
Lactate dehydrogenaseLactate + 2 NAD+ + 2 reduced Fd ↔ pyruvate + 2 NADH + 2 oxidized Fd
The enzyme uses flavin-based electron confurcation to drive endergonic lactate oxidation with NAD+ as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin to NAD+
Pyruvate is transformed to acetyl-CoA and further to acetate with the release of ATPSee Table 2, Part B
Reduction of ferredoxin by NADH by reverse electron flow in a reaction catalyzed by Rnf complexSee Part F
Carbon dioxide is reduced to acetate via the Wood-Ljungdahl pathwaySee Table 2, Part C

Table 3.

The selected enzymes of acetogenic step of anaerobic digestion. F and G refer to the processes indicated in Figure 1.

EnzymeReaction/processEC number
MFR—methanofuran, H-S-CoM—coenzyme M, H-S-CoB—coenzyme B, H4MPT—tetrahydromethanopterin, F420—5’deazaflavin, H4SPT—tetrahydrosarcinapterin
Hydrogenotrophic pathway
Formylmethanofuran dehydrogenaseCO2 + MFR + 2 reduced Fd + 2H+ ↔ formyl-MFR + H2O + 2 oxidized FdEC
Formylmethanofuran-H4MPT formyltransferaseFormyl-MFR + H4MPT ↔ MFR + formyl-H4MPTEC
Methenyl-H4MPT cyclohydrolaseFormyl-H4MPT + H+ ↔ methenyl-H4MPT + H2OEC
F420-dependent methylene-H4MPT dehydrogenaseMethenyl-H4MPT + reduced F420 ↔ methylene-H4MPT + oxidized F420EC
H2-forming methylene-H4MPT dehydrogenaseMethenyl-H4MPT + H2 ↔ methylene-H4MPT + H+EC
F420-dependent methylene-H4MPT reductaseMethylene-H4MPT + reduced F420 ↔ CH3-H4MPT + oxidized F420EC
Methyl-H4MPT:coenzyme M methyl-transferaseCoenzyme M + methyl-H4MPT + 2 Na+/in ↔ 2-methyl-coenzyme M + 2 Na+/out + H4MPTEC
Methyl-CoM reductaseCH3-S-CoM + H-S-CoB ↔ CoM-S-S-CoB + CH4EC
Heterodisulfide reductaseCoM-S-S-CoB + dihydromethanophenazine ↔ CoB + CoM + methanophenazineEC
Acetotrophic pathway
Acetate kinase-phosphotransacetylase system in Methanosarcina; acetate thiokinase in MethanosaetaAcetate + CoA ↔ acetyl-CoA + H2OEC
CO-methylating acetyl-CoA synthaseAcetyl-CoA + a [Co(I) corrinoid Fe-S protein] ↔ CO + CoA + [methyl-Co(III) corrinoid Fe-S protein]EC
5-Methyltetrahydrosarcinapterin:corrinoid/iron-sulfur protein Co-methyltransferase[Methyl-Co(III) corrinoid Fe-S protein] + tetrahydrosarcinapterin ↔ a [Co(I) corrinoid Fe-S protein] + 5-methyltetrahydrosarcinapterinEC
Anaerobic carbon monoxide dehydrogenaseCO + H2O + 2 oxidized Fd ↔ CO2 + 2 reduced Fd + 2 H+EC
Methyl H4SPT: coenzyme M methyltransferaseCH3 H4SPT + H-S-CoM ↔ CH3-S-CoM + H4SPTEC 2.1.1.-
Methyl-CoM reductaseCH3-S-CoM + H-S-CoB ↔ CoM-S-S-CoB + CH4EC
Heterodisulfide reductaseCoM-S-S-CoB + dihydromethanophenazine ↔ CoB + CoM + methanophenazineEC
Methylotrophic pathway
Methanol:corrinoid protein Co-methyltransferaseMethanol + Co(I) corrinoid protein ↔ Methyl-Co(III) corrinoid protein + H2OEC
[Methyl-Co(III) corrinoid protein]:coenzyme M methyltransferaseCoenzyme M + Methyl-Co(III) corrinoid protein ↔ 2-(methylthio)ethanesulfonate + Co(I) corrinoid proteinEC
Methylamine:corrinoid protein Co-methyltransferaseMethylamine + [Co(I) methylamine-specific corrinoid protein] ↔ a [methyl-Co(III) methylamine-specific corrinoid protein] + NH3EC
Dimethylamine:corrinoid protein Co-methyltransferaseDimethylamine + [Co(I) dimethylamine-specific corrinoid protein] ↔ a [methyl-Co(III) dimethylamine-specific corrinoid protein] + methylamineEC
Trimethylamine:corrinoid protein Co-methyltransferaseTrimethylamine + a [Co(I) trimethylamine-specific corrinoid protein] ↔ a [methyl-Co(III) trimethylamine-specific corrinoid protein] + dimethylamineEC
[Methyl-Co(III) methylamine-specific corrinoid protein]:coenzyme M methyltransferase[Methyl-Co(III) methylamine-specific corrinoid protein] + CoM ↔ methyl-CoM + a [Co(I) methylamine-specific corrinoid protein]EC
Methyl-CoM reductaseCH3-S-CoM + H-S-CoB ↔ CoM-S-S-CoB + CH4EC
Heterodisulfide reductaseCoM-S-S-CoB + dihydromethanophenazine ↔ CoB + CoM + methanophenazineEC

Table 4.

The selected enzymes of methanogenic step of anaerobic digestion [48, 49].


2. Selected enzymes of anaerobic digestion

Figure 1 shows a scheme of AD and Tables 14 present a summary of the selected enzymes and enzymatic reactions involved in decomposition of organic matter to methane and carbon dioxide. Tables 14 are an extension of Figure 1, and in Figure 1, there are the links to Tables 14.

The key groups of hydrolases involved in the process of degradation of organic matter are esterases, glycosidases, and peptidases, which catalyze the cleavage of ester bonds, glycoside bonds, and peptide bonds, respectively (Table 1). Table 1 also includes other classes of hydrolases such as acting on carbon-nitrogen bonds, other than peptide bonds.

In the acidogenic stage of AD, the key step is pyruvate formation from carbohydrates (Table 2, Part A) or other compounds and further pyruvate transformations toward short-chain fatty acids and ethanol (Table 2, Part B). The Part C of the Table 2 also considers transformation of gaseous and nongaseous products of acidic fermentations, resulting from nonsyntrophic nutritional interaction between bacteria. The Parts D and E present the enzymes of glycerol and amino acid transformations, respectively. The latter requires syntrophic cooperation between microorganisms.

The enzymes catalyzing oxidation of nongaseous products of acidogenesis mainly butyrate, propionate, acetate, lactate, ethanol including the enzymes of reverse electron transfer (process responsible for energy conservation in syntrophically growing acetogens) are shown in Table 3.

The enzymes of the three recognized pathways of methanogenesis such as acetotrophic, hydrogenotrophic, and methylotrophic are listed in Table 4.

The data were prepared on the basis of detailed analysis of AD research. The enzyme nomenclature comes from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database resource.

3. Conclusion

Biomass conversion to methane and carbon dioxide is the effect of complex interactions between microorganisms. These processes occur due to the microbial enzymatic machinery involved in specific metabolic pathways. Meta-omic analyses of microbial communities involved in AD reveal (i) dependence of microbial communities on the type of feedstock and operational conditions and (ii) describe interactions within microbial communities and ecophysiological functions of the specific taxa. Searching for the gene presence, gene expression, and protein expression, as well as linking structure and function of microbial communities, allows to develop a fundamental understanding of AD. This chapter is believed to contribute to the studies on the enzymatic road map of anaerobic digestion. However, it is only the tip of the iceberg of processes occurring in the microbial cells/microbial communities.


We acknowledge the support of The National Science Centre, Poland, through grant UMO-2015/17/B/NZ9/01718 and The National Centre for Research and Development, Poland, through grant BIOSTRATEG2/297310/13/NCBiR/2016.

Conflict of interest

The authors declare that there are no conflicts of interest.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Anna Sikora, Anna Detman, Damian Mielecki, Aleksandra Chojnacka and Mieczysław Błaszczyk (November 5th 2018). Searching for Metabolic Pathways of Anaerobic Digestion: A Useful List of the Key Enzymes, Anaerobic Digestion, J. Rajesh Banu, IntechOpen, DOI: 10.5772/intechopen.81256. Available from:

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