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

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.


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 CO 2 with H 2 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].
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  Tables 1-4: 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 in Table 2; F, G refer to the title rows in Table 3.
Analysis of many studies on metagenomes of microbial communities from anaerobic digesters shows that (i) contribution of methanogens in the methaneyielding 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. 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.

Selected enzymes of anaerobic digestion
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. Without syntrophy with H 2 -scavenging microorganism: Stickland Reaction-coupled oxidationreduction 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 + 3H 2 Table 2.

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

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.  Table 4.