4.1 Gut Methanogens
Methanogens are microorganisms initially exclusively thought to be from the phylum Euryarchaeota. From the recent documentation, there has been advancement to other different phyla. Some of the phyla already agreed upon are Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota (TACK), Nonarchaeota, Bathyarchaeota, Geoarchaeota, Marsarchaeota, and Verstraetearchaeota (Berghuis et al., 2019; Vanwonterghem et al., 2016; Evans et al., 2015; Kelly et al., 2011). This study identified species from TACK, Euryarchaeota, and Nonarchaeota. Phyla Euryarchaeota and Crenarchaeota were noted to be dominant among archaeal methanogens and a few presences of methanogens from the phyla Thaumarchaeota, Korarchaeota and Nanoarchaeota. The same methanogen abundance was noted by Jia et al., (2017). Jia et al., (2017) further noted that the phylum Euryarchaeota (specifically those from the genus Methanosarcina) was notably responsible for a variety of functionalities related to methane biosynthesis. The genus Methanosarcina possibly will produce methane with the help of enzymes and protein constituents in the methyl nutrient pathway, acetic acid, and CO2 reduction (Thauer, 2011). Methanogens occupy various diverse environments ranging from hostile environments to favorable conditions. They are acidophilic mesophiles and/or psychrophiles (Evans et al., 2015). Their vast environmental exposure would cause them to utilize different types of metabolism for their nourishment. Some of the phyla were not identified during this study because of the high temperature requirement that the gut of the bovines do not offer as their optimal conditions are between 38.8°C ± 0.5°C (Chen et al., 2018) and/or their full information and categorization has not been fully documented. The Phylum Bathyarchaeota, for example, which was initially identified as Miscellaneous Crenarchaeotal group, has a small proportion of its species cultured and their genomic information completed, calling for advance work (Dayu et al., 2020; Meng et al., 2014; Lloyd et al., 2013).
Metharchaeal methanogens have been exclusive studied in comparison to other phyla. They have been recorded to have 155-200 isolated species which are clustered into 4 classes, 7 orders, 14 families, and 29-35 diverse genera (Singh et al., 2011). This study showed presence for 3 taxonomic classes, 6 orders, 12 families, 24 genera and 37 individual species of the Metharchaeal methanogens. This is an illustration of a high representation of the metharchaeal methanogens in the study areas. Methanogens in the rumen and rectal area of the ruminant species vary in their population’s organization, ecology, and their substrate sources (Knapp et al., 2014). Such variation could be because of the prevailing physicochemical properties (Dayu et al., 2020). The most dominant species in the fore and hindgut was noted to be Methanocorpusculum labreanum (~17%), followed by other hydrogenotrophic methanogens which is in agreement with a study by Auffret et al., (2017) and Chen et al., (2014). The dominance of Methanocorpusculum can be explained by their ability to utilize a wide range of substrates such as acetate, H2 + CO2, formate, ethanol, 2-propanol, 2- butanol, or cyclo-pentanol. Methanocorpusculum relies on acetate as a growth feature and on peptone, tungstate, and nickel for their stimulatory (Rosenberg et al., 2014). Moreover, this genus can survive in a wide environment, temperature range of 15-60℃ and pH of 6.1-8.0 (Liu and Whitman, 2008), all of which can be achieved in the rumen.
Methanococcus are methanogens that were thought to be only isolated from the sea. However, that opinion since changed when they were found in other environments that are not like marine conditions (Tumbula and Whitman, 2003). These species are not associated with any disease on their hosts and are firmly anaerobic and hydrogenotrophic. They have distinct abilities to undertake sulfur metabolism (Liu et al., 2009). Most are mesophilic (require a temperature of between 20-45°C), and others are thermophilic (41-122°C) or hyperthermophilic (above 60°C) (Stetter, 2006). In this study, mesophilic species: Methanococcus aeolicus, Methanococcus maripaludis, Methanococcus vannielii, and Methanococcus voltae were identified. The species identified in this study were similar to those identified by Goyal et al., (2016).
Methanosarcina metarchaeals utilize the substrates acetate, H2 +, CO2, CO, methanol, methylamines, and metylmercaptoproprionate dismetylsulfide. They grow on a variety of substrates because they are notably cytochrome bound (Buan et al., 2011). The methanogens here operate in a temperature range of between 1°C - 70°C and a pH range of 4-10 and adapt to low hydrogen availability (Liu and Whitman, 2008). Methanosarcinas have a flexible metabolic pathway making their growth genes on one substrate easily deleted without affecting their subsequent growth on another available substrate(s). With this capability, methanosarcina’s genetic analysis can be used to investigate how methanogens grow and participate in methane production along the known methane metabolism pathways (Kulkarni et al., 2009). Methanosarcina acetivorans, Methanosarcina barkeri, and Methanosarcina mazei were identified during this study.
The methanogen Methanocorpusculum labreanum species that hailed from Methanocorpusculum was identified during this study. This species was found both among the rumen fluid samples and dung samples from the Kenyan dairy cattle. This was in contrast with a study by Liu et al., (2012) in a study on Chinese sheep that identified sequences related to Methanocorpusculum species from sheep droppings only but absent in the rumen of the same sheep. In another study by Luo et. al., (2013), the authors found out that Methanocorpusculum were dominant (60%) in the hindgut of captive Ceratotheriumsimum. Methanocorpusculum utilizes the substrate acetate, H2 +CO2, formate, ethanol, 2-propanol, 2- butanol, or cyclo-pentanol. Species from this genus were also identified from a wastewater bio digester (Oren, 2014). Identification of this species in the rumen indicated that they may have a wider niche than previously thought.
4.2 Functional enzymes associated with methane metabolism
Enzymes catalyze chemical reactions that are key for life functionality such as metabolism and digestion (Blanco and Blanco, 2017). Over the years, there is an advancement of knowledge on methane metabolism between methanogens and methanotrophic archaea with a universal display of the Methyl-coenzyme M reductase complex as a main enzyme in their pathways (Evans et al., 2019). The study identified the function of the enzyme Glycine/Serine hydroxy methyltransferase in the aspects of Amino acid transportation and metabolism/biosynthesis from module entry K00600. This was identified from the Enzyme commission (EC) EC: 126.96.36.199 and was associated with the structural gene glyA. Glycine/Serine hydroxy methyltransferaseis a vitamin reliant enzyme that catalyzes the reversible and conversion of L serine to glycine and tetrahydrofolate to 5, 10-Methylenetetrahydrofolate. Upon completion of the enzymatic reaction, it leads to the delivery of substantial carbon units to the cell (Edgar, 2005). Other studies have noted that this enzyme as well catalyzes glycine and acetaldehyde to form L-threonine with 4-trimethylammoniobutanal to form 3-hydroxy N6, N6, N6-trimethyl-L-lysine (Schweitzer et al., 2009). Methanogens like Methanococcus jannaschii, which was identified in this study, have been shown to use the enzyme for amino acid biosynthesis (Tsoka et al., 2004).
Formylmethanofuran-tetrahydromethanopterin-N-formyl transferase enzyme was noted from the EC: 188.8.131.52 (Formerly EC. 184.108.40.206). The module entry involved was K00672. The enzyme was notably involved in energy production and conversion. Entry K00123 that is involved in the anaerobic selenocysteine-containing dehydrogenase was also noted and needed for energy production and conversion. The gene responsible for the enzyme is Ftr. Structural genes associated with the functionality of the enzyme were fdoG, fdhF, and fdwA. Formylmethanofuran-tetrahydromethanopterin N-formyltransferase enzyme catalyzes two notable substrates; methanofuran and 5-formyl-5-6-7-8-tetrahydromethanopterin (Wagner et al. 2016). Methanofuran is the key for methane formation from CO2 by methanogens. CO2 as a methanogenic substrate is initially reduced and activated to formyl-methanofuran (Wagner et al., 2016). Mesophilic methanogen (Methanosarcina barkeri) and thermophilic methanogens (Methanopyrus kandleri), that were also identified in this study, have shown functionality for the enzyme (Enzmann et al., 2018).
Anaerobic carbon monoxide dehydrogenase enzyme facilitates the metabolism of methanogens by the reversible interconversion between carbon monoxide and CO2. The catalyzed reaction is vital for energy conservation and carbon fixation among methanogens, especially during the Wood-Ljungdahl pathway (King and Weber, 2007; Borrel et al., 2016). Enzyme anaerobic carbon monoxide dehydrogenase of the catalytic subunit was noted during this study from the EC: 220.127.116.11 pathway, formerly EC. 18.104.22.168. The involved module entry was K00198. Hydroxylamine reductase (hybrid-cluster protein) together with the enzymatic function of anaerobic carbon monoxide dehydrogenase are vital for inorganic ion transport and metabolism energy production and conversion. The key genes involved were cooS and acsA.
Catalase-peroxidase enzyme is documented to be an inconsequential material in the antioxidant system in methanogens even for those aerotolerant, including species such as Methanosarcina acetivorans (Jennings et al., 2014). The catalase-peroxidase enzyme is a sturdy catalase with H2O2 as the contributor which releases O2 (Vlasits et al., 2010) and molecules of water in a two-step reaction (Nandi et al., 2019). Methanogens in their environments are exposed to oxygen occasionally in aerobic situations, this would call in for the functionality of antioxidants to facilitate lowering levels of oxygen (Angel et al., 2012). Ma and Lu (2011) pointed out that some methanogens can withstand some levels of oxygen for some hours. The enzyme possessed by methanogens (although they are noted to be limited in the numbers- restricted to Methanosarcina species and Methanobrevibacter species) has been noted to have been acquired through a gene (katG) transferred laterally (Zamocky et al., 2012a). When catalase peroxidase enzyme has functionality in EC: 22.214.171.124, thereby it acts on hydrogen peroxide as an acceptor and not the functionality under EC. 126.96.36.199 of both catalase and peroxidase. Module entry K03782 was involved. Specifically, the Catalase peroxidase I is involved in catalyzing inorganic ion transport and metabolism. The gene involved is katG.
In conclusion, this study broadens our understanding on dominant methanogen species in Kenya and Tanzanian among smallholder cattle and functional enzymes associated with methane metabolism and production. The methanogen species abundances from these study areas in numbers/kind can be utilized exclusively or jointly as indirect selection criteria for methane mitigation. This calls for interdisciplinary cohesion and collaboration for fruitful achievements. Studies should be carried out to taxonomically categorize species missing out of place. Furthermore, every part of the gut (either fore or the hindgut) was capable of hosting methanogens. Targets for methanogens should entirely be towards the whole gastrointestinal tract. Furthermore, there is a need to target functional genes of the microbes and those of animals to achieve a friendly environment without affecting the animal’s functionality. Animals who are less methane emitters should be bred to cut on their methane release from the gut. Further studies should be carried out to target pathways for tolerable methane emitter dairy cattle without hindering other necessary metabolic processes. The fraction of the methanogens that are yet to be fully classified should be carried out and a thorough study of their temperature, substrate(s), and pH should be noted.