Table 3.

Benefits and challenges of methanogen biotechnology.

Benefits	Challenges
Methanogens are some of the fastest-replicating organisms, particularly members of Methanococcus (Jones et al., 1983; Goyal et al., 2016; Long et al., 2017) and Methanopyrus (Takai et al., 2008) genus. (Jones et al., 1983; Takai et al., 2008; Goyal et al., 2016; Long et al., 2017).	Strain differences in growth rate and carrying capacity. Growth is flux-controlled depending on substrate feed rates. Gas-phase fermentation presents similar problems as oxygenation in traditional fermentations (Mosier and Ladisch, 2011; Chen, 2012; Luo and Angelidaki, 2012).
Methanogens can grow on inexpensive substrates including negative value substrates such as wastewater (Daniels et al., 1977; Schiraldi et al., 2002; McGenity, 2010; Ferry, 2012; Costa and Leigh, 2014; Borrel et al., 2016; Buan, 2018; Chadwick et al., 2022) Methanogens already scaled up worldwide for water treatment and biogas production.	Process disfavors growth of aerobic pathogens. Co-product can be water ready for discharge to aquifers and waterways.
Can be coupled directly or indirectly to electrodes for carbon capture by electrosynthesis or for electricity generation from biomass (Ragab et al., 2020).	Surface-to-area, substrate solubility, and other challenges commensurate with microbial fuel cell technologies.
Oxygenation not required. Can grow on non-gas substrates. No contamination by aerobic organisms.	Methanogens require specialized culture environments to maintain anaerobicity (Balch et al., 1979; Rouviere and Wolfe, 1988; Buan, 2018).
Mesophilic and thermophilic strains available to tailor to the desired product and process needs.	Methanogen chassis organisms may need different optimization strategies.
Novel metabolic pathways are constantly being discovered (Costa and Leigh, 2014; Guan et al., 2015; Borrel et al., 2016; Mayumi et al., 2016; Buan, 2018; Yan and Ferry, 2018; Chadwick et al., 2022; Zhou et al., 2022).	Methanogen genetics and biochemistry are less characterized than other model organisms.
Synthetic biology pathways often use archaeal or methanogen genes to improve yields and reduce feedback inhibition.
Bacterial synthetic biology and genetic strategies have been successfully translated to methanogens.
Methanogens have a high substrate to volume ratio with low accumulation of biomass relative to products (Thauer et al., 2008; Ferry, 2012; Buan, 2018).	High titers of intracellular products may be difficult to obtain unless accumulated into vacuoles or secreted extracellularly.
Multiple validated genetic tools available including tools for Methanosarcina spp., (Metcalf et al., 1997; Buan et al., 2011; Nayak and Metcalf, 2017) Methanococcus maripaludis,(Blank et al., 1995; Bao and Scheller, 2021) and Methanothermobacter thermautotrophicus (Buan et al., 2011; Sarmiento et al., 2011; Nayak and Metcalf, 2017; Bao and Scheller, 2021; Fink et al., 2021).	Variability in genome copy number can present challenges when performing chromosomal modifications (Hildenbrand et al., 2011; Aldridge et al., 2021).
The lack of cell wall and envelope in most methanogens ensures that products generated through methanogen fermentations are not contaminated with peptidoglycan or endotoxin (Jones et al., 1987; Claus and König, 2010).	Some methanogen species produce pseudomurein cell walls or extracellular polysaccharide capsules, although these are generally non-or weakly immunogenic (Sirohi et al., 2010; Subedi et al., 2021).
Methanoarchaea are non-pathogenic, though there have been studies suggesting a link between methanogens and other microbes in dysbiotic anaerobic abscesses (Drancourt et al., 2017; Sogodogo et al., 2019).	Not currently recognized as a GRAS (Generally Regarded as Safe) organism.