TABLE 2.

Summary of the advantages and disadvantages of different methods to determine the importance of different methanogenesis pathways

Method	Advantages	Disadvantages
Isotope-based methods	Can clearly distinguish some pathways based on natural 13C abundance	Can be ambiguous, as some substrates have overlapping signatures
	Can clearly track carbon from substrate to product with 14C label	Can be influenced by other biochemical processes/pathways such as methanotrophy that affect natural 13C abundances
		Can be ambiguous if different CH4 pools in the environment mix physically before measurement
Substrate quantification	Identifies which substrates are present	Can be difficult for some substrates and require specialized analytical methods
	Quantifies concentration of substrate to help interpret its relevance	Measures substrate pools rather than production and consumption rates
Nucleic acid sequencing	Enables high-throughput processing of many samples	Can be affected by organisms and genes that are present but not active or expressed (DNA)
	Provides a deeper understanding of the ecology and taxonomy	Can be affected by relic DNA
	Facilitates comparisons to databases and other studies	Can be difficult for RNA, which is easily degraded
	Can assess which genes are actively expressed (RNA)
	Provides a clear relationship between substrate concentration and CH4 produced
Substrate addition experiments	Enable manipulating other environmental variables of interest	Are performed under laboratory conditions that cannot completely mimic field conditions
	Can be performed on microcosms or isolates	Test potential rates of methanogenesis, not the actual field rates
	Can utilize inhibitors to help indicate importance of a pathway
	Can be combined with isotope-based methods to clearly link substrate to methane
	Can be combined with microscopy-based methods to show the spatial arrangement of microorganisms