**6. CH4 emission in flooding areas: biogeochemistry, isotopic signature, and metagenomics**

Understanding the level of sensitivity of flooding areas as a response to climate change also requires efforts to be better achieved. This demands more knowledge at all levels, ranging from single-cell ecophysiology to *in situ* and *ex situ* biogeochemical functioning. Thus, efforts to cultivate recalcitrant microorganisms, evaluations of the microbiota with independent methods of cultivation, and the combination of these approaches with isotopic determinations and physical and chemical characterization of soil and water are necessary to achieve an understanding of the processes mediated by microorganisms in these environments [67].

It is a challenge for researchers to design experiments and adopt methods that can detect C cycling in alluvial plains. One of the approaches used is the characterization of microbial communities in space and time from the sequencing of DNA or RNA and the construction of correlation matrices of relative abundances of microbial taxa or functional groups with environmental variables [68]. Regardless of the study method, the characterization needs to reflect the correct scale for the issue and should contain enough replicates to provide meaningful data [69].

Metagenomic studies indicate high functional redundancy in flooding areas. Although we know that microbial communities are diverse, DNA-based methods can artificially inflate functional redundancy estimates [69]. This fact is due to the DNA-based approach not being able to distinguish between dormant and active cells [70].

Different responses have been found when assessing the effect of increased salinity on the composition of the total (DNA) and active (RNA) microbial community in an anaerobic reactor [71]. The concentration and exposure time most strongly affected the microbial community, and especially the *Archaea* phylotypes at the RNA level, both in terms of global diversity and specific phylotypes.

The evaluation of the survival conditions of *Archaea* methanogenic under aerated conditions in floodplains with rice crops revealed the composition of the active community, indicating that unknown mechanisms maintain the stability of the community in these environments until 1 year after the start of drainage [72].

Stable isotopes have long been used as a tool to investigate environmental processes and their relationships with microorganisms, which can be established through metabolic pathways [73]. The merit of C isotopes lies in their relatively slow exchange rate for many minerals containing C and in relatively large fractionations, even at high temperatures. These two properties make C isotopes an excellent recorder of geological processes and allow a better understanding of C sources and related-volatile flows in geological time scales [74]. We are undergoing a reformulation of isotopic approaches based on the increase in genomic and transcriptomic databases, the latest technologies with improved instrumental and mass spectrometric data acquisition, processing, and evaluation [73]. For Coyotzi et al. [75], the incorporation of stable isotopes into the microbial biomass allows the recovery of labeled nucleic acids from active microorganisms. The combination of stable isotopes with metagenomics provides access to the genomes of microorganisms involved in metabolic processes of interest.

The CH4 fluxes in terrestrial and aquatic environments have been evaluated in several ways, but in general, the monitoring of gas accumulation in flow chambers has been the predominant methodology to date. This methodology is conceptually simple and does not require expensive field equipment, but is laborious, based mainly on manual sampling [17].
