# Microbial Activity in Subterranean Ecosystems: Recent Advances
## 2 The Control of Greenhouse Gas Fluxes by Cave Microorganisms
The potential methanothophy in four Spanish caves was assessed by tracking the presence of methane-oxidizing bacteria using the particulate methane monooxygenase gene \(pmoA\), which is a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment [35]. The study revealed the presence of the proteobacteria _Methylocaps aurea_, _Methylomicrobium album_, _Methylococcus capsulatus_, and methanotrophs of the K1-1 and K3-16 groups in samples from Altamira, Sidron, and Ojo Guarefa caves, mainly in locations where CH\({}_{4}\) usually reaches concentrations near to the atmospheric background levels. These soil bacteria oxidize the atmospheric CH\({}_{4}\)[36]. However, the analyses did not detect methanotrophs in remote subterranean locations or poorly ventilated caves, such as Castafarar de Ibor Cave, where CH\({}_{4}\) is absent or present in minimal concentrations (below the accuracy threshold) throughout the year. [PERSON] et al. [34] suggested that complete consumption of CH\({}_{4}\) was favored in the subsurface atmosphere under near vapor-saturation conditions without significant intervention of methanotrophic bacteria. This led to the assumption that CH\({}_{4}\) oxidation was induced by ions and \(\bullet\)OH generated by the radioactive decay of radon (\({}^{222}\)Rn). In fact, one of the important \(\bullet\)OH sources in cave air may be from radioactive \({}^{222}\)Rn decay [34]. However, further research verified that the mechanism of CH\({}_{4}\) consumption was seasonally changing and methane-oxidizing bacteria were primarily responsible for the widespread observations of CH\({}_{4}\) depletion in subterranean environments, discarding any evidence of radiolysis contribution [37; 38; 39]. [PERSON] et al. [37] tested, in controlled laboratory experiments, whether radiolysis could rapidly oxidize CH\({}_{4}\) in sealed air with different relative humidity and elevated levels of radiation from Rn isotopes. No evidence of CH\({}_{4}\) oxidation by radiolysis was found. On the contrary, a rapid loss of CH\({}_{4}\) was found when moist soil in the absence of Rn was added to the container. This was consistent with the presence of methane-oxidizing bacteria, which were responsible for the widespread observations of CH\({}_{4}\) depletion in subterranean environments. Since the pioneering work of [PERSON] et al. [34], a few authors, based on studies in caves from Australia, the USA, Vietnam, and Spain, additionally supported CH\({}_{4}\) oxidation by methanotrophic bacteria [38; 39; 40; 41; 42]. [PERSON] et al. [38] reported that the concentrations and stable isotopic compositions of CH\({}_{4}\), CO\({}_{2}\), and Rn in cave air overlapped and diverged from those of the atmosphere, as the majority of cave air samples were depleted in CH\({}_{4}\) and enriched in CO\({}_{2}\) relative to the local atmosphere. These differences indicate that atmospheric and internal cave processes influenced the composition of cave air. Therefore, the authors, on the basis of CH\({}_{4}\) concentrations, \(\delta^{13}\)C\({}_{\text{CH}4}\), and \(\delta^{2}\)H\({}_{\text{CH}4}\) values measured in 33 caves in the USA and three caves in New Zealand, suggested that microbial methanotrophy within caves is the primary CH\({}_{4}\) consumption mechanism. Furthermore, the stable isotopic composition of CH\({}_{4}\) in the studied caves suggested that, in addition to atmospheric CH\({}_{4}\), at least two additional CH\({}_{4}\) sources were present in some caves: CH\({}_{4}\) produced from acetate fermentation, and from CO\({}_{2}\) reduction, processes occurring over a wide scale in the environment. [PERSON] et al. [39] also proposed that biological processes, largely oxidation by methanotrophic bacteria, cause a depletion of CH\({}_{4}\) in caves.