# Microbial Activity in Subterranean Ecosystems: Recent Advances
## 2 The Control of Greenhouse Gas Fluxes by Cave Microorganisms
The analyses were conducted by taking advantage of technological advances in high-precision field-deployable CRDS and FTIR spectrometers, which allowed to measure target tracer gases (NO\({}_{2}\), N\({}_{2}\)O, CH\({}_{4}\), and CO\({}_{2}\)) and \(\delta^{13}\)C of both carbon-GHGs in situ. The \(\delta^{13}\)C\({}_{\text{CO2}}\) data (\(-\)4.5 to \(-\)7.5%) suggested a mantle-rooted CO\({}_{2}\) likely generated by the thermal decarbonation of underlying marine carbonates, combined with degassing from CO\({}_{2}\)-rich groundwater. CH\({}_{4}\) molar fractions and their 8D (\(-\)77 to \(-\)48%) and \(\delta^{13}\)C values (\(-\)52 to \(-\)30%) indicated that the CH\({}_{4}\) reaching Vapor Cave is the remnant of a larger and deep-sourced CH\({}_{4}\), which was likely generated by the microbial reduction in carbonates. This CH\({}_{4}\) was affected by a postgenetic depletion during its migration through the cave environment as a component of the rising warm air. CH\({}_{4}\) concentrations and \(\delta^{13}\)CH\({}_{4}\) varied with depth. At \(-\)80 m, higher concentrations were found but above \(-\)30 m depth lower CH\({}_{4}\) concentrations were found and heavier \(\delta^{13}\)C values were found near the cave entrance. This was consistent with a methane oxidation mediated by microorganisms and in fact, next generation sequencing (NGS) analysis of sediments showed a relative abundance of _Candidatus_ Methylomirabilis 4 to 5 times higher in the deepest sample (\(-\)80 m) with respect to \(-\)30 and \(-\)15 m. _Candidatus_ Methylomirabilis oxyfera (_Rokubacteria_) is an anaerobic denitrifying methanotroph [46]. It must be noticed that [PERSON] et al. [47] found that members of the uncultivated candidate phylum _Rokubacteria_ responded positively to elevate CO\({}_{2}\) concentrations. In a similar way, [PERSON] et al. [48] studied an area of agricultural soils in Italy with anomalously high temperatures (up to \(\cong\) 50 \({}^{\circ}\)C) and found emissions of biogenic CO\({}_{2}\) linked to CH\({}_{4}\) oxidation at a depth of 0.7 m from the surface. A strong biological methane-oxidizing activity in these soils was found and an examination of the _pmoA_ clone libraries revealed the large biodiversity of methanotrophs including _Methylomonas_, _Methylococcus_, _Methylocystis_, and _Methylocalatum_. Regarding the nitrogen gases, [PERSON] et al. [44] stated that the analysis of the ecological functions and metabolism of the microbiota from cave sediments suggested that N\({}_{2}\)O is mainly produced in the deepest areas of Vapor Cave (below \(-\)15 m depth). In these areas, high CO\({}_{2}\) concentrations and low O\({}_{2}\) levels within the sediments determine a prevailing hypoxic and acidic environment that promotes the release of nitrite, nitric oxide, and hydroxylamine as products of the metabolism of ammonia-oxidizing archaea and nitrate reduction. In fact, at \(-\)15 m depth, the archaeal communities were dominated by the class _Nitrososphaeria_ (69.0% of the total _Archaea_), with a majority of uncultured members and only two identified genera, _Nitrososphaera_ and _Nitrosotenuis_. This is consistent with the abundant occurrence of these _Archaea_ in deep sediments and better survival under conditions of low dissolved oxygen. To summarize, considerable advances have been reached in recent years regarding processes of production, consumption, and storage of greenhouse gases (CO\({}_{2}\), CH\({}_{4}\) and N\({}_{x}\)O\({}_{x}\)) by cave microorganisms in subterranean vadose ecosystems. Recent and current research has shown that cave _Actinobacteria_ are active agents in the fixation of CO\({}_{2}\), capturing CO\({}_{2}\) from air and forming calcium carbonate polymorphs [8]. In particular, direct CO\({}_{2}\) flux measurements in areas heavily colonized by bacteria indicate that they were promoting the uptake of this gas.