The work performed over thirteen months of research, is organised in two work packages.
(1) To characterize the most significant microbial communities, to identify the dominant microbial agents, and to approach the specific metabolic pathways triggering the CO2 and CH4 microbial fluxes (uptake-fixation-production processes). 4 fixed places for in situ gas monitoring, geochemical tracing gas sampling and microbial sampling was selected.
Trimestral systematic microbial sampling was developed in the cave. We developed the structural and functional characterization by meta-barcoding analyses of bacteria 16S rRNA genes and Shotgun Metagenomics. We developed also a full characterization of microbial microstructure by using electron microscopy, transmission electron microscopy, combined with geochemical analysis. We observed three-dimensional structures and microbe-mineral interactions as well as analysed by-products compounds and also effects by microbial-mineral interaction (bio-precipitation, corrosion, etc.).
(2) To assess the CO2 and CH4 fluxes from microbial processes directly exchanged with the cave atmosphere, in the several temporal scales (daily, seasonal, annual pattern).
We developed in situ real-time automatic monitoring of CO2 and CH4 cave-substrates fluxes during the trimestral campaigns. Daily continuous monitoring of CO2 and CH4 fluxes was conducted by a closed chamber-based gas exchange system (LICOR Automated Soil Gas Flux System LI-8100A), equipped with a Long-Term Chamber 8100-104), in conjunction with a compatible Gasmet FTIR gas analyser (Gasmet DX4015). We used SoilFluxPROTM to compute CO2 and CH4 flux measurements and analyse data results. To the best of our knowledge, direct measures of carbon fluxes from substrates/microbial colonies inside caves do not exist in literature. During the quarterly field campaigns, we also developed a geochemical trace sampling of the atmosphere, soil, cave air and of the closed chamber air for microbial fluxes at each of the 4 placed fixed. We analysed the CO2 and CH4 molar fractions and stable carbon isotopic δ13C in both gases by using cavity ring-down spectroscopy (CRDS, G2201-I Picarro spectrometer), to allow a genetic diagnosis of CH4 and CO2 in the air and fluxes for each period. In addition, in each campaign, we collected data from the autonomous monitoring network of cave main microenvironmental parameters of the local subsurface-soil-atmosphere system, in order to evaluate the effect of microclimate conditions ant the states of ventilation inside the cavity. The organisation and processing of all the data collected were carried out.
In this first year of research, we have verified negative CH4 fluxes (uptake) from microbial communities, simultaneously linked to positive CO2 fluxes (emission) directly related to microbial methanotrophy. The most recent data from direct measurements of gas exchange fluxes indicate that both gases are inextricably linked in these microbial-induced processes.
The phylogenetic characterisation of bacteria in the cave substrates has shown a major presence of Proteobacteria, Acidobacteria, Planctomycetes and Chloroflexi phyla, with an increase of Actinobacteria in the most superficial ones.
The in situ real-time monitoring of CO2 fluxes has shown a predominance of CO2 emission, resulting from respiration by chemolithotrophic microorganisms. Uptake CO2 fluxes have been measured on moonmilk speleothems. Crossiella found in moonmilk have the ability to capture CO2 from the underground atmosphere, resulting in precipitation of calcium carbonate as a by-product of the action of carbonic anhydrase.
The results also revealed simultaneous CH4 uptake fluxes in all locations and under several ventilation conditions, on a daily scale at significant rates. In this case, methanotrophy is assigned to members of the phylum Rokubacteria (NC10), that mediates the anaerobic oxidation of CH4 (AOM) coupled to nitrite reduction.
