Periodic Reporting for period 1 - MIFLUKE (Microbial fluxes of greenhouse gases (CO2 and CH4) in karst ecosystems: comprehensive assessment and biogeochemical modelling)
Berichtszeitraum: 2019-09-01 bis 2021-08-31
The assessment of carbon cycle in the Earth-climate system is one the highest challenge in science nowadays. It still remains some key knowledge gaps and uncertainties concerning the budgets of greenhouse gases (GHGs) at ecosystem scale. Karst ecosystems cover up to 25 % of the land surface and they are acting as rapid CH4 sink and as alternately CO2 source or sink. Microbial action could be playing a crucial role on it, but there are many knowledge gaps. Previous studies in subterranean environments had shown that some of microbial communities (mostly Actinobacteria) are active agents in the fixation of CO2, and that oxidizing bacteria (MOBs) seems to be the main responsible for in-situ removal of CH4 from cave air, but this had been inferred mainly by indirect methods.
MIFLUKE overall research aim is to elucidate the role of karst microbiota in the main GHGs -CO2 and CH4- content and fluxes in underground atmospheres, as a key challenge to clarify the accurate effective contribution of karst ecosystems to global carbon cycle (global balances) and help to resolve uncertainties in climate feedback dynamics.
We apply an innovative and highly interdisciplinary combination of a broad suite of cutting-edge technologies -GHG flux monitoring, isotopic geochemical tracing, biogeochemistry, metagenomics, etc.—to quantify the GHG fluxes controlled by microbial-induced processes and directly exchanged with the cave atmosphere in several temporal scales (daily, seasonal, annual pattern). After thirteen months of research, and with the analyses still in progress, the results absolutely reveal that cave microbiota is acting as net up taker of CH4 and as an emitter / sink of CO2 alternately. Their uptake and emissions rates appear to be meaningfully high. It quantitatively confirms that cave microbiota is playing an outstanding role in the processes of production and consumption of CO2 and CH4, that is determining the strong variations of these major GHGs in natural subterranean ecosystems. This research line is crucial to achieve a more accurate assessment of the effective contribution of karst ecosystems to the global carbon cycle.
MIFLUKE overall research aim is to elucidate the role of karst microbiota in the main GHGs -CO2 and CH4- content and fluxes in underground atmospheres, as a key challenge to clarify the accurate effective contribution of karst ecosystems to global carbon cycle (global balances) and help to resolve uncertainties in climate feedback dynamics.
We apply an innovative and highly interdisciplinary combination of a broad suite of cutting-edge technologies -GHG flux monitoring, isotopic geochemical tracing, biogeochemistry, metagenomics, etc.—to quantify the GHG fluxes controlled by microbial-induced processes and directly exchanged with the cave atmosphere in several temporal scales (daily, seasonal, annual pattern). After thirteen months of research, and with the analyses still in progress, the results absolutely reveal that cave microbiota is acting as net up taker of CH4 and as an emitter / sink of CO2 alternately. Their uptake and emissions rates appear to be meaningfully high. It quantitatively confirms that cave microbiota is playing an outstanding role in the processes of production and consumption of CO2 and CH4, that is determining the strong variations of these major GHGs in natural subterranean ecosystems. This research line is crucial to achieve a more accurate assessment of the effective contribution of karst ecosystems to the global carbon cycle.
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.
(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.
The results of this research project will have direct implications on current challenges of the scientific community, potentially triggering important socio-economic impacts and the wider societal implications, such as: (1) Characterize subterranean microbial communities by –omic approaches, providing valuable insights into the functional ecology and interactions of the communities, focused mainly on those involved in the carbon cycle; (2) Get a more accurate assessment of GHGs balance at ecosystem scale through studying the role of the microbial communities’ components from subterranean environments, whose potential bi-directional feedback on climate change is unaccounted so far; (3) The adaptation of subsurface microorganisms to extreme environmental conditions can also yield insights about the origins of life on Earth and other planets and, specifically, searching microbe-mineral interactions and biosignatures detection in subterranean environments will have a potential use as extra-terrestrial analogues for space exploration; (4) A better understanding of the biogeochemical processes involved in conservation of subterranean sites including natural cultural heritage, will serve to configure the most appropriate strategies for the conservation of subterranean sites with a valuable heritage.