Integrating microbial METAbolism into CLOUD chemistry: from" omics" to a model – METACLOUD

Task 1: Sampling and characterization of cloud water (ICCF, LaMP)
This involves collecting cloud water at the top of the puy de Dôme, an internationally recognized observatory. For this it is necessary to sample a very large quantity of cloud water, which implies the development and manufacture of high volume impactors. This cloud water must be chemically and biologically characterized (analytical chemistry and molecular biology).
Task 2: Incubations and preparation of cellular extracts (ICCF, LaMP).
After tangential filtration to concentrate the biomass, the samples are incubated in microcosms mimicking at best two contrasting atmospheric conditions (light + H2O2 17 ° C for the day; black, no H2O2, 5 ° C for the night) in a artificial cloud medium containing formaldehyde. For this, photo-bioreactors must be built. After incubation, cell extracts are produced to obtain metabolites or mRNAs which will be analyzed in tasks 3 and 4.
Task 3: Metametabolomics and Metatranscriptomics (ICCF, GMGM)
The metabolites extracted from the microorganisms generated in task 2 will be analyzed by mass spectrometry using a metabolomic approach. The messenger RNAs will be analyzed by bioinformatics using a transcriptomic approach. These studies will highlight the modulation of metabolic pathways induced by the two atmospheric scenarios.
Task 3: Metafluxomics (LISBP)
For this task, incubations in microcosms will be carried out with formaldehyde enriched in 13C. The NMR and MS analyzes of the different isotopomers present in the microbial extracts will make it possible to trace the involved metabolic pathways and to measure the rates of transformation of the various intermediates of these pathways (fluxomics approach).
Task 4: Modeling (LaMP, LISBP)
The metabolic pathways corresponding to the biotransformation of formaldehyde will be integrated into an atmospheric chemistry model (CLEPS). Several scenarios will be taken into account to configure the model with transformation rates determined in task 3.

Task 1: Sampling and characterization of cloud water
• Construction of 5 cloud impactors. Three of them were already operational, two new are now (not foreseen in the initial plan).
• Use of a high volume rain collector and first rain collection (not initially planned).
• Collection of the aerosol phase; the number of microorganisms collected was found to be too low despite the air volumes sampled, this approach will be abandoned (not initially planned).
• Collection of the cloud water phase: several unsuccessful attempts in the fall of 2021. The period of November 2021 is expected to allow the collection of a high volume sample.
Task 2: Incubations and preparation of cellular extracts
• Purchase and installation of a tangential centrifuge (planned), now operational, test on rain and aerosol samples. The cell concentration factor is 3 to 4 times (also an exchange of the natural environment by artificial medium).
• Construction of 26 photo-bioreactors for incubations in microcosms (planned).
• Implementation and optimization of a protocol for the incubation in microcosms of a consortium of microbial strains in two contrasting atmospheric conditions (summer day, light, H2O2, 17 ° C vs winter night, without light ni, H2O2 5 ° C). The idea is to replace for this optimization step, the microflora of a real cloud, difficult to obtain during the COVID period, by a 'synthetic' consortium composed of 4 bacteria (2 Pseudomonas, 1 Sphingomonas, 1 Rhodococcus ) and 1 yeast (Dioszegia). These strains were isolated from the clouds at the Puy du Dôme site and are characteristic of this environment. The strains were studied separately and then in a consortium, the degradation rates of H2O2 and formaldehyde could be measured, the optimal incubation times for the samples required for task 3 and 4 («omics«) are therefore determined.
• The in-depth study of these strains made it possible to characterize the genome of the yeast Dioszegia, a genus little described in the literature (not planned).
Task 3: Metametabolomics and Metatranscriptomics
• In the absence of cloud samples, non-targeted LC-MS analyzes of the metabolome (not initially planned) had to be performed on rain and microbial cell samples (individual and consortium) to assess the threshold of sensitivity. detection of metabolites (established at 10x8 cells extracted).
• The study of the sensitivity threshold for extracting mRNA from microbial cells is ongoing (not initially planned).
Task 4: Metafluxomics
• The same samples (extracts from rain and microbial strains) were successfully analyzed by LC-MS in a targeted manner (amino acids, sugars, central metabolism intermediary). NMR analyzes were also attempted (not initially planned). However, the concentration of metabolites seems too low for this technique.

We have faced many challenges related to COVID19. Indeed we were not able to perform laboratory experiments for many months. Access to the puy de Dôme site for samples was also impossible. On the other hand, we recently had to deal with a timing issue for manufacturing two of the cloud impactors, which postponed the high volume, this postpone the samplings originally planned. All our experiments (incubations, meta-metabolomics, meta-fluxomics and meta-transcriptomics) being initially based on these cloud samples, we thoroughly revised our project and started alternative research routes. The idea is to keep the structure of the project with the different work packages, to work with microbial biomass representative of the atmosphere, and to change the nature of the samples:

1) Atmospheric samples: We have developed techniques for collecting high volume natural atmospheric samples: aerosols and rain.
2) Consortium of microbial strains (4 bacteria and 1 yeast) isolated from clouds: we have developed the incubation protocols in the microcosms.
For these two types of samples we tested the limits of detection of intracellular metabolites by LC-MS and NMR.
These alternative approaches, especially with a consortium of strains, constitute our plan B in case we fail to collect clouds within a timeframe compatible with the timing of the project. We have planned a collection campaign in November / December 2021; the conditions being favorable to the samples and having no more constraints related to COVID. We are aware of the delay, but we are arriving in optimal conditions for high volume cloud collection; however, the project may switch to plan B, especially with strains in the event of failure on cloud samples.

International
Revues à comité de lecture Article commun ICCF et GMGM, en collaboration avec le DOE Joint Genome Institute : F. Bringel, D. Jarrige, L. Eck, S. Haridas, M. Joly, J.M. Vyskocil, T. Nadalig, S. Vuilleumier, I. Grigoriev, P. Amato, A.M. Delort. A high-quality genome assembly of the cloud isolated basidiomycetous Dioszegia hungarica PDD-24b-2.
Communications (conférence) 1. J.M. Vyskocil, M. Joly, P. Amato, F. Rossi, C. Jousse, L. Deguillaume, A.M. Delort (ICCF +LaMP). Meta-Omics Analyses Of Cloud Microbial Metabolism Of C1 Compounds. European Meeting on Environmental Chemistry (EMEC21), 30 Nov –3 Dec 2021, Novi Sad, Serbia.

France

Communications (conférence) 1. F. Bringel, Y. Louhichi, T. Nadalig, S. Vuilleumier, J.-C. Portais, L. Deguillaume, P. Amato, A.M. Delort. (présentation poster) Metatranscriptomic exploration of the role of methylotrophic microbial communities in cloud chemistry. Journée métatranscriptomique complexe: de la dual transcriptomique à la métatranscriptomique: challenges et perspectives”, Nancy France 10 décembre 2019

Actions de diffusion
Articles de vulgarisation
1. A.M. Delort et L. Deguillaume (ICCF + LaMP)
« Dans l'intimité des nuages »,
Sciences et Avenir, sept 2021, N° 895.
2. Sixième Sciences : shows.acast.com/sixieme-science/episodes/ce-laboratoire-unique-au-monde-analyse-les-nuages
3. L. Deguillaume : Article INSU CNRS www.insu.cnrs.fr/fr/cnrsinfo/150-ans-de-lopgc-150-ans-de-donnees-sur-le-climat



Liste des publications monopartenaires (impliquant un seul partenaire)
International
Revues à comité de lecture 1. Millard P, Sokol S, Kohlstedt M, Wittmann C, Létisse F, Lippens G, Portais JC. IsoSolve: An Integrative Framework to Improve Isotopic Coverage and Consolidate Isotopic Measurements by Mass Spectrometry and/or Nuclear Magnetic Resonance. Anal Chem. 2021 Jul 13;93(27):9428-9436. doi: 10.1021/acs.analchem.1c01064.

Communications (conférence)
1. A.M. DELORT (conférence invitée) « The cloud microbiota:
Microorganisms - H2O2 interactions »
2. From Interactions To Global Environmental Challenges, Israel's Microbial Ecology Meeting (Visioconference), 26-28 avril 2021.
4. A.M. DELORT (conference invitée) « The cloud microbiota: Microorganisms - H2O2 interactions »
5. European Meeting on Environmental Chemistry (EMEC21), 30 Nov –3 Dec 2021, Novi Sad, Serbia.

France

Communications (conférence)
1. AM DELORT, ICCF (conférence invitée) « Quel rôle des microorganismes dans la chimie des nuages ? le cas de H2O2. »
Atelier LEFE-AEROCLIM, 20-22 octobre 2021, Lille

Actions de diffusion Articles de vulgarisation
1. A.M. Delort, ICCF, « La chimie des nuages » dans le Livre « Etonnante chimie », CNRS éditions (EAN13 : 9782271136527), avril 2021.
2. Conférences de vulgarisation 1. A.M. Delort, ICCF, Présentation du livre « Etonnante chimie », 29 sept 2021, Maison de la chimie, Paris

Clouds play a major role in atmospheric chemical reactivity. They are complex multiphase environments of gas, liquid and solid particles. In the last decade, considerable progress has been made in understanding the reactivity of these multiphase systems, notably with the discovery of active microbial communities potentially involved in organic matter transformations in clouds. Nonetheless, nighttime atmospheric chemistry remains poorly understood, especially since biological activity may drive the chemical transformations at night. The role of microbial activity needs to be considered in numerical models of cloud chemistry. To date, no numerical models have taken into account these biological processes.

The objective of METACLOUD is to improve the fundamental understanding of the cloud system, including its chemistry and biodiversity. More specifically, we want to assess the potential role of microbial metabolism in C1 chemistry in clouds, particularly at night. This will be achieved by: i) obtaining novel and detailed data on cloud microbiome metabolic networks and their modulations in contrasting atmospheric scenarios, in particular with detailed "meta-fluxome maps" to reveal the dynamics of the system; ii) integrating this new knowledge, in particular biodegradation rates of C1 compounds, into new explicit numerical models of cloud multiphase chemistry.

This project focuses on C1 compounds, the major end products of radical chemistry, and in particularly, on formaldehyde. Formaldehyde is found at highly concentrations in clouds (microM) and is at the intersection of several metabolic pathways in methylotrophic microorganisms.

Large cloud water samples will be collected from the puy de Dôme observatory (France), an international reference site for atmospheric research, using cloud droplet collectors constructed in 2018. The chemical content and microbial biodiversity will be characterized. After tangential filtration concentration, the endogenous microorganisms will be incubated in microcosms. These microcosms were designed to mimic contrasted "summer day" (light, H2O2, 17°C) or "winter night" (dark, no H2O2, 5°C) scenarios, representing reference scenarios of atmospheric chemistry. The behaviour of the microbial communities will be assessed by transcriptomics and metabolomics under these two atmospheric scenarios. The integration of these "omics" data will give a completely new "metabolic network map" that will be exploited to validate the Meta-fluxome map of C1 compounds determined using 13C-formaldehyde. Metabolic fluxes will be measured from 13C-labelled compounds and the transformation rates implemented, along with the corresponding pathways, in a newly developed cloud chemistry numerical model.

This project is ambitious and innovative both for its integrative, multidisciplinary concept ("biogeochemistry" of clouds, integration of biological data in atmospheric chemistry models) and the proposed methodologies (meta-fluxomics performed on a whole ecosystem is particularly new).
The originality of the project relies on collaboration between LaMP and ICCF one of the few groups worldwide working on cloud microorganisms and their implication in atmospheric chemistry. It also benefits from the GMGM team with recognized expertise of microbial C1 metabolism and from the LISBP team’s unique expertise in developing fluxomics tools. The interdisciplinary nature of the project, linking cloud chemistry, modelling, and the study of the microbial behavior by "omics" approaches, is also, to the best of our knowledge, absolutely unique worldwide.

Results will be disseminated in high impact factor reviews and journals, communications in major international conferences, an organized international workshop, communications to the general public, active participation in education (summer schools, "Les cours d’eau H2O" for scholars), and production of a dedicated open source software.