Methods to Investigate the Global Atmospheric Microbiome

The interplay between microbes and atmospheric physical and chemical conditions is an open field of research that can only be fully addressed using multidisciplinary approaches. The lack of coordinated efforts to gather data at representative temporal and spatial scales limits aerobiology to help understand large scale patterns of global microbial biodiversity and its causal relationships with the environmental context. This paper presents the sampling strategy and analytical protocols developed in order to integrate different fields of research such as microbiology, –omics biology, atmospheric chemistry, physics and meteorology to characterize atmospheric microbial life. These include control of chemical and microbial contaminations from sampling to analysis and identification of experimental procedures for characterizing airborne microbial biodiversity and its functioning from the atmospheric samples collected at remote sites from low cell density environments. We used high-volume sampling strategy to address both chemical and microbial composition of the atmosphere, because it can help overcome low aerosol and microbial cell concentrations. To account for contaminations, exposed and unexposed control filters were processed along with the samples. We present a method that allows for the extraction of chemical and biological data from the same quartz filters. We tested different sampling times, extraction kits and methods to optimize DNA yield from filters. Based on our results, we recommend supplementary sterilization steps to reduce filter contamination induced by handling and transport. These include manipulation under laminar flow hoods and UV sterilization. In terms of DNA extraction, we recommend a vortex step and a heating step to reduce binding to the quartz fibers of the filters. These steps have led to a 10-fold increase in DNA yield, allowing for downstream omics analysis of air samples. Based on our results, our method can be integrated into pre-existing long-term monitoring field protocols for the atmosphere both in terms of atmospheric chemistry and biology. We recommend using standardized air volumes and to develop standard operating protocols for field users to better control the operational quality

Structuring factors of microbial communities in the atmospheric boundary layer

La couche limite planétaire est la couche atmosphérique la plus basse qui est en interaction directe et constante avec les surfaces terrestres et marines sur lesquelles se concentrent les activités humaines, les cultures et divers écosystèmes. Comprendre l’origine de sa composition à la fois chimique et microbiologique est fondamental dans notre étude approfondie de la biosphère. Alors que les microorganismes de la couche limite planétaire – retrouvés jusqu’à 106 cellules par mètre cube d’air – semblent varier significativement à l’échelle spatiale et temporelle en termes de concentration et de diversité, ils restent largement méconnus. L’objectif principal de cette thèse est de comprendre comment se structurent les communautés microbiennes dans la troposphère, et en particulier dans la couche limite planétaire, ainsi que d’identifier les facteurs de contrôle majeurs. En travaillant sur des échantillons collectés pendant plusieurs semaines sur neuf sites répartis sur la planète, et en utilisant les technologies de séquençage ADN haut-débit, nous avons étudié la composition taxonomique et fonctionnelle des communautés microbiennes de la phase gazeuse et solide de l’atmosphère (c’est-à-dire non associés aux nuages).Nos premiers résultats sur la taxonomie des communautés microbiennes révèlent que les surfaces proches des sites sont les contributeurs principaux de distribution des communautés microbiennes atmosphériques, malgré l’occurrence potentielle du transport longue-distance des microorganismes atmosphériques. Egalement, les conditions météorologiques combinées à la diversité des surfaces locales terrestres ou océaniques jouent un rôle important dans la variation temporelle de la structure des communautés microbiennes de la couche limite planétaire. Une deuxième étude nous a permis d’étudier davantage la variation temporelle des communautés microbiennes atmosphériques sur un site continental montagneux en France (1465 m d’altitude) sur une année complète. Cette étude révèle l’importance des conditions de surface des paysages aux alentours dans la composition taxonomique des communautés atmosphériques. L’évolution au cours de l’année des terres agricoles et de la végétation, qui composaient en majeure partie le paysage du site, était responsable du changement temporel observé dans la composition taxonomique des communautés microbiennes atmosphériques. Finalement, nous avons étudié la composition fonctionnelle des communautés microbiennes de la couche limite planétaire afin d’identifier si les conditions physiques et chimiques de l’atmosphère jouaient un rôle dans la sélection ou adaptation microbienne des microorganismes atmosphériques. L’analyse comparative de données métagénomiques ne révèle pas de signature atmosphérique spécifique du potentiel fonctionnel des communautés microbiennes atmosphériques. La composition fonctionnelle semble avant tout liée aux écosystèmes locaux. Toutefois, nous avons observé que les champignons étaient plus dominants relativement aux bactéries dans l’air comparativement aux autres écosystèmes. Ce résultat suggère un processus de sélection des champignons durant l’aérosolisation et/ou le transport aérien. Les champignons pourraient survivre davantage l’aérosolisation et le transport aérien comparativement aux bactéries du fait de leur résistance naturelle aux conditions physiques stressantes de l’atmosphère. Nos résultats ont apporté une meilleure compréhension des facteurs déterminants (c’est-à-dire les surfaces locales, les sources distantes, les conditions météorologiques locales, les conditions physiques stressantes de l’atmosphère) et de leur contribution dans la structuration des communautés microbiennes de la couche limite atmosphérique. Nos investigations constituent une base importante pour de nouvelles études sur la prévision et le contrôle des communautés microbiennes atmosphériques, afin de répondre à des questions majeures dans les domaines de la santé publique et de l’agronomie.Up to 106 microbial cells per cubic meter are found in suspension in the planetary boundary layer, the lowest part of the atmosphere. Direct influences of the planetary boundary layer on humans, crops and diverse ecosystems like soils and oceans make the full understanding of its composition, both chemical and microbiological, of utmost importance. While microbial communities of the planetary boundary layer vary significantly at different temporal and spatial scales, they remain largely unexplored. The main goal of this thesis was to understand how airborne microbial communities are structured in the troposphere with special emphasis on the planetary boundary layer and to identify their main controlling factors. We investigated both the taxonomic and functional composition of airborne microbial communities in the dry phase (i.e. not cloud-associated) over time at nine different geographical sites around the world using high throughput sequencing technologies.Our investigation that focused on microbial taxonomy showed that local landscapes were the main contributors to the global distribution of airborne microbial communities despite the potential occurrence of long-range transport of airborne microorganisms. We also observed that meteorology and the diversity of the surrounding landscapes played major roles in the temporal variation of the microbial community structure in the planetary boundary layer. We further explored the temporal variation of airborne microbial communities at a continental and mountainous site in France (1465 m above sea level) over a full-year. This study demonstrated the importance of the surface conditions (i.e. vegetation, snow cover etc.) of the surrounding landscapes on the taxonomic composition of airborne microorganisms. The seasonal changes in agricultural and vegetated areas, which represented a significant part of the site’s surrounding landscape, were correlated to the shifts in the taxonomic composition of airborne microbial communities during the year. Finally, we investigated the functional composition of microbial communities of the planetary boundary layer to identify whether the physical and chemical conditions of the atmosphere played a role in selection or microbial adaptation of airborne microorganisms. The comparative metagenomic analysis did not show a specific atmospheric signature in the functional potential of airborne microbial communities. To the contrary, their functional composition was mainly correlated to the underlying ecosystems. However, we also showed that fungi were more dominant relatively to bacteria in air as compared to other (planetary bound) ecosystems. This result suggested a selective process for fungi during aerosolization and/or aerial transport and that fungi might likely survive aerosolization and/or aerial transport better than bacteria due to their innate resistance to stressful physical conditions (i.e. UV radiation, desiccation etc.). Our results provide a clearer understanding of the factors (i.e. surrounding landscapes, distant sources, local meteorology, and stressful physical atmospheric conditions) that control the distribution of microbial communities in the atmospheric boundary layer. Our investigations provide a basis for further studies on the prediction and even control of airborne microbial communities that would be of interest for public health and agriculture

Facteurs de structuration des communautés microbiennes de la couche limite atmosphérique

Up to 106 microbial cells per cubic meter are found in suspension in the planetary boundary layer, the lowest part of the atmosphere. Direct influences of the planetary boundary layer on humans, crops and diverse ecosystems like soils and oceans make the full understanding of its composition, both chemical and microbiological, of utmost importance. While microbial communities of the planetary boundary layer vary significantly at different temporal and spatial scales, they remain largely unexplored. The main goal of this thesis was to understand how airborne microbial communities are structured in the troposphere with special emphasis on the planetary boundary layer and to identify their main controlling factors. We investigated both the taxonomic and functional composition of airborne microbial communities in the dry phase (i.e. not cloud-associated) over time at nine different geographical sites around the world using high throughput sequencing technologies.Our investigation that focused on microbial taxonomy showed that local landscapes were the main contributors to the global distribution of airborne microbial communities despite the potential occurrence of long-range transport of airborne microorganisms. We also observed that meteorology and the diversity of the surrounding landscapes played major roles in the temporal variation of the microbial community structure in the planetary boundary layer. We further explored the temporal variation of airborne microbial communities at a continental and mountainous site in France (1465 m above sea level) over a full-year. This study demonstrated the importance of the surface conditions (i.e. vegetation, snow cover etc.) of the surrounding landscapes on the taxonomic composition of airborne microorganisms. The seasonal changes in agricultural and vegetated areas, which represented a significant part of the site’s surrounding landscape, were correlated to the shifts in the taxonomic composition of airborne microbial communities during the year. Finally, we investigated the functional composition of microbial communities of the planetary boundary layer to identify whether the physical and chemical conditions of the atmosphere played a role in selection or microbial adaptation of airborne microorganisms. The comparative metagenomic analysis did not show a specific atmospheric signature in the functional potential of airborne microbial communities. To the contrary, their functional composition was mainly correlated to the underlying ecosystems. However, we also showed that fungi were more dominant relatively to bacteria in air as compared to other (planetary bound) ecosystems. This result suggested a selective process for fungi during aerosolization and/or aerial transport and that fungi might likely survive aerosolization and/or aerial transport better than bacteria due to their innate resistance to stressful physical conditions (i.e. UV radiation, desiccation etc.). Our results provide a clearer understanding of the factors (i.e. surrounding landscapes, distant sources, local meteorology, and stressful physical atmospheric conditions) that control the distribution of microbial communities in the atmospheric boundary layer. Our investigations provide a basis for further studies on the prediction and even control of airborne microbial communities that would be of interest for public health and agriculture.La couche limite planétaire est la couche atmosphérique la plus basse qui est en interaction directe et constante avec les surfaces terrestres et marines sur lesquelles se concentrent les activités humaines, les cultures et divers écosystèmes. Comprendre l’origine de sa composition à la fois chimique et microbiologique est fondamental dans notre étude approfondie de la biosphère. Alors que les microorganismes de la couche limite planétaire – retrouvés jusqu’à 106 cellules par mètre cube d’air – semblent varier significativement à l’échelle spatiale et temporelle en termes de concentration et de diversité, ils restent largement méconnus. L’objectif principal de cette thèse est de comprendre comment se structurent les communautés microbiennes dans la troposphère, et en particulier dans la couche limite planétaire, ainsi que d’identifier les facteurs de contrôle majeurs. En travaillant sur des échantillons collectés pendant plusieurs semaines sur neuf sites répartis sur la planète, et en utilisant les technologies de séquençage ADN haut-débit, nous avons étudié la composition taxonomique et fonctionnelle des communautés microbiennes de la phase gazeuse et solide de l’atmosphère (c’est-à-dire non associés aux nuages).Nos premiers résultats sur la taxonomie des communautés microbiennes révèlent que les surfaces proches des sites sont les contributeurs principaux de distribution des communautés microbiennes atmosphériques, malgré l’occurrence potentielle du transport longue-distance des microorganismes atmosphériques. Egalement, les conditions météorologiques combinées à la diversité des surfaces locales terrestres ou océaniques jouent un rôle important dans la variation temporelle de la structure des communautés microbiennes de la couche limite planétaire. Une deuxième étude nous a permis d’étudier davantage la variation temporelle des communautés microbiennes atmosphériques sur un site continental montagneux en France (1465 m d’altitude) sur une année complète. Cette étude révèle l’importance des conditions de surface des paysages aux alentours dans la composition taxonomique des communautés atmosphériques. L’évolution au cours de l’année des terres agricoles et de la végétation, qui composaient en majeure partie le paysage du site, était responsable du changement temporel observé dans la composition taxonomique des communautés microbiennes atmosphériques. Finalement, nous avons étudié la composition fonctionnelle des communautés microbiennes de la couche limite planétaire afin d’identifier si les conditions physiques et chimiques de l’atmosphère jouaient un rôle dans la sélection ou adaptation microbienne des microorganismes atmosphériques. L’analyse comparative de données métagénomiques ne révèle pas de signature atmosphérique spécifique du potentiel fonctionnel des communautés microbiennes atmosphériques. La composition fonctionnelle semble avant tout liée aux écosystèmes locaux. Toutefois, nous avons observé que les champignons étaient plus dominants relativement aux bactéries dans l’air comparativement aux autres écosystèmes. Ce résultat suggère un processus de sélection des champignons durant l’aérosolisation et/ou le transport aérien. Les champignons pourraient survivre davantage l’aérosolisation et le transport aérien comparativement aux bactéries du fait de leur résistance naturelle aux conditions physiques stressantes de l’atmosphère. Nos résultats ont apporté une meilleure compréhension des facteurs déterminants (c’est-à-dire les surfaces locales, les sources distantes, les conditions météorologiques locales, les conditions physiques stressantes de l’atmosphère) et de leur contribution dans la structuration des communautés microbiennes de la couche limite atmosphérique. Nos investigations constituent une base importante pour de nouvelles études sur la prévision et le contrôle des communautés microbiennes atmosphériques, afin de répondre à des questions majeures dans les domaines de la santé publique et de l’agronomie

Microbial Ecology of the Planetary Boundary Layer

International audienceAerobiology is a growing research area that covers the study of aerosols with a biological origin from the air that surrounds us to space through the different atmospheric layers. Bioaerosols have captured a growing importance in atmospheric process-related fields such as meteorology and atmospheric chemistry. The potential dissemination of pathogens and allergens through the air has raised public health concern and has highlighted the need for a better prediction of airborne microbial composition and dynamics. In this review, we focused on the sources and processes that most likely determine microbial community composition and dynamics in the air that directly surrounds us, the planetary boundary layer. Planetary boundary layer microbial communities are a mix of microbial cells that likely originate mainly from local source ecosystems (as opposed to distant sources). The adverse atmospheric conditions (i.e., UV radiation, desiccation, presence of radicals, etc.) might influence microbial survival and lead to the physical selection of the most resistant cells during aerosolization and/or aerial transport. Future work should further investigate how atmospheric chemicals and physics influence microbial survival and adaptation in order to be able to model the composition of planetary boundary layer microbial communities based on the surrounding landscapes and meteorology

Microbial functional signature in the atmospheric boundary layer

International audienceMicroorganisms are ubiquitous in the atmosphere, and some airborne microbial cells were shown to be particularly resistant to atmospheric physical and chemical conditions (e.g., ultraviolet – UV – radiation, desiccation and the presence of radicals). In addition to surviving, some cultivable microorganisms of airborne origin were shown to be able to grow on atmospheric chemicals in laboratory experiments. Metagenomic investigations have been used to identify specific signatures of microbial functional potential in different ecosystems. We conducted a comparative metagenomic study on the overall microbial functional potential and specific metabolic and stress-related microbial functions of atmospheric microorganisms in order to determine whether airborne microbial communities possess an atmosphere-specific functional potential signature as compared to other ecosystems (i.e., soil, sediment, snow, feces, surface seawater etc.). In the absence of a specific atmospheric signature, the atmospheric samples collected at nine sites around the world were similar to their underlying ecosystems. In addition, atmospheric samples were characterized by a relatively high proportion of fungi. The higher proportion of sequences annotated as genes involved in stress-related functions (i.e., functions related to the response to desiccation, UV radiation, oxidative stress etc.) resulted in part from the high concentrations of fungi that might resist and survive atmospheric physical stress better than bacteria

Geographical distribution of bacterial communities in the atmosphere and controlling factors

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Holobiont responses of mesophotic precious red coral Corallium rubrum to thermal anomalies

Abstract Marine heat waves (MHWs) have increased in frequency and intensity worldwide, causing mass mortality of benthic organisms and loss of biodiversity in shallow waters. The Mediterranean Sea is no exception, with shallow populations of habitat-forming octocorals facing the threat of local extinction. The mesophotic zone, which is less affected by MHWs, may be of ecological importance in conservation strategies for these species. However, our understanding of the response of mesophotic octocoral holobionts to changes in seawater temperature remains limited. To address this knowledge gap, we conducted a study on an iconic Mediterranean octocoral, the red coral Corallium rubrum sampled at 60 m depth and 15 °C. We exposed the colonies to temperatures they occasionally experience (18 °C) and temperatures that could occur at the end of the century if global warming continues (21 °C). We also tested their response to extremely cold and warm temperatures (12 °C and 24 °C). Our results show a high tolerance of C. rubrum to a two-month long exposure to temperatures ranging from 12 to 21 °C as no colony showed signs of tissue loss, reduced feeding ability, stress-induced gene expression, or disruption of host-bacterial symbioses. At 24 °C, however, we measured a sharp decrease in the relative abundance of Spirochaetaceae, which are the predominant bacterial symbionts under healthy conditions, along with a relative increase in Vibrionaceae. Tissue loss and overexpression of the tumor necrosis factor receptor 1 gene were also observed after two weeks of exposure. In light of ongoing global warming, our study helps predict the consequences of MHWs on mesophotic coralligenous reefs and the biodiversity that depends on them

Comparaison des populations bactériennes en suspension dans l'air déterminées par échantillonnage passif et actif au puy de Dôme, France

International audienceBioaerosols have impacts on atmospheric processes, as well as ecosystem and human health. Common bioaerosol collection methods include impaction, liquid impingement, filtration, and electrostatic precipitation. These methods are used by active samplers that require an air mover and power, but this requirement can also represent a major constraint in field studies. Alternatively, passive samplers do not require power and can operate for long times. In this study, the Rutgers Electrostatic Passive Sampler (REPS), which captures particles by electrostatic attraction and gravitational settling, was deployed at the summit of puy de Dôme (1465 m a.s.l., France) alongside an active PM10 sampler (~1000 L min-1) collecting aerosols on a quartz fiber filter. The diversity of the airborne bacteria captured by both samplers across six weekly sampling periods was examined by 16S rRNA gene amplicon sequencing. The dominant phyla observed by both samplers were similar and included Firmicutes, Proteobacteria, and Actinobacteriota. Overall, 12 to 63% of the total bacterial richness at the genus level was shared between the two samplers, depending upon a paired sample, i.e., sampling week. The PM10 sampler and REPS detected the same dominant genera, including Lysinibacillus and Sphingomonas, although their relative abundances for each paired sampler varied. The observed bacterial richness and diversity, as estimated through Shannon's and Simpson's indexes, were significantly greater in REPS samples compared to the PM10 samples. The results suggest that REPS could be used for simple and convenient sampling of bioaerosols, especially in remote areas and other locations with limited power access

Global airborne microbial communities controlled by surrounding landscapes and wind conditions

International audienceAbstractThe atmosphere is an important route for transporting and disseminating microorganisms over short and long distances. Understanding how microorganisms are distributed in the atmosphere is critical due to their role in public health, meteorology and atmospheric chemistry. In order to determine the dominant processes that structure airborne microbial communities, we investigated the diversity and abundance of both bacteria and fungi from the PM10 particle size (particulate matter of 10 micrometers or less in diameter) as well as particulate matter chemistry and local meteorological characteristics over time at nine different meteorological stations around the world. The bacterial genera Bacillus and Sphingomonas as well as the fungal species Pseudotaeniolina globaosa and Cladophialophora proteae were the most abundant taxa of the dataset, although their relative abundances varied greatly based on sampling site. Bacterial and fungal concentration was the highest at the high-altitude and semi-arid plateau of Namco (China; 3.56 × 106 ± 3.01 × 106 cells/m3) and at the high-altitude and vegetated mountain peak Storm-Peak (Colorado, USA; 8.78 × 104 ± 6.49 × 104 cells/m3), respectively. Surrounding ecosystems, especially within a 50 km perimeter of our sampling stations, were the main contributors to the composition of airborne microbial communities. Temporal stability in the composition of airborne microbial communities was mainly explained by the diversity and evenness of the surrounding landscapes and the wind direction variability over time. Airborne microbial communities appear to be the result of large inputs from nearby sources with possible low and diluted inputs from distant sources

Sequencing Depth Has a Stronger Effect than DNA Extraction on Soil Bacterial Richness Discovery

Although Next-Generation Sequencing techniques have increased our access to the soil microbiome, each step of soil metagenomics presents inherent biases that prevent the accurate definition of the soil microbiome and its ecosystem function. In this study, we compared the effects of DNA extraction and sequencing depth on bacterial richness discovery from two soil samples. Four DNA extraction methods were used, and sequencing duplicates were generated for each DNA sample. The V3–V4 region of the 16S rRNA gene was sequenced to determine the taxonomical richness measured by each method at the amplicon sequence variant (ASV) level. Both the overall functional richness and antibiotic resistance gene (ARG) richness were evaluated by metagenomics sequencing. Despite variable DNA extraction methods, sequencing depth had a greater influence on bacterial richness discovery at both the taxonomical and functional levels. Sequencing duplicates from the same sample provided access to different portions of bacterial richness, and this was related to differences in the sequencing depth. Thus, the sequencing depth introduced biases in the comparison of DNA extraction methods. An optimisation of the soil metagenomics workflow is needed in order to sequence at a sufficient and equal depth. This would improve the accuracy of metagenomic comparisons and soil microbiome profiles