Slavomíra Husárová

Mária Matulová
email: maria.matulova@savba.sk

Peter Capek

Martine Sancelme

Anne-Marie Delort

Production of exopolysaccharides by Bacillus sp. 3B6 on various saccharide sources

ACKNOWLEDGEMENT

I would like to thank all my friends for caring they provided.

Last but not least I would like to thank my parents and my husband Branislav for the enormous support, patience, and love.

SUMMARY

The discovery of the presence of living and active microorganisms in clouds is rather recent. Their survival ability under such hostile environment and their potential role in atmospheric processes are open questions in the scientific community. The objective of this thesis was to bring some answers to this recent topic. The main goal of PhD thesis was to study the metabolism of Bacillus sp. 3B6, isolated from cloud water, by in situ NMR on different substrates. Substrates represented two classes: C1 compounds (formaldehyde and methanol) and carbohydrates.

Formaldehyde and methanol have been chosen since they represent major atmospheric volatile organic carbons (VOCs) and carbohydrates serve as a source of carbon for bacteria. Many of them are present in the atmosphere. Metabolism of chosen C1 compounds by Bacillus sp. 3B6 was compared with other three bacterial strains isolated from cloud water: Frigoribacterium sp., Pseudomonas graminis, and P. syringae. The metabolic routes were similar to photodegradation pathways and in a particular case C3 compounds were formed from a C1 compound In addition, a comparison of obtained biodegradation rates with rates related to the reactivity of organic species with free radicals • OH (daytime chemistry) and NO 3 • (night-time chemistry) showed that the microbiological oxidation of organic compounds has to be considered as an alternative route to radical chemistry in cloud water especially at night. Carbohydrate metabolism study of Bacillus sp. 3B6 followed two axes: basic metabolism of chosen sugars as carbon sources and exopolymeric substances (EPSs) production. Two substrate (glucose and SUMMARY sucrose) afforded different types of EPSs. On sucrose the production of levan, fructooligosaccharides of levan and inulin type, and dianhydride of fructose DFA IV were identified in the incubation medium (IM). IM on glucose afforded a glycoprotein precipitate, 1,6-linked α-galactan, and some polyhydroxyalkanoate (PHAs) structure of which could not be identified from in situ NMR spectra. In fractions afforded by gradual precipitation from IM 3-hydroxybutyrate, butyric acid, glycerol, and ester of glycerols were also identified. Besides biotechnological application, identified EPSs, could have also an impact on the atmospheric environment. EPSs, which are highly functionalised molecules could influence the cloud condensation nuclei (CCN) formation by interacting with water, or due to their ability to change the "wettability" of the cell surface. In this latter case they will act by changing the CCN properties of bio-aerosols.

ABSTRAKT

Prítomnosť živých a aktívnych mikroorganizmov v oblakoch bola objavená len pomerne nedávno. Ich schopnosť prežívať v tomto nehostinnom prostredí, ako aj ich potenciálna úloha v atmosférických procesoch nie sú doteraz uspokojivo vysvetlené. Cieľom prezentovanej dizertačnej práce bolo prispieť k riešeniu tejto témy prostredníctvom štúdia metabolizmu baktérie Bacillus sp. 3B6 izolovanej z oblačnej vody. Jej metabolizmus bol študovaný vo väčšine prípadov priamou analýzou inkubačných zmesí získaných na rôznych substrátoch prostredníctvom in situ NMR spektroskopie. Použité substráty možno rozdeliť do dvoch tried: i) C1 zlúčeniny (metanol, formaldehyd) ako hlavní znečisťovatelia ovzdušia, ii) rôzne sacharidy, ktoré slúžia ako zdroj uhlíka pre baktérie a ktorých prítomnosť v atmosfére bola dokázaná. Metabolizmus metanolu a formaldehydu u Bacillus sp. 3B6 bol porovnaný s ďalšími tromi bakteriálnymi kmeňmi izolovanými z oblačnej vody: Frigoribacterium sp., Pseudomonas graminis a P. syringae. Metabolické dráhy boli podobné fotodegradácii a to hlavne v prípade tvorby C3 zlúčenín z C1 substrátu.

Porovnanie takto získaných rýchlostí biodegradácií s rýchlosťami reaktivít voľných radikálov • OH (denné chemické procesy) a NO 3 • (nočné chemické procesy) ukázalo, že mikrobiologické oxidácie organických látok, musia byť považované za alternatívny proces k radikálovej chémii prebiehajúcej v oblačnej vode prevažne v noci. Štúdium metabolizmu sacharidov u baktérie Bacillus sp. 3B6 sledovalo dve samostatné experimentálne línie: i) základný metabolizmus cukrov zvolených ako zdroj uhlíka, ii) produkcia exopolymérnych látok (EPSs). Rast baktérie na dvoch rôznych sacharidových substrátoch (glukóza a sacharóza) viedol k produkcii rôznych typov EPSs. Analýzou inkubačného média (IM) na sacharóze boli identifikované: polysacharid levan, fruktooligosaccharidy levanového a inulínového typu a dianhydrid fruktózy DFA IV. V IM na glukóze boli identifikované: glykoproteín, 1,6-α-galaktán a polyhydroxyalkanoáty (PHA), ktorých štruktúru nebolo možné z in situ NMR spektier bližšie identifikovať. Vo frakciách, ktoré vznikli postupným vyzrážaním nerozpustných zložiek zmesi z IM, bol identifikovaný 3-hydroxybutyrát, kyselina maslová, glycerol a estery glycerolu. Okrem biotechnologických aplikácií, identifikované EPSs môžu zohrávať úlohu v atmosférických procesoch. EPSs, ako vysoko funkcionalizované molekuly, by mohli mať vplyv na tvorbu oblačných kondenzačných jadier (CCN) prostredníctvom ich interakcií s vodou, alebo v dôsledku ich schopnosti meniť zmáčavosť povrchu buniek. V druhom prípade sa tento efekt deje zmenou CCN vlastností bioaerosólov. Table 3. Ambient concentrations of saccharides, sugar-alcohols, and vanillin compounds during the haze and none-haze days.. Table 4. Lifetimes of some dicarboxylic acids present in the atmosphere.

LIST OF FIGURES

LIST OF SCHEMES

LIST OF TABLES

Table 5. Estimated rates of photo-and biodegradation in artificial cloud water at 17 °C. Table 6. The summary of published EPS structure elucidation.

INTRODUCTION

Clouds represent an extreme environment for living cells, characterised by low temperatures, acidic pH, UV radiation, and lack of nutriments.

Recent studies showed that microbial communities are present and metabolically active there and provoked new investigations of their structure and functioning. This field of the research is quasi virgin and innovative. Clouds are a heterogeneous medium (solid, liquid, and gaseous phases) containing a very complex mixture of organic and inorganic compounds, and can be considered as a large reactor where many chemical reactions take place. Up to now only radical chemistry induced by solar light has been taken into account to describe atmospheric chemistry. However, microorganisms are potentially very active biocatalysts and the proposed project would like to find the answers to some questions concerning their potential role in atmospheric chemistry as an alternative to photochemical transformations, and more generally their implication in atmospheric processes.

The presented PhD work has been carried out in the frame work of a cotutelle and of a collaborative work between a French team (SEESIB: Synthèse et Etude de Systèmes à Interêt Biologique in Clermont-Ferrand) and a Slovak team (Institute of Chemistry in Bratislava).

INTRODUCTION

The studied bacteria were isolated from cloud water collected at the summit of the Puy de Dôme in free troposphere by the French team, a pioneer in this field. This site is a European place for cloud studies.

The main focus of this PhD work was to study the metabolism of Bacillus sp. 3B6 in detail, as it is present in many atmospheric samples. These metabolic studies have been performed using a main tool, Nuclear Magnetic Resonance (NMR, which is the domain of expertise of both French and Slovak teams). Two types of substrates have been studied: carbohydrates and C1 compounds, both of them are potentially present in cloud waters.

This study is quite interdisciplinary and requires the knowledge in various areas. This is why the bibliography presented here gives the state of the art of different fields involved in this work: clouds, microbial community in clouds, NMR in microbiology and metabolism studies; with the main focus on Bacillus species.

BIBLIOGRAPHIC STUDY

2.1

Microorganisms in Clouds: Their Role in Atmospheric Processes

Life Cycle of Microorganisms via the

Atmosphere

Microorganisms are ubiquitous on the Earth including water, soil and vegetation, and they can experience a life cycle via the atmosphere as

shown in Figure 1. The first step consists in the aerosolisation of microorganisms into the air. Detailed mechanisms involved in this process remain still unknown. However, some hypotheses have been highlighted:

In the case of water, especially sea water, microorganisms are ejected by bubbling. Concerning soil, wind is obviously the major factor that brings microorganisms in the air. Bacteria can be present on soil particulates and blown up as dust. Spores of some fungi present in conidia can be easily aerosolised by wind depending on meteorological factors [START_REF] Jones | The effects of meteorological factors on atmospheric bioaerosol concentrations -a review[END_REF]. Finally, microorganisms highly present in the phyllosphere can be ejected in the air by the action of wind depending on factors such as humidity and temperature, which allow plant leaves to dry and produce particles. Aerosols can also result from fragmentation of dried biofilms at the leaf surface where bacteria are embedded [START_REF] Lindemann | Aerial Dispersal of Epiphytic Bacteria over Bean Plants[END_REF][START_REF] Hirano | Bacteria in the leaf ecosystem with emphasis on pseudomonas syringae -a pathogen, ice nucleus, and epiphyte[END_REF]. Once in the atmosphere, microorganisms are exposed to hostile conditions including solar radiation (especially UV), desiccation when they are in the air, cold, interaction with oxidants (H 2 O 2 , iron, etc…), low quantity of nutrients, acidity, difference of osmolarity. Microorganism physiologies must be thus adapted to the life in this rather extreme environment. They can be incorporated into cloud droplets by nucleation scavenging as they have cloud condensation nuclei (CCN) or ice nuclei (IN) potential and they come back to the Earth as a part of raindrops and snowflakes by washout processes of the air or as a possible result of ice nucleation processes (Möhler et al., 2007;[START_REF] Morris | Ice nucleation active bacteria and their potential role in precipitation[END_REF][START_REF] Sun | Atmospheric organic and bio-aerosols as cloud condensation nuclei (CCN): A review[END_REF].

The precise mechanisms involved in CCN and IN processes are still under controversy in the scientific community (Möhler et al., 2007). The transport of microorganisms in the atmosphere has already been simulated as well as the factors influencing their residence time and the CCN and IN activities [START_REF] Burrows | Bacteria in the global atmosphere -Part 2: modeling of emissions and transport between different ecosystems[END_REF].

The revelation of living microorganisms integrated in cloud water recently initiated a fundamental and new scientific question by Ariya's group [START_REF] Ariya | Microbiological degradation of atmospheric organic compounds[END_REF][START_REF] Ariya | New Directions: The role of bioaerosols in atmospheric chemistry and physics[END_REF][START_REF] Côté | Microbial and "de novo" transformation of dicarboxylic acids by three airborne fungi[END_REF] and our group [START_REF] Amato | Microbial population in cloud water at the Puy de Dôme: Implications for the chemistry of clouds[END_REF](Amato et al., , 2007a)): Do microorganisms play a role in atmospheric processes?

Number, Type, Activity, and Specificities of

Microorganisms in Clouds

Clouds can be defined as atmospheric air masses, in which water is condensed around particles in solid (ice crystals) or liquid form. They represent low-temperature "aquatic" environments involved in the largescale transport of matter and energy, and so participate in the aerial connection between ecosystems. It is assumed that the liquid and supercooled water, from which the free tropospheric clouds are composed, provides a better temporary habitat for living airborne cells than dry air, in which desiccation can be a limiting factor for growth.

There are numerous studies on the composition of organic part of primary biological particles in aerosols (PBPAs). PBPAs comprise pollens, bacteria, spores, fungi, algae, protozoa, fragments of vegetal debris to name few. It was shown that bacteria and fungal spores account at the same percentage level as plant debris in atmospheric aerosols [START_REF] Bauer | The contribution of bacteria and fungal spores to the organic carbon content of cloud water, precipitation and aerosols[END_REF]. Studies on PBPAs structure and origin indicate that cloud droplets may provide a medium, in which bacterial cells can divide [START_REF] Fuzzi | Fog droplets-an atmospheric source of secondary biological aerosol particles[END_REF][START_REF] Dimmick | Evidence that bacteria can form new cells in airborne particles[END_REF][START_REF] Sattler | Bacterial growth in supercooled cloud droplets[END_REF][START_REF] References Ahern | Fluorescent pseudomonads isolated from Hebridean cloud and rain water produce biosurfactants but do not cause ice nucleation[END_REF][START_REF] Bowers | Characterization of airborne microbial communities at a high-elevation site and their potential to act as atmospheric ice nuclei[END_REF]. Very recently, [START_REF] Womack | Biodiversity and biogeography of the atmosphere[END_REF] introduced the concept of biogeography of the atmosphere, suggesting a specific ecosystem of this very specific environment, which is transitory and totally opened contrary to soils and waters. To date, very few data are available concerning the microbial population in the air and clouds. The reported total number of microorganisms in cloud water ranges from about 10 3 to 10 5 cells mL -1 . Table 1 reports more precisely these numbers depending on the sampling sites. Several research groups searched for cultivable cells in fog water at low altitude, and retrieved only three bacterial genera (Pseudomonas, Bacillus, and Acinetobacter), and several fungi and yeasts [START_REF] Fuzzi | Fog droplets-an atmospheric source of secondary biological aerosol particles[END_REF]. In tropospheric clouds more diversified cultivable populations, consisting of many bacterial phyla, were observed, namely Alpha-, Betaand Gamma-Proteobacteria, Bacteroidetes, Actinobacteria, and

Firmicutes belonging mainly to the genera Pseudomonas, Sphingomonas, Staphylococcus, Streptomyces, Bacillus, Rhodococcus, and Arthrobacter [START_REF] Amato | Microbial population in cloud water at the Puy de Dôme: Implications for the chemistry of clouds[END_REF](Amato et al., , 2007b). An important diversity was also found among fungal (Cladosporium and Trametes) and yeast (Cryptococcus) communities [START_REF] Amato | Microbial population in cloud water at the Puy de Dôme: Implications for the chemistry of clouds[END_REF] Firmicutes. These phyla belonged to those described before, except Cyanobacteria, which are described for the first time.

Although less than 1% of the bacteria and less than 50% of the fungi and yeasts isolated at the Puy de Dôme station could be cultivated; the measuring of the adenosine triphosphate (ATP) concentration in cloud samples showed that a large majority of cells counted by microscopy are actually alive (Amato et al., 2007b).

Cloud water presents some specific environment with acidic pH (generally from 3 to 7), high oxidative capacity, the presence of toxic compounds such as formaldehyde, intensive light (including UV) exposure, and relatively low temperatures (5 °C in average). As a consequence, cells show specific physiological properties, which enable them to remain alive in such environment. Many bacterial colonies isolated from cloud water at the Puy de Dôme were pigmented or spore-forming, psychrophiles and some were closely related to strains isolated from cold environments. These red, orange, and yellow pigments are actually carotenoids, which protect cells against UV damage. That decreases the fluidity of their membrane to protect them against cold conditions (Amato et al., 2007b).

The Role of Microorganisms in Cloud

Microphysics

It is well known that atmospheric aerosols influence a cloud formation and precipitation development, and thus they have important impact on the global climate, water cycling and atmospheric chemical reactions.

Less is known about the contribution of Primary Biological Aerosols (PBAs) to these phenomena. PBAs comprise either living and dead cells (microorganisms, pollens, plant fragments, fungal spores, ...) or any kind of organic substances deriving from biomolecules and contributing to aerosol masses. PBAs similarly to particles of other origin, influence cloud formation and precipitation processes by participation in the following pathsway: i) phase change from vapour to liquid, ii) acceleration of coalescence by large particles; and iii) phase change from vapour or liquid to ice. Details of these processes are described in several textbooks and articles [START_REF] Pruppacher | Microphysics of clouds and precipitation[END_REF][START_REF] Young | Microphysical processes in clouds[END_REF][START_REF] Santachiara | Atmospheric particles acting as Ice Forming Nuclei in different size ranges[END_REF] and their description is beyond the scope of the study.

PBAs are a part of the organic carbon fraction of aerosols formed by mixture of thousands of organic compounds. These aerosols exhibit a wide range of molecular structures and could have a major influence on the physico-chemical, biological, climate, and health related behaviours of atmospheric aerosols [START_REF] Jacobson | Organic atmospheric aerosols: Review and state of the science[END_REF][START_REF] Turpin | Measuring and simulating particulate organics in the atmosphere: problems and prospects[END_REF][START_REF] Seinfeld | Organic atmospheric particulate material[END_REF][START_REF] Fuzzi | Critical assessment of the current state of scientific knowledge, terminology, and research needs concerning the role of organic aerosols in the atmosphere, climate, and global change[END_REF]. Although they are capable to form nucleating cloud droplets and ice particles via physical processes (Möhler et al., 2007), the quantification of their role in the real atmosphere, compared to that of other aerosols is not known and is under investigation by many international groups.

Clouds are formed by condensation of water vapour on the aerosol particles surface when the relative humidity (in %) exceeds the saturation with respect to the pertinent phase (water or ice). This is a kinetic process that is controlled by the size, chemical composition, and surface properties of particles. Particles that serve as nuclei for liquid cloud formation at a specified relative humidity are referred as CCN (Cloud Condensation Nuclei), while those that serve as nuclei for ice formation are called IN (Ice Nuclei). Concerning the role of microorganisms in nucleation processes, the story started a long time ago [START_REF] Vali | Comments on "Freezing nuclei derived from soil particles[END_REF], however, experimental evidence is still missing to give final conclusions (Möhler et al., 2007;[START_REF] Sun | Atmospheric organic and bio-aerosols as cloud condensation nuclei (CCN): A review[END_REF][START_REF] Georgakopoulos | Microbiology and atmospheric processes: biological, physical and chemical characterization of aerosol particles[END_REF].

First, microorganisms can be considered as CCN, as a particular case of aerosol particles presenting some specific physico-chemical properties due to their biological nature. Second, IN properties of microorganisms were studied focusing on highly specialised bacteria, namely INA (Ice Nucleation Active) bacteria belonging mainly to Pseudomonas genus.

Insoluble biological particles, capable to retain water on their surface behave as CCN due to the presence of lipopolysaccharides or exopolymeric substances composed of polysaccharides (PS) and proteic structures, which can enhance the hydrophilic behaviour of the cell surface and thus "wettability" and hygroscopy. Data obtained by comparison of different "wettable" bacteria have shown that bacteria could participate to cloud droplet activation at similar critical supersaturation (relative humidity -100%) conditions (Möhler et al., 2007;[START_REF] Morris | Microbiology and atmospheric processes: An upcoming era of research on bio-meteorology[END_REF]Petters and Kreidenweis, 2007). On the contrary, the production of surfactants, especially if they remain at the cell surface (typically for Rhodococcus strains), the presence of carotenoids or unsaturated fatty acids in the bacterial membrane can increase the hydrophobic nature of the bacterial surface and thus decrease its hydroscopic property. Biosurfactants lead to a significant decrease of critical supersaturation when present in sufficient quantities [START_REF] Prisle | Surfactants in cloud droplet activation: mixed organic-inorganic particles[END_REF][START_REF] Ekström | Possible role of ground based microorganisms on cloud formation in the atmosphere[END_REF]. The term supersaturation refers to a solution containing higher quantity of the dissolved material than the solution formed under normal circumstances. It can also refer to a vapour of a compound that has a higher partial pressure than the vapour pressure of this compound.

Only a few studies exist, which are dealing with an activation of cloud condensation nuclei by primary biological aerosol particles and they concern bacteria. [START_REF] Levin | Possible application of bacterial condensation freezing to artificial rainfall enhancement[END_REF] showed rapid growth of freeze-dried bacteria in saturated environment by absorption of water vapour. These data suggest that a substantial proportion of bacteria in the atmosphere act as wettable or hygroscopic particles at their sizes and they are probably frequently scavenged into drops by nucleation during the cloud formation. [START_REF] Franc | Cloud activation characteristics of airborne Erwinia carotovora cells[END_REF] found different fractions of various strains of plant pathogenic Erwinia carotovora bacteria to activate CCN over a wide range of supersaturations. Some bacterial strains, such as Bacillus subtilis (Gram-positive) or Escherichia coli (Gram-negative),

showed relatively large CCN capacity. On the surface of these cells water vapour condensation starts from a relative humidity of 85% [START_REF] Lee | Hygroscopic growth of E. coli and B. subtilis bioaerosols[END_REF][START_REF] Bauer | Airborne bacteria as cloud condensation nuclei[END_REF] tested bacteria cultivated from aerosol and water samples for their ability to act as CCN. As their sizes were smaller than Kelvin diameters for the respective supersaturations, results of this study indicated that the CCN activity must have been enhanced by physico-chemical properties of bacterial outer cell walls.

Ice can be formed in the atmosphere either from the vapour or from the liquid phases of water -these are distinguished as "deposition" and "freezing" [START_REF] Vali | Nucleation terminology[END_REF]. In this case, the ice nucleation pathway is catalyzed by some particle -heterogeneous nucleation. Homogenous freezing nucleation is observed at temperatures approaching -40 °C, when supercooled liquid will freeze spontaneously [START_REF] Tabazadeh | Surface crystallization of supercooled water in clouds[END_REF].

Heterogeneous ice nucleation can take on further variations in clouds depending on the mode how a particle gets into the liquid. Three modes of freezing nucleation were identified experimentally: immersion-, condensation-, and contact-freezing. Nucleation activity can be further influenced by many other effects such as pre-activation or prior exposure to low temperatures and/or humidity conditions.

Both inorganic and organic particles have been identified as atmospherically relevant ice nuclei. Mineral dusts are a general category of inorganic particles that are reported as a strong source of ice nuclei in the atmosphere [START_REF] Isono | The nature and the origin of ice nuclei in the atmosphere[END_REF][START_REF] Demott | African dust aerosols as atmospheric ice nuclei[END_REF]. Biological particles, including certain bacteria, pollen, and decayed organic material have also been identified as ice nuclei [START_REF] Cantrell | Production of ice in tropospheric clouds: A review[END_REF][START_REF] Morris | Ice nucleation active bacteria and their potential role in precipitation[END_REF][START_REF] Schnell | Atmospheric Ice Nuclei from Decomposing Vegetation[END_REF]. Studies of certain epiphytic bacteria of Pseudomonas, Xanthomonas, and Erwinia have shown that these microorganisms bear a membrane protein InaZ, which is encoded by a unique gene. INaZ is the lipoglycoprotein in the external membrane that mimics the structure of ice crystals and it starts a phenomenon of water crystallisation at the temperature dependent on the arrangement of the protein at the cell surface (-2 °C for some Pseudomonas syringae strains, for example, against -40 °C for pure water in homogenous nucleation) [START_REF] Maki | Ice nucleation induced by Pseudomonas syringae[END_REF][START_REF] Vali | Biogenic ice nuclei -2. Bacterial sources[END_REF][START_REF] Szyrmer | Biogenic and anthropogenic sources of ice-forming nuclei: A review[END_REF][START_REF] Cochet | Ice crystallization by Pseudomonas syringae[END_REF].

Only the structural integrity of the protein is responsible for the IN activity, a dead cell is as effective as a living cell. There are Ina + bacteria in the genera Erwinia (species ananas and herbicola), Pantoa (species ananas), Pseudomonas (species syringae and fluorescens), and Xanthomonas (species campestris). The species Pseudomonas syringae concerns the majority of the most active Ina + strains. This species is phytopathogenic as INaZ protein promotes the freezing of water on the plant surface, deteriorating walls and plant to facilitate bacterial attack.

In fungi species, Fusarium avenaceum is almost as effective as Ina + bacteria. This property gives them better protection against desiccation and, in addition, the greater cellulolytic efficiency against their hosts.

The presence of microorganisms with so unusual physical properties in clouds can suggest their significant role in the droplets and crystals Finally, [START_REF] Pratt | In situ detection of biological particles in cloud ice-crystals[END_REF]

The Role of Bacteria in Cloud Chemistry

Airborne microorganisms may have a potential role in cloud chemistry as biocatalysts because they contain multitude of enzymes organised in different metabolic pathways. As it was shown, they can be activated into cloud droplets (Möhler et al., 2007) and once there, they could interact with nitrogen, sulphur, and organic compounds present in aqueous phase.

Thus, they can modify multiphase cloud chemistry by entering into a competition with photochemistry and multiphase processes (dissolution, reactivity, scavenging, and deposition).

The aqueous phase of clouds is actually a very complex chemical reactor.

Table 2 presents the average concentrations of main carboxylic acids and inorganic compounds present in cloud waters collected at the Puy de Dôme summit [START_REF] Marinoni | Cloud chemistry at the Puy de Dôme: variability and relationships with environmental factors[END_REF]. Puy de Dôme mountain is a reference site to study clouds since the top of the mountain is often cloudy and it offers the possibility to sample air masses from various origins. Clouds are classified as a function of their origins (two main categories: continental and marine) and present significant differences in their chemical composition. For instance the pH is lower for higher NO 3 -, NH 4 + , and SO 4 2-concentrations in continental air masses compared to oceanic air ones. It can be noted that pH can be rather acidic for microbial life.

Organic and inorganic compounds present in cloud water result from various multiphase processes than are summarised in Figure 3. Chemical compounds that are present in the atmospheric aqueous phase can result from various sources. First, chemicals present in the solid phase (aerosol particles that acted as cloud nuclei) can be dissolved in the aqueous phase of clouds. Second, soluble chemicals present in the gaseous phase can exchange with the liquid phase of clouds and thus be transferred to this phase. These gaseous chemicals can be primary emitted from the natural or anthropogenic sources and/or are produced through atmospheric gaseous reactivity. For example, VOCs (Volatile Organic Carbons) are soluble in the cloud droplet and photoreactivity in the gaseous atmospheric phase can lead to the production of a myriad of oxygenated compounds that can enter in the aqueous phases. These compounds in the gas phase can also lead to the formation of so-called "Secondary Organic Aerosols" (SOAs) (Hallquist et al., 2009) that can potentially act as CCN and be transferred in the atmospheric aqueous phase. Finally, in the liquid phase, chemical reactions can also occur; clouds are oxidative media and the reactivity is driven by two main radicals ( • OH, NO 3 • ) produced by the presence of strong oxidants such as iron and H 2 O 2 and by the mass transfer from the gas and liquid phases [START_REF] Deguillaume | Transition metals in atmospheric liquid phases: Sources, reactivity, and sensitive parameters[END_REF][START_REF] Tilgner | SPACCIM: Simulations of the multiphase chemistry occurring in the FEBUKO hill cap cloud experiments[END_REF][START_REF] Tilgner | Radical-driven carbonyl-to-acid conversion and acid degradation in tropospheric aqueous systems studied by CAPRAM[END_REF]. These radicals result from photochemical reactions or from the Fenton-type reactions.

In this context, the organic matter represents a significant fraction of the soluble matter in cloud droplets [START_REF] Hadi | Particulate and dissolved organic carbon in cloud water in southern Scotland[END_REF][START_REF] Saxena | Water-soluble organics in atmospheric particles: a critical review of the literature and application of thermodynamics to identify candidate compounds[END_REF][START_REF] Fuzzi | Soluble organic compounds in fog and cloud droplets: what have we learned over the past few years[END_REF]. Among them, carboxylic acids represent 9% of the dissolved organic carbon in the cloud droplets [START_REF] Marinoni | Cloud chemistry at the Puy de Dôme: variability and relationships with environmental factors[END_REF]. The carboxylic acids can be produced in the gaseous phase and dissolved in the aqueous phase (main source of acetic and formic acid); they can also result from the dissolution of soluble particles (main source of acids oxalic, succinic, malonic, and maleic acids, ...); or produced via the aqueous phase reactions [START_REF] Chebbi | Carboxylic acids in the troposphere, occurrence, sources, and sinks: A review[END_REF][START_REF] Goldstein | Known and unexplored organic constituents in the Earth's atmosphere[END_REF]Sellegri et al., 2003;[START_REF] Ervens | A modeling study of aqueous production of dicarboxylic acids: 1. Chemical pathways and speciated organic mass production[END_REF]. Due to the presence of free radicals such as • OH in the aqueous phase, the oxidation of organic matter can be considered as an important source of carboxylic acids; they also represent one of their main sinks [START_REF] Ervens | Temperaturedependent rate constants for hydroxyl radical reactions with organic compounds in aqueous solutions[END_REF][START_REF] Herrmann | Kinetics of aqueous phase reactions relevant for atmospheric chemistry[END_REF]Herrmann, , 2010;;[START_REF] Vel Leitner | Mechanism of the reaction between hydroxyl radicals and glycolic, glyoxylic, acetic and oxalic acids in aqueous solution: Consequences on hydrogen peroxide consumption in the H 2 O 2 /UV and O 3 /H 2 O 2 system[END_REF][START_REF] Warneck | In-cloud chemistry opens pathway to the formation of oxalic acid in the marine atmosphere[END_REF].

Among these VOCs, methanol is the second most abundant organic molecule in the atmosphere after methane and it is the predominant oxygenated compound in the mid to the upper troposphere [START_REF] Singh | Distribution and fate of selected oxygenated organic species in the troposphere and lower stratosphere over the Atlantic[END_REF][START_REF] Singh | Evidence from the Pacific troposphere for large global source of oxygenated organic compounds[END_REF]. 11 to 20% of the methanol in the atmosphere is of anthropogenic origin and from oxidation taking place in the atmosphere, while the major part (80 to 90%) is of biogenic origin, mainly issued from plant metabolism [START_REF] Tie | Biogenic methanol and its impacts on tropospheric oxidants[END_REF][START_REF] Brunner | Methanol exchange between grassland and the atmosphere[END_REF]. Formaldehyde is also a key atmospheric VOC since it is an important indicator of so-called Non-Methane Volatile Organic Compound (NMVOC) emissions and photochemical activity. Formaldehyde is a primary emission product from biomass burning and fossil fuel combustion, but the photochemical oxidation of methane and non-methane hydrocarbons is its main source in the atmosphere.

Methanol and formaldehyde are soluble in water and thus they are efficiently transferred into atmospheric waters (Henry's law constants equal to 2.2 × 10 2 M atm -1 and 10 3 M atm -1 respectively) [START_REF] Sander | Compilation of Henry's Law Constants for Inorganic and Organic Species of Potential Importance in Environmental Chemistry[END_REF].

Very few papers reported methanol concentrations in atmospheric waters. It was found at 0.7 µM concentrations in rain [START_REF] Snider | Tropospheric light alcohols, carbonyls and acetonitrile: Concentrations in the southwestern United States and Henry's low data[END_REF]. Formaldehyde concentration varies in a very wide range of values: from 0.001 µM in rainwater in rural areas [START_REF] Kieber | Rainwater formaldehyde: concentration, deposition, and photochemical formation[END_REF] to 710 µM in highly polluted fog [START_REF] Jacob | A field investigation of physical and chemical mechanisms affecting pollutant concentrations in fog droplets[END_REF]. The average concentration measured in cloud water at the Puy de Dôme station is 2.9 µM (Parazols, 2007) and depends on the air mass origins. [START_REF] Pashynska | Development of a gas chromatography/ion trap mass spectrometry method for determination of levoglucosan and saccharidic compounds in atmospheric aerosols. Application to urban aerosols[END_REF] have shown that compounds belonging to various classes of saccharides are important constituents of ambient atmospheric aerosols.

Although the concentration of these saccharides has not been measured in cloud waters, their occurrence is highly likely due to their of low polarity and high solubility in water. The origin of saccharides present in atmospheric aerosols can be different. Monosaccharides as glucose, fructose, xylose and disaccharides sucrose and trehalose can be released into the atmosphere by microorganisms, plants, and animals [START_REF] Pashynska | Development of a gas chromatography/ion trap mass spectrometry method for determination of levoglucosan and saccharidic compounds in atmospheric aerosols. Application to urban aerosols[END_REF]. Fungi, lichens, and bacteria were found to produce sugar alcohols, such as arabitol, mannitol, and glucitol [START_REF] Simoneit | Sugars -Dominant water soluble organic compounds in soils and characterization as tracers in atmospheric particulate matter[END_REF][START_REF] Medeiros | Sugars as source indicators of biogenic organic carbon in aerosols collected above the Howland Experimental Forest, Maine[END_REF]. Levoglucosan derived from cellulose, galactans and mannans, all are released by burning of biomass [START_REF] Medeiros | Sugars as source indicators of biogenic organic carbon in aerosols collected above the Howland Experimental Forest, Maine[END_REF][START_REF] Simoneit | Levoglucosan, a tracer for cellulose in biomass burning and atmospheric particles[END_REF]. Type and quantities of sugars present in the aerosol were found changing depending on the year period [START_REF] Medeiros | Sugars as source indicators of biogenic organic carbon in aerosols collected above the Howland Experimental Forest, Maine[END_REF]. Moreover, biogenic organic carbon in aerosols can be produced also by photochemical reactions with volatile hydrocarbons and thus it can form a secondary organic aerosol. In recent years, an increased interest in characterisation of polar organic constituents in atmospheric particulate samples -including acids, alkanols, aldehydes, and sugar derivatives, has been observed. Short chain dicarboxylic acids, carbohydrates and their alcohols were two largest identified groups of compounds. The presence of carbohydrates in PBPAs have been shown

by [START_REF] Wang | Molecular, seasonal, and spatial distributions of organic aerosols from fourteen Chinese cities[END_REF], [START_REF] Duarte | Twodimensional NMR studies of water-soluble organic matter in atmospheric aerosols[END_REF], Jia et al. (2010aJia et al. ( , 2010b)).

Composition of PBPAs fixed attention of scientists and thus numerous studies appeared dealing with analytical methods enabling detection and quantification of water soluble organics in atmospheric aerosols. For example sugar compounds have been detected in atmospheric aerosols by LC-MS liquid chromatography combined with positive electrospray ionisation mass spectrometry [START_REF] Wan | Determination of sugar compounds in atmospheric aerosols by liquid chromatography combined with positive electrospray ionization mass spectrometry[END_REF], High-performance anion-exchange chromatography coupled with amperometric detection (HPAEC-PAD) [START_REF] Caseiro | Determination of saccharides in atmospheric aerosol using anion-exchange high-performance liquid chromatography and pulsed-amperometric detection[END_REF] and GC-MS [START_REF] Pietrogrande | GC-MS analysis of watersoluble organics in atmospheric aerosol: Response surface methodology for optimizinf silyl-derivatization for simultaneous analysis of carboxylic acids and sugars[END_REF]. A critical review of advanced analytical techniques for watersoluble organic matter from atmospheric aerosols, which includes also FT-IR and NMR spectroscopy, was published by [START_REF] Duarte | A critical review of advanced analytical techniques for water-soluble organic matter from atmospheric aerosol[END_REF].

Table 3 summarises the ambient concentration of sacharides and other compounds in the atmosphere presented by [START_REF] Wang | Molecular composition and size distribution of sugars, sugar-alcohols and carboxylic acids in airborne particles during severe urban haze event caused by wheat straw burning[END_REF]. The presence of metabolically active air-borne microorganisms suggests organic compound transformation in clouds and thus a scientific emerging question rose about their role in the chemistry of clouds [START_REF] Ariya | New Directions: The role of bioaerosols in atmospheric chemistry and physics[END_REF]Delort et al., 2010).

Few studies tried to analyse biodegradation efficiency under cloud conditions. [START_REF] Herlihy | Bacterial utilization of formic and acetic acid in rainwater[END_REF] were the first who studied the degradation of formic and acetic acid by bacteria in rainwater. [START_REF] Ariya | Microbiological degradation of atmospheric organic compounds[END_REF] and [START_REF] Côté | Microbial and "de novo" transformation of dicarboxylic acids by three airborne fungi[END_REF] observed that several dicarboxylic acids detected in the atmosphere can be efficiently degraded by airborne bacteria and fungi using 13 C NMR. [START_REF] Amato | Microbial population in cloud water at the Puy de Dôme: Implications for the chemistry of clouds[END_REF] showed that bacterial strains present in cloud water contained the enzymatic equipments required for the degradation of some atmospheric compounds including formate, acetate, lactate, methanol, and formaldehyde. A more detailed study (Amato et al., 2007c) was based on 60 microbial strains incubated at 27 °C as pure cultures with one single substrate. Using in situ 1 H NMR it could be shown that their metabolic pathways presented high similarities with chemical reactions common to atmospheric radical chemistry (Figure 4). This work showed that microbial metabolism can be a sink but also a source of organic compounds for the atmospheric chemistry. These first papers mainly showed the ability of microbial strains to degrade organic compounds in clouds. However, in order to evaluate their effective contribution in atmospheric chemistry a quantitative comparison of the contribution of this microbial activity with that of radical chemistry is necessary.

Biodegradation rates of dicarboxylic acids served to [START_REF] Ariya | New Directions: The role of bioaerosols in atmospheric chemistry and physics[END_REF] for the estimation of their lifetimes (few days, see Table 4). They were comparable to that issued from the reactivity of the major atmospheric oxidants ( • OH, O 3 , and HO 2 • ). However, it should be noted that the experimental conditions used by [START_REF] Ariya | Microbiological degradation of atmospheric organic compounds[END_REF] were rather unclear and far from real cloud environment: the temperature was not controlled and close to room temperature, the isolated microorganism were from the air and not from cloud waters.

In order to be closer to cloud water conditions, [START_REF] Vaïtilingom | Rôle des microorganismes des nuages dans la chimie atmosphérique. Comparaison avec la chimie radicalaire[END_REF], used artificial cloud water that mimicked the composition of real cloud water collected at the Puy de Dôme station. P. graminis, a strain isolated from cloud water, was incubated at 5 °C and 17 °C. The colder temperature (5 °C) corresponds to the mean annual temperature measured at the Puy de Dôme summit, while 17 °C is approximately the maximal temperature observed there when a cloud forms (see http://wwwobs.univ-bpclermont.fr/SO/mesures/pdd.php).

P. graminis

strain was able to degrade acetate, succinate, and formate but not oxalate. The relative contribution of microbial metabolism to the degradation of acetate, succinate, and formate was compared with that of radical chemistry involving • OH and NO 3 • reactivity [START_REF] Ervens | Temperaturedependent rate constants for hydroxyl radical reactions with organic compounds in aqueous solutions[END_REF][START_REF] Herrmann | Kinetics of aqueous phase reactions relevant for atmospheric chemistry[END_REF]. Figure 5 shows that during the day • OH activity is dominating; bacteria have only a small influence. On the contrary, during the night, when • OH are no longer present, microorganisms could be the main actors of cloud chemistry.

Table 4. Lifetimes of some dicarboxylic acids present in the atmosphere resulting from the reactivity of • OH, O3, and HO2 • and from the metabolism of atmospheric microorganisms [START_REF] Ariya | Microbiological degradation of atmospheric organic compounds[END_REF]. [START_REF] Vaïtilingom | Rôle des microorganismes des nuages dans la chimie atmosphérique. Comparaison avec la chimie radicalaire[END_REF].

Carboxylic acid Oxidant Kinetic constant k [M -1 s -1 ] Lifetime [days] Reference • OH (aq) Oxalic 1.0 ×
Very recently, Vaïtilingom et al. (2011) has extended this approach to a series of 17 strains incubated in artificial cloud waters mimicking both marine and continental clouds (Table 5). They showed that by the change of temperature from 17 °C to 5 °C, the biodegradation rate decreased from a factor 2 to 5. The composition of cloud water and more specifically the pH did not have an influence on biodegradation rates. The obtained biodegradation rates were then compared with those obtained from photochemical reaction performed in the same artificial media in two different irradiation systems (P1 and P2), or from theoretical calculations

17°C

using two different • OH concentrations. Table 5 shows results obtained at 17 °C. They show that oxalate is not degraded by microorganisms, only by photochemical processes. The rates of photodegradation and biodegradation are in the same range of order. Depending on the chosen photochemical scenarios, the microbial activity has a major or a less important contribution to the transformation of organic acids in cloud water.

Finally, in order to validate their results concerning the biodegradation rates, Vaïtilingom et al. (2011) conducted experiments using real cloud water with complex composition containing the whole endogenous microflora without any strain selection. The biodegradation rates obtained in real cloud water were very similar to those obtained in artificial cloud water.

Table 5. Estimated rates of photo-and biodegradation in artificial cloud water at 17 °C (Vaïtilingom et al., 2011). 1.1 (± 1.8) × 10 -11 1.9 (± 3.2) × 10 -11 0 Continental P1 0 7.4 (± 2.2) × 10 -10 0 3.4 (± 0.9) × 10 -10 P2 0 1.5 (± 0.4) × 10 -10 0 1.9 (± 0.7) × 10 -10 Microbial * 6.7 (± 1.4) × 10 -11 1.4 (± 2.3) × 10 -10 1.5 (± 1.8) × 10 -11 0

Rate in artificial cloud (M s

Marine and continental

• OH (1 × 10 -12 M) 1.2 × 10 -9 † 3.2 × 10 -8 ⁂ 6.6 × 10 -10 ⁂ 3.2 × 10 -10 ⁂ • OH (1 × 10 -14 M) 1.2 × 10 -11 †
3.2 × 10 -10 ⁂ 6.6 × 10 -10 ⁂ 3.2 × 10 -12 ⁂ * Cell concentration used was 8 × 10 7 cells L -1 (mean value in the cloud water sampled at the Puy de Dôme station (Amato et al., 2007c)) † [START_REF] Chin | A temperature-dependent competitive kinetics study of the aqueous phase reactions of OH radicals with formate, formic acid, acetate, acetic acid and hydrated formaldehyde[END_REF] ⁂ [START_REF] Ervens | Temperaturedependent rate constants for hydroxyl radical reactions with organic compounds in aqueous solutions[END_REF] All these results are consistent with a potential contribution of microorganisms to atmospheric chemistry. However, to know a real contribution of microbiological processes relative to photochemical processes in cloud chemistry, an application of numerical process models is necessary [START_REF] Deguillaume | Microbiology and atmospheric processes: Chemical interactions of primary biological aerosols[END_REF]. These process models will take into account the exchange existing between the interstitial phase (gases and particles), cloud droplets and crystals. To simulate more realistic clouds, these models will introduce both biological and radical reactions and modify different parameters (kinetic constants of degradation, number of cells, chemical composition, temperature, pH, light, flux, …) (Georkakopoulos et al., 2009;[START_REF] Leriche | Numerical quantification of sources and phase partitioning of chemical species in cloud: application to wintertime anthropogenic air mases at the Puy de Dôme station[END_REF][START_REF] Deguillaume | Effect of iron dissolution on cloud chemistry: from laboratory measurements to model results[END_REF].

Metabolism of Carboxylic Acids and C 1

Compounds, Major VOCs Present in Atmospheric Waters

This chapter is dedicated to a brief description of metabolic routes related to the degradation of the major carboxylic acids and C 1 compounds of interest, present in cloud water. Only metabolisms of aerobic heterotrophic microorganisms will be considered here.

Metabolism is defined as an interconnection of complex biochemical reactions catalysed by enzymes. Heterotrophic aerobic organisms use all nutrition obtained from their solid or liquid environment, and use oxygen as final electron acceptor from breathing. Different substrates or intermediates of microbial metabolism have been found in clouds, and they might be potentially further used as substrates by cells. Some metabolic pathways represent the central part of the energy production, such as the glycolysis and the Krebs cycle (also called the citrate cycle or cycle of tricarboxylic acid, TCA).

Metabolism of Carboxylic Acids

Metabolic pathways, which could be involved in the metabolism of bacteria in the clouds, are presented here (See Schemes 1, 2, 3, and 4).

Metabolism of C 1 Compounds

Some microorganisms can transform single carbon compounds (C 1 compounds). Among them, methylotrophs are microorganisms, which metabolism is based on their assimilation of C 1 compounds. Most of them are aerobic and compatible with droplets in clouds. Methylotrophs are microorganisms very disseminated in the environment (aquatic and terrestrial media) and they are very important in humid zones, tundras.

Methylotrophs should be found among the Alpha-, Beta-, and Gamma-Proteobacteria and among Gram-positive bacteria. In addition, many microorganisms are facultative methylotrophs, and therefore according to conditions they can operate on the same substrates as obligatory methylotrophs. For the energy production purposes, if C 1 compounds more reduced than formaldehyde (methane, methanol, methylated amines, ...) are metabolised by bacteria, they are generally oxidised to formaldehyde, then to formate, and finally to CO 2 . Formaldehyde can also join central metabolism of the cell and participate in the synthesis of cell constituents. Each C 1 compound is susceptible to be found in the cloud waters, if formaldehyde is present. Formaldehyde is in the centre of this type of metabolism. C 1 compound metabolic routes can be found in KEGG, however, we have decided to describe each step of this metabolism in detail later in this chapter as these compounds will be studied in this work. The different enzymes, co-enzymes, and their modes of action are described in detail.

Oxidation of Methane to Methanol and Formaldehyde

The methanotrophs are methylotrophs, which are able to use methane as a sole source of carbon. The methanotrophs can form lipidic intracytoplasmic membranes, which are unusual for bacteria. Depending on their physiology, the arrangement of these membranes and the path of formaldehyde assimilation, two categories of methanotrophes have been established. The methanotrophes type I (Methylomonas, Methylococcus, Methylobacter) using ribulose monophosphate (RuMP) pathway, and type II (Methylosinus, Methylocystis) using serine pathway. Figure 6 shows details of the oxidation of methane into methanol, which is oxidised to formaldehyde and afterward it joins the metabolism of single carbon compounds [START_REF] Hanson | Methanotrophic bacteria[END_REF]. dehydrogenase [START_REF] Hanson | Methanotrophic bacteria[END_REF].

The oxidation of methane into methanol can be performed by two forms of enzyme, a soluble form, the sMMO, and a form bound to intracytoplasmic membranes, the pMMO (for "particulate" MMO). MMO requires the presence of oxygen and only the sMMO is dependent on the presence of NAD + . The methanol dehydrogenase (MDH) oxidises methanol to formaldehyde. A cytochrome c is required as electron acceptor [START_REF] Jiang | Methanotrophs: Multifunctional bacteria with promising applications in environmental bioengineering[END_REF].

Formaldehyde Assimilation

Formaldehyde can be assimilated by the cell and participates in the formation of the cellular structures. The cyclic pathways of RuMP and serine can be used (Figure 7). In general, RuMP pathway is more effective and more common than that of serine. The latter is known in methanotrophs type II and in facultatively methanotrophs, which is activated in the presence of methanol [START_REF] Trotsenko | Metabolic aspects of aerobic obligate methanotrophy[END_REF]. MTK -maleate thiokinase; MCL -malyl coenzyme A lyase [START_REF] Hanson | Methanotrophic bacteria[END_REF].

Formaldehyde Disproportionation

The disproportionation of formaldehyde to methanol and formate can proceed in 2 ways (Figure 8). The first involves a NAD + dependent oxidoreductase. It results either to the oxidation of formaldehyde to formate by simultaneous reduction of NAD + , or to methanol by reduction of NAD + . The second way, also called Cannizaro's reaction is catalysed by formaldehyde dismutase [START_REF] Kato | Dismutation and cross-dismutation of aldehydes, and alcohol: aldehyde oxidoreduction by resting-cells of Pseudomonas putida F61-a[END_REF] and it does not require the presence of cofactor. It is induced by the presence of formaldehyde in the medium [START_REF] Hektor | Nicotinprotein methanol dehydrogenase enzymes in Gram-positive methylotrophic bacteria[END_REF][START_REF] Mitsui | Formaldehyde-limited cultivation of a newly isolated methylotrophic bacterium, Methylobacterium sp. MF1: enzymatic analysis related to C1 metabolism[END_REF].

Reaction of Formaldehyde with Methanol

Formaldehyde can recombine with methanol leading to the formation of methylformate, following the reaction [START_REF] Mason | Formaldehyde metabolism by Escherichia coli. Detection by in vivo carbon-13 NMR spectroscopy of S-(hydroxymethyl)glutathione as a transient intracellular intermediate[END_REF][START_REF] Delort | Use of NMR to study in situ bioconversion of gaseous compounds[END_REF]:

HCHO + CH 3 OH → CH 3 O-CH 2 OH → CH 3 O-CHO

Oxidation of Formaldehyde to Formate

The oxidation of formaldehyde to formate takes place in the cytoplasm by action of both, free or to the membrane bound enzymes. There are several pathways that can lead to the formation of formate from formaldehyde. Three of them are described in the review of [START_REF] Vorholt | Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria[END_REF]. They are shown in Figure 9. Each of them requires a specific cofactor, which binds to the molecule of formaldehyde and NAD(P) + . FGH -formyl-glutathione hydrolase; X -unknown cofactor [START_REF] Vorholt | Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria[END_REF].

The path H 4 F-dependent requires the presence of tetrahydrofolate. It is mainly present in bacteria, for which the serine pathway represents only the part of formaldehyde assimilation, and is therefore relatively little frequent. The H 4 MPT-dependent path is associated with the presence of dephosphorylated tetrahydromethanopterin. This path was observed in anaerobic methanogenic bacteria, sulfate-reductors, and in some Archaea. The third path, GSH-dependent, is mostly widespread, both in bacteria that in yeasts, plants, and mammals, and most often it is used to detoxify the organism from formaldehyde. It requires the presence of glutathione (GSH) or mycothiol (myshi).

In each case, the oxidation reaction is initiated by the binding between the cofactor and formaldehyde. This activation is spontaneous in the case of the H 4 F-dependent path. For the other two paths, this binding is gluthation-dependent in certain bacteria, or it is caused by an activation of other formaldehyde activation enzymes: gluthatione-dependent or H 4 MPT-dependent. These activation enzymes allow creating complexes with free formaldehyde in the cell and thus to prevent its interaction with molecules that can cause damages (e.g., DNA, etc.).

Above-mentioned facts give the evidence that there is no universal way how formaldehyde is used by microorganisms. Several ways exist and often coexist in the same cell, whether for carbon assimilation, energy production, and detoxification.

NMR as a Tool to Investigate the

Metabolism of Cloud Microorganisms

Previous studies related to the metabolism of molecules of interest found in the atmosphere have been performed using Nuclear Magnetic Resonance (NMR) [START_REF] Ariya | New Directions: The role of bioaerosols in atmospheric chemistry and physics[END_REF][START_REF] Amato | Microbial population in cloud water at the Puy de Dôme: Implications for the chemistry of clouds[END_REF]2007c). We propose to give some insight in this technique, which is a method of choice to study microbial metabolism.

NMR is a non destructive method, enabling thus the analysis of complex intracellular or incubation media (in situ NMR) but also the analysis of the metabolism of living cells (in vivo NMR) and its monitoring over the time. NMR offers an important alternative to most commonly used analytical techniques such as mass spectroscopy, HPLC, GPC, enzymatic assays, or the use of radio markers ( 14 C, 3 H). 1 H, 13 C, 31 P, and 15 N are the most frequently observed nuclei in the metabolism studies either due to their natural abundance or the availability of enriched substrates.

However, some other nuclei such as 23 Na and 39 K can be used for special purposes such as the measurement of trans-membrane gradients.

Numerous NMR studies of microorganism metabolism have been reviewed [START_REF] Grivet | NMR for microbiology: In vivo and in situ applications[END_REF][START_REF] Grivet | NMR and microbiology: from physiology to metabolomics[END_REF][START_REF] Portais | Carbohydrate cycling in microorganisms: what can 13 C-NMR tell us?[END_REF]. The majority of them were performed on supernatants (incubation media) of bacterial cultures or cell extracts.

For in situ studies, the pre-treatment of biological samples is often necessary, e.g. separation of cellular fractions, pH adjustment, freezedrying and subsequent dissolution in D 2 O, partial purification of certain metabolite, etc. When in vivo NMR is performed, specific apparatus must be used to maintain the cells alive in the NMR spectrometer. Figure 10 presents the system of perfusion described by [START_REF] Chorao | 2-Aminobenzothiazole degradation by free and Ca-alginate immobilized cells of Rhodococcus rhodochrous[END_REF] that has been used in this work. saturated with air. The perfusion rate is controlled by a peristaltic pump (flow rate 20 mL min -1 ). Bacterial suspension circulates continuously in the system. Benzene is used as an internal reference to calibrate chemical shifts (128.39 ppm relative to TMS (0 ppm)) in a sealed capillary placed in a 10 mm sample tube. Presaturation methods [START_REF] Hoult | Solvent peak saturation with single phase and quadrature fourier transformation[END_REF] individual advantages and drawbacks, and may not be applicable in all situations depending on the details of underlying pulse sequence and characteristics of the sample itself [START_REF] Nguyen | SOGGY: Solvent-optimized double gradient spectroscopy for water suppression. A comparison with some existing techniques[END_REF].

1 H NMR

Modern high-resolution NMR probes contain besides the RF coils additional coils that can be fed by a direct current. The coils are built so that a pulse (~1 ms long) of direct current creates a gradient of the B 0 field along z (x or y, these refer to the lab coordinate system), as if the z (x/y) shims were mis-set so that the line-widths would be ~100 kHz. A signal of 100 kHz line width will be completely dephased after 1 ms. 200 μs after the gradient pulse, the B 0 field is homogeneous again but the magnetisation is still dephased. Therefore, if one applies another gradient after some time, the magnetisation can be refocussed if the gradients have the right ratio. [START_REF] Mo | Pre-SAT180, a simple and effective method for residual water suppression[END_REF] have improved pulse sequence for residual water suppression -WET180 (WET with 180° pulse-toggling), which is proposed to cancel the water contribution to the residual solvent signal.

This pulse sequence uses shaped, selective pulses and magnetic field gradient pulses to suppress one or more solvent signals. It provides very efficient suppression with excellent selectivity. The WET sequence was originally developed for the suppression of the effects of T 1 and B 1 inhomogeneity during the in vivo spectroscopy [START_REF] Frahm | Improvements in localized proton NMR spectroscopy of human brain. Water suppression, short echo times, and 1 ml resolution[END_REF][START_REF] Bottomley | Spatial localization in NMR spectroscopy in vivo[END_REF]. The WET technique uses a series of variable-tip-angle solvent selective RF pulses, where each selective RF pulse is followed by a dephasing field-gradient pulse [START_REF] Smallcombe | WET solvent suppression and its applications to LC-NMR and high-resolution NMR spectroscopy[END_REF]. The B 1 insensitivity shares the same refocusing mechanism. For the two-pulse B 1 insensitive WET sequence, positive longitudinal magnetisation components are generated by the 79.7° excitation pulse and then start to refocus near the null after being inverted by the 110.2° pulse [START_REF] Ogg | WET, a T 1 -and B 1intensitive water suppression method for in vivo localized 1 H NMR spectroscopy[END_REF].

WATERGATE (WATER suppression by GrAdient Tailored Excitation)

sequence is very efficient in suppression of water signal. The sequence comprises of selective inverse pulse surrounded by two gradients of equal magnitude and duration. Its drawback is the suppression of signals surrounding the water signal. The sequence is frequently used in protein and nucleic acids structural studies [START_REF] Piotto | Gradient-tailored excitation for single quantum NMR spectroscopy of aqueous solutions[END_REF][START_REF] Sklenář | Gradienttailored water suppression for 1 H-15 N HSQC experiments optimized to retain full sensitivity[END_REF]. [START_REF] Zheng | Solvent signal suppression in NMR[END_REF][START_REF] Cook | Coupling NMR to NOM[END_REF][START_REF] Tsang | Some practical aspects of doubleresonance techniques in solution-state NMR studies of high-molecular-weight systems[END_REF].

In microbiology, 1 H NMR has been used in numerous of studies concerning carbohydrate metabolism but also xenobiotic degradation (pollutants, food additives) [START_REF] Grivet | NMR for microbiology: In vivo and in situ applications[END_REF]. In the case of xenobiotics, proton is often the only nucleus to be used due to low concentrations of these compounds (Figure 11) and the unavailability of commercially labelled compounds. Formaldehyde resonance is hidden by the huge residual water signal. TSPd4 is the internal reference (from [START_REF] Amato | Microbial population in cloud water at the Puy de Dôme: Implications for the chemistry of clouds[END_REF].

1 H NMR is also extremely useful in indirect 13 C (or X) label detection in produced compounds and the 13 C/ 12 C ratio quantification as well as the 13 C label distributions. Obtained information is often used in studies of metabolic fluxes (see [START_REF] Grivet | NMR for microbiology: In vivo and in situ applications[END_REF] for review) and it is a key point to carbohydrate metabolic cycles studies for instance [START_REF] Portais | Carbohydrate cycling in microorganisms: what can 13 C-NMR tell us?[END_REF].

Finally, 1 H NMR covers also growing area of metabolomics [START_REF] Grivet | NMR for microbiology: In vivo and in situ applications[END_REF]. This approach allows comparing the whole metabolome of genetically different microbial strains (biodiversity, mutants, etc.) but also to investigate the global response of microorganisms to various stresses (case of environment microbes) or to different diets (case of gut microbes).

13 C NMR

A limiting factor for the use of 13 Numerous examples of the microbial metabolism studied by 13 C NMR were shown in several reviews [START_REF] Grivet | NMR and microbiology: from physiology to metabolomics[END_REF][START_REF] Grivet | NMR for microbiology: In vivo and in situ applications[END_REF].

Since C 1 compound (methanol and formaldehyde) metabolism is of major interest in this PhD work, we would like to give here some examples of 13 C NMR studies using 13 C labelled substrates. Most of them are cited by [START_REF] Delort | Use of NMR to study in situ bioconversion of gaseous compounds[END_REF].

Figure 12 presents the transformation of 13 C-formaldehyde (~82 ppm) into different compounds, including 13 C-formate (~70 ppm) and 13 Cmethanol (~50 ppm) by E. coli [START_REF] Hunter | Formaldehyde metabolism by Escherichia coli. In vivo carbon, deuterium, and twodimensional NMR observations of multiple detoxifying pathways[END_REF] et al., 1984).

Spectra, in the Figure 13 show the 13 C labelled methanol degradation by B. methanolicus. Simultaneously with the methanol consumption the three peaks of metabolites appeared: 13 C-formaldehyde, 13 C-methanol, and 1-[ 13 C]hexose-phosphate (Figure 13). The assimilation of methanol into the RuMP pathway is presupposed according to the presence of 1-[ 13 C]hexose-phosphate, to which methanol is incorporated during the first step of this pathway [START_REF] Pluschkell | Dissimilation of[END_REF]. [START_REF] Pluschkell | Dissimilation of[END_REF].

31 P NMR

High sensitivity of 31 P due to a high natural abundance of this isotope is an advantage for the studying of a great number of biological processes by NMR: Energetic state of biological systems (in vivo and in vitro analysis), measurement of intracellular pH, and quantification of phosphorylated metabolites [START_REF] Grivet | NMR and microbiology[END_REF][START_REF] Grivet | NMR for microbiology: In vivo and in situ applications[END_REF][START_REF] Grivet | NMR and microbiology: from physiology to metabolomics[END_REF]. Identification of some intracellular phosphorylated metabolites including polyphosphates, nucleotide triphosphates (NTPs)

(mainly ATP and sugar phosphates) can be carried out directly by in vivo

15 N NMR

In general, the major drawback in observing 15 N nucleus is the insensitivity inherent with a natural abundance of 0.4%. Thus, 15 N NMR studies are mostly limited to 15 N enriched substrates utilisations. On the other hand, such a low natural abundance background makes it easy to detect an incorporation of a 15 N-enriched source and depletion of this isotope if it is diluted and replaced by an excess of 14 N fixed into the same species. However, the 15 N NMR spectra analysis is complicated by negative NOE factors and sometimes long T 1 values. Moreover, they can have a marked dependence on their physico-chemical environment (pH, ionic charge, viscosity, etc.) making difficult the quantitative analysis of cell extracts from experiments using labelling. Some of these dependences were studied by Nassima Houyou et al. (2006) in order to find conditions, in which quantitative analysis of some major metabolites (amino acids, ammonium, nitrate, and urea) could be performed reliably and with sufficient signal intensity. In methanogens, the free amino acids turnover was studied using labelled substrates [START_REF] Roberts | Free amino acid turnover in methanogens measured by 15 N NMR spectroscopy[END_REF].

2 H NMR

Due to the relatively low magnetogyric ratio and the magnetic properties of the quadrupole nucleus, the detection of 2 H by NMR suffers from its low sensitivity. The considerable line broadening, associated with its quadrupolar nature can be reduced to some extent by measuring at higher temperatures. Besides a precursor/roduct relationships establishment, 2 H NMR is especially useful for studying cyclisations, rearrangements, and the mechanistic aspects of metabolic reactions [START_REF] Schneider | Nuclear magnetic resonance spectroscopy in biosynthetic studies[END_REF].

An elegant application of in vivo 2 H NMR concerns the biotransformation of 2 H formaldehyde by bacterial strains [START_REF] Mason | In vivo enzymology: a deuterium NMR study of formaldehyde dismutase in Pseudomonas putida F61a and Staphylococcus aureus[END_REF]. As it was already shown in Chapter 2.2.2, formaldehyde can be reduced to methanol by two ways: via an NAD-oxidoreductase activity, or via a dismutase activity (see below):

In the first case (oxido-reductase), the methanol obtained contains both 2 H and 1 H atoms, the resulting CHD 2 signal is thus a doublet in 2 H NMR spectra due to the J H-D coupling. In the second case (dismutase), methanol is totally 2 H labelled and thus the 2 H NMR signal of the methyl group is a singlet. Figure 15 shows the identification of the mechanisms of enzymatic reaction directly in in vivo experiments with various bacterial strains.

NMR Experiments Used for Structure Elucidations

The resolution in 1D NMR is often inadequate for structure identification of complex mixtures. Enhancement of the resolution can be achieved by spreading of desired data into a second or more dimension(s). Depending on the type of experiment, needed information can be thus extracted more easily. A drawback of 2D NMR measurements is that they typically require much longer experimental times than 1D spectra acquisition depending on the sensitivity of the observed nucleus, metabolite concentration, and the resolution desired.

Homonuclear 2D Experiments

Two common types of 1 H-1 H homonuclear 2D methods utilise scalar coupling between protons to display the covalent bonding network. The This disadvantage is overcomed in ROESY (Rotating frame nuclear Overhauser Effect SpectroscopY) experiment in which ROE is evolving in the weak magnetic field of rotating frame and it is not dependent on the molecular mass of the compound. However, as the spin lock is used in ROESY TOCSY-type cross-peak appear in the spectra making their interpretation more difficult.

Both TOCSY and NOESY exist as a 1D variant. In the case when signals in 1 H NMR spectra are sufficiently resolved enabling their selective irradiation and more precise information could be obtained in reasonable shorter times.

Heteronuclear Correlations

COSY and TOCSY spectra are used for the assignment of proton spin systems. Although, many common metabolites can be reliably identified with them, many others remain ambiguous, particularly in strongly overlapped regions of the 1 H spectrum, such as in the 3-5 ppm region occupied by various sugars. Hz depending on the α or β configuration, and aromatic CH protons typically have 1 J CH in the range of 180 Hz and higher. Thus, in a single HSQC experiment, the transfer efficiency cannot be optimised for all groups simultaneously. Thus, two separate experiments, each with a 1 J CH setting optimised for aliphatic or aromatic/anomeric protons, are usually needed. In addition, a spectral editing in HSQC spectra is possible on the basis of the different evolution of 1 J CH , 1 J CH2 , a 1 J CH3 couplings which allow a discrimination between CH/CH 3 (positive) and CH 2 (negative) signals. In the HMBC experiment cross-peaks due to long range interactions between protons and hetero nuclei are detected.

Many more information dealing with the best strategy for structure elucidation is available in numerous textbooks and reviews. As an example Fan and Lane (2007) should be mentioned.

2.4

Bacillus, a Bacterial Genus Present in Clouds 2.4.1 Generalities

Bacillus Genus

As shown in [START_REF] Amato | Microbial population in cloud water at the Puy de Dôme: Implications for the chemistry of clouds[END_REF], Bacillus strains have been frequently isolated from cloud water. This chapter will give thus some information about properties and metabolism of this genus.

The term "aerobic endospore-forming bacteria" is used to embrace Bacillus species and related genera, for which the production of resistant endospores in the presence of oxygen remains the defining feature. They are also expected to possess Gram-positive cell wall structures (but staining reactions, even in young cultures, may be Gram-variable or frankly Gram-negative), and may be aerobic or facultative anaerobic.

These features formed part of the definition of this group for many years, but some exceptions have emerged. Bacillus infernus and Bacillus arseniciselenatis are strictly anaerobic, and spores have not been detected in Bacillus infernus, Bacillus subterraneus, and Bacillus thermoamylovorans.

Representatives of this genus are widely distributed in the soil, air, and water where they are involved in a range of chemical biotransformations.

Although primarily saprophytes, at least one species (Bacillus schlegelii),

show facultatively chemolithotrophic activity in O 2 /CO 2 /H 2 or O 2 /CO 2 atmosphere [START_REF] Schenk | Bacillus schlegelii, a new species of thermophilic, facultatively chemolithoautotrophic bacterium oxidizing molecular-hydrogen[END_REF], and two species (Bacillus macerans and Bacillus polymyxa) are able to fix nitrogen [START_REF] Hino | Nitrogen fixation by a facultative Bacillus[END_REF][START_REF] Witz | Nitrogen fixation by growing cells and cell-free extracts of the Bacillaceae[END_REF]. The ability of certain strains to tolerate high or low temperature and pH has made them a particularly important source of commercial enzymes [START_REF] Norris | The Prokaryotes: A Handbook on Habitats, Isolation and Identification[END_REF]. The metabolic diversity of Bacillus sp., together with reported low incidence of pathogenicity, has led to representatives of this group being used in wide range of industrial processes. Nowadays, strains of Bacillus are used for the production of four main types of product: i) enzymes, ii) antibiotics,

iii) fine biochemicals including flavor enhancers and food supplements, iv) insecticides [START_REF] Schallmey | Developments in the use of Bacillus species for industrial production[END_REF].

Carbon Catabolism in Bacillus Species

Limitation of the carbon source in clouds and other extreme environments is a potential reason why in bacteria efficient and competitive ways of its utilisation has been developed. Pathways of carbon source utilisation in Bacilli and the different aspects of their regulation have been the subjects of several reviews [START_REF] Sauer | The PEP-pyruvate-oxalacetate mode as the switch point for carbon flux distribution in bacteria[END_REF][START_REF] Fisher | Control of carbon and nitrogen metabolism in Bacillus subtilis[END_REF][START_REF] Holmberg | Glycerol catabolism in Bacillus subtilis: nucleotide sequence of the genes encoding glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD)[END_REF][START_REF] Priest | Extracellular enzyme-synthesis in genus Bacillus[END_REF]. An exhaustive description of the mechanisms of carbon catabolism and its regulation in Bacillus species was made by [START_REF] Stülke | Regulation of carbon catabolism in Bacillus species[END_REF].

Microbial Exopolymeric Substances

Generalities

Initially, the term EPS was used to describe extracellular polysaccharides (PS), but recent studies showed that polymeric matrices have complex structures (lipopolysaccharides, glycolipids, lipids, proteins, nucleic acids) [START_REF] Decho | Exopolymer microdomains as a structuring agent for heterogeneity within microbial biofilm[END_REF]. Microorganisms convert very efficiently nutriments in exopolymeric substances (EPS) to create a stock of carbon and energy.

They can optimise their utilisation also in the case when xenobiotic compounds are provided as a sole carbon source [START_REF] Wolfaardt | Microbial exopolymers provide a mechanism for bioaccumulation of contaminants[END_REF].

The ability to synthesise and excrete exopolysaccharides (PS) through rigid cell wall or membrane is widespread among microorganisms (Sutherland, 2004). In some cases, it is a feature of all isolates of a given genus and PS are synthesised virtually under all conditions. In spite of very few data reporting the PS production by Bacillus species, large number of microorganisms is able to produce them (Dedonder, 1966;Han and Clarke, 1990). Production of PS results from specific nutritional conditions or as a response on the stress. In some prokaryote it may also result from development or morphological changes. In Gram-positive genera of Bacillus and Clostridium it may be due to the sporulation. PS may be firmly attached to a microbial surface or it might be excreted into the incubation medium as an amorphous mass of slime, which can be separated from bacteria by centrifugation. Many of bacterial strains might have the capacity to produce simultaneously more than one type of PS. The chemical composition of PS depends on the genetics of the microbial cells and the physico-chemical environment, in which a biofilm matrix develops (Sutherland, 2001). Generally, monosaccharide composition and structure of PS are independent on the carbon substrate available. Some bacterial genera seldom yield PS producing species. Only few Gram-positive Bacillus species produce PS.

Microbial PS are multifunctional and can be divided into intracellular polysaccharides, structural and extracellular PS. They are produced by both prokaryotes (eubacteria and archaebacteria) and eukaryotes (phytoplankton, fungi, and algae). The bacterial PS represent a wide range of chemical structures. Chemically, they may have homo-or heteropolymeric composition of high molecular weight (10 to 30 kDa).

They have newfangled applications due to the unique properties they possess. Owing to this, exopolysaccharides have found various applications in the food, pharmaceutical, and other industries (Kumar et al., 2007). They also play a role in the environmental field as biofloculants for instance (Suh et al., 1997)

Polysaccharides

The production of PS by many Bacillus strains was described, however, in • PS produced by Bacillus circulans reported by [START_REF] Isobe | Structural study of an exocellular polysaccharide of Bacillus circulans[END_REF] was composed of β-L-rhamnopyranose (Rha), α-D-mannopyranose (Man), α-D-Gal, and α-D-glucopyranuronic acid (GlcA).

• Bacillus circulans st. AM7 isolated from soil produced two novel cyclic oligosaccharides during the incubation on amylose: cyclomaltopenta-and cyclomaltohexaose, in which 1,4-linked α-Glc were closed to the ring by α-1,6-linkage [START_REF] Watanabe | )-α-D-Glcp-(1→4)α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}[END_REF]. [START_REF] Lee | Optimization of fermentation conditions for production of exopolysaccharide by Bacillus polymyxa[END_REF] optimised the fermentation condition for production of exopolysaccharide by Bacillus polymyxa. Its monomer composition made by TLC and HPLC after hydrolysis of the polymer, showed the presence of Glc, Gal, Man, GlcA, and fucose (Fuc). [START_REF] Pfiffner | Isolation of halotolerant, thermotolerant, facultative polymerproducing bacteria and characterization of the exopolymer[END_REF] isolated PS produced on sucrose under anaerobic conditions by encapsulated Grampositive microorganism similar to Bacillus species. Purified polymer contained Man, Glc, arabinose (Ara), ribose (Rib), allose (All) and glucosamine (Glc-NH 2 ). [START_REF] Muhammadi | Characterization of exopolysaccharide produced by Bacillus strain CMG1447[END_REF][START_REF] Muhammadi | Isolation and Characterization of Exopolysaccharide Produced by Indigenous Soil Bacterium Bacillus Strain CMG1403[END_REF] showed that in two strains of Bacillus (strain CMG1447 and CMG1403) produced acidic hetero-PS slightly differing in sugar: Gal and Man (~92%) and GlcA (~8%). The only difference between them was the presence of a small quantity of Rha in PS produced by CGM1447 strain. Optimal conditions (C/N ratio of the growth medium, pH, and temperature) for EPS production by Bacillus megaterium, isolated from the infected plant leaf of Aralia species, were determined by [START_REF] Gandhi | Exopolymer production by Bacillus species[END_REF].

Many Bacillus strains have been described to produce bioflocculants, a specific class of EPS produced by microorganisms during their growth.

Synthetic flocculants are widely used in industrial fields (e.g. for water purification) but they have been shown to be harmful to the environment as a dangerous source of pollution. On the contrary, bioflocculants produced by microorganisms are expected to be eco-friendly because of their biodegradability.

Main Bacillus bioflocculant structural features are summarised here and in Table 7 • Bioflocculant DP-152 (molecular weight of >2 × 10 3 kDa) produced by Bacillus sp. DP-152 contained Glc, Man, Gal, and Fuc in a molar ratio of 8:4:2:1 (Suh et al., 1997).

• Bacillus sp. F19 produced the bioflocculant named MBFF19. Its chemical analyses of the indicated that it was a sugar-protein derivative, composed of neutral sugar (3.6%, w/w), uronic acid (37.0%, w/w), amino sugars (0.5%, w/w), and protein (16.4%, w/w).

The two neutral sugar components were Man and Glc in the molar ratio 6:5 (Zheng et al., 2008).

• Bacillus megaterium TF10 isolated from soil, produced bioflocculant, study of which was performed by [START_REF] Yuan | Identification of key constituents and structure of the extracellular polymeric substances excreted by Bacillus subtilis TF10 for their flocculation capacity[END_REF].

The relationship between structure constituents and their flocculation capacity have been shown. Their results suggested that the bridging mechanism is responsible for the flocculation. GC-MS and NMR analyses demonstrated that the PS had long chain composed of Rha, Glc, and Gal with the side chain containing uronic acids, acetyl amino sugars, and the proteins.

• Bacillus thermantarcticus produced two PSs with typical mucous character. PS1 repeating unit was constituted of four differently linked α-Man and three β-Glc units with a structure close to some xantan polymers. PS2 with molecular weight 3 kDa was found to be a mannan with traces of pyruvic acid with repeating unit composed of four differently linked α-Man units [START_REF] Nicolaus | Polysaccharides from extremophilic microorganisms[END_REF]. where Bacp2N4NAc means N-acetylbacillosamine (4-acetamido-2amino-2,4,6-trideoxy-D-glucose), Py (pyruvate) [START_REF] Schäffer | Purification and structure elucidation of the Nacetylbacillosamine-containing polysaccharide from Bacillus licheniformis ATCC 9945[END_REF].

• Bacillus licheniformis of marine origin produced PS which was characterised by [START_REF] Singh | Isolation and characterization of exopolysaccharides from seaweed associated bacteria Bacillus licheniformis[END_REF]. Advanced analytical methods such as MALDI-TOF-TOF MS, FT-IR, UV-VIS, and NMR spectroscopy revealed its complex and heterogeneous nature of PS with high thermal stability (330 °C) with the monosaccharidic composition Gal, Glc, Man, and Ara.

• Bacillus licheniformis isolated from bottled spoiled cider produced in Normandy, was found to be composed of Man (80% of total polysaccharide) and Glc (6.5%) (based on HPLC and NMR analysis) [START_REF] Larpin | Biosynthesis of exopolysaccharide by a Bacillus licheniformis strain isolated from ropy cider[END_REF].

• Bacillus licheniformis from a shallow marine hot spring of Vulcano Island, Italy was isolated by [START_REF] Arena | Antiviral and immunoregulatory effect of a novel exopolysaccharide from a marine thermotolerant Bacillus licheniformis[END_REF] showed antiviral and immunomodulatory effects. A tetrasaccharide composed of Man units was found as a repeating unit of this PS.

• Curdlan is a water-insoluble homopolysaccharide composed exclusively of β-1,3-linked glucose residues. Most of the reports on curdlan production are on Agrobacterium and Alcaligenes sp.

However, [START_REF] Gummadi | Production of extracellular water insoluble β-1,3-glucan (Curdlan) from Bacillus sp. SNC07[END_REF] reported for the first time that Gram-positive bacterium Bacillus sp. SNC 107 is also capable to produce extracellular curdlan in appreciable amounts. • Bacillus subtilis bioflocculant PS was described recently by Patil et al. (2009). Bacterium was incubated in the media composed of different carbohydrates. The best bioflocculant production was observed on Glc and sucrose (Suc). The PS was found to be composed of Glc, Gal, and fructose (Fru). Chemical analysis showed that sugar/protein ratio was 94:6.

• Production of bioflocculants composed of γ-glutamic acid by a strain of Bacillus subtilis has been described [START_REF] Yokoi | Flocculation properties of poly(γ-glutamic acid) produced by Bacillus subtilis[END_REF].

Polyhydroxyalkanoates

Exopolymeric substances produced by microorganisms are not limited only to polysaccharides. Many bacterial strains produce polyhydroxyalkanoates (PHA). PHA are polyesters of 3-, 4-, 5-, and 6hydroxyalkanoic acids produced by a variety of bacterial species when their growth is restricted by an essential nutrient (as nitrogen, phosphorus, sulphur, magnesium, potassium, or iron) and if a carbon source is available in excess (Anderson and Dawes, 1990;Oeding and Schlegel, 1973;Steinbüchel and Schlegel, 1989). These water insoluble storage polymers are biodegradable, exhibit thermoplastic properties and can be produced from renewable sources. PHAs serve as energy and they are stored as granular inclusion bodies in the cytoplasm of many bacteria and archea (Lemoigne, 1926;Anderson and Dawes, 1990;Steinbüchel, 1991). Accumulation of intracellular storage polymers has been The first identified PHA was polyhydroxybutyrate (PHB) from Bacillus megaterium (Lemoigne, 1926). Until now a great number of many other different constituents have been described (Steinbüchel and Valentin, 1995), however, PHB occurs as an inclusion surrounded by a phospholipid monolayer. Proteins, referred as a "granulate-asssociated proteins", are associated with the membrane and located only at the surface of the PHB granules (Hocking et al., 1994;Huisman et al., 1991;Steinbüchel et al., 1995). Recent studies have shown that the polymer is in fluid state inside the cell and crystallises to form granules when is released from the cells (Kawaguchi and Doi, 1990).

In a screening study made by Valappil et al. (2007), Bacillus cereus was identified as the best PHAs producing strain using various carbohydrate substrates (Glc, Fru, Suc, various fatty acids, and gluconate).

Putative Importance of EPS for Atmospheric Environment

The production of EPS has a specific importance for a microorganism living in the atmospheric environment because it is closely related to the ability of microorganisms to form biofilms. Biofilms are generally formed by bacteria, which are intimately attached to some surface and aggregate into a heterogeneous hydrated polymeric matrix themselves synthesised.

Formation of biofilms seems to be the typical way how bacterial cells grow in nature. Each microcolony in the biofilm is surrounded by extracellular polymeric substances (EPS) (predominantly exopolysaccharides, DNA, and proteins). EPS layer exhibits secondary, sometimes even tertiary structure [START_REF] Chapell | Surface chemistry and function of microbial biofilms[END_REF]. There are more reasons for biofilm formation: First, biofilms are important for bacterial self-defence and survival of cells (chance to resist to physical and chemical external stresses are higher for cells in biofilms). Second, biofilm formation allows cells to remain in a favourable niche (bacterial cells are fixed in location where nutrients are more abundant or are constantly replenished). Third, biofilms allows bacteria to live in close association with other ones and thus their survival chances are increased.

Moreover, when cells are in close proximity to each other, opportunities for genetic exchanges are more available [START_REF] Madigan | Metabolic diversity and microbial ecology[END_REF]. Also, the presence of a gelled PS layer around the cell may have paramount effects on the diffusion properties, both into and out of the cell [START_REF] Dudman | The role of surface polysaccharides in natural environment[END_REF].

For instance, cells buried within a polymer matrix would be inaccessible to antibiotics. The ability of a microorganism to surround itself with a highly hydrated exopolysaccharide layer may provide it with protection against desiccation and predation by protozoa. The environmental conditions are the most important factor influencing the physico-chemical properties of biofilms (such as porosity, density, water content, charge, sorption and ion exchange properties, hydrophobicity, and mechanical stability).

If we now consider the atmosphere, the production of EPS by microorganisms present in this environment could be of special interest.

First, this structure surrounding the cells could protect them against desiccation, UV exposure, • OH and NO 3 • radicals, etc. and could help them to survive in this extreme environment. Second, such functionalised and very hydroscopic structures could influence the formation of CCN by modification of the bacterial cells surface bioaerosols. Finally, EPS could be released in the atmosphere and could be thus considered as SOAs.

THESIS OBJECTIVES

The objective of this thesis is to continue and deepen the knowledge about the metabolism of bacteria isolated from clouds. We will try to understand their possible role in atmospheric processes and their ability to survive in such stressing environment.

The work will be mainly focused on the strain Bacillus sp. 3B6 as it was

shown to be very active and also because Bacillus genus is frequently present in cloud water.

In order to achieve this goal the following strategy was chosen:

1.

Study of the carbohydrate metabolism of Bacillus sp. 3B6

In situ NMR spectroscopy was chosen as a main tool to monitor the biodegradation kinetics of various saccharides and to identify the major metabolites.

A large range of carbohydrates have been chosen for the study: Different saccharides (aldoses, ketoses) covering penta-and hexasaccharides in acyclic and cyclic forms as well as oligo-and polysaccharides. Obtained results will give on one side an extensive overview of the metabolic abilities of this strain, which is unknown. On the other side, they will have an impact on the knowledge about cloud chemistry because some of these compounds are present in the atmosphere.

A special attention will be paid to the synthesis of EPSs as it is well known that these polymers are essential for bacteria living in a changing environment. In the specific context of atmospheric environment, where this strain has been isolated, the synthesis of such substances could have consequences on the survival of the strain and on precipitation formation.

Study of the metabolism of formaldehyde and methanol by

Bacillus sp. 3B6 and other typical strains present in cloud waters

Formaldehyde and methanol have been chosen as their represent major atmospheric VOCs and because virtually no results are reported about their biodegradability in clouds.

Their biodegradation rates will be measured by in situ and in vivo NMR using both unlabelled and 13 C labelled compounds. Obtained data on their microbiological transformations will be then compared with those due to radical chemistry processes.

Different scenarios will be used by taking into account the temperature changes (winter/summer), the degree of pollution, and the day/night alternance. The idea is to evaluate quantitatively the relative importance of biodegradation processes compared to radical processes, even though the chosen experimental conditions here are still too far from real cloud environment.

The long term goal, which is beyond the scope of the proposed project, is the utilisation of knowledge about metabolic pathways and kinetic constants in atmospheric chemistry numeric models (collaboration with physicists of the atmosphere in Clermont-Ferrand, France). These models will be the only way to simulate realistic clouds and thus really know whether microorganisms play a role in atmospheric chemistry. Also any data concerning the formation of compounds that would impact on the microphysics of clouds will be taken into account by our colleagues from atmospheric sciences.

RESULTS

NMR Study of Carbohydrate Metabolism

Introduction

Bacillus genus is frequently present in the atmosphere. For Bacillus strains isolated from cloud water no detailed study of their carbohydrate metabolism have been reported to date. We focussed our attention on the strain Bacillus sp. 3B6 as it has been shown to be very active towards the degradation of pollutants [START_REF] Durand | First isolation and characterization of a bacterial strain that biotransforms the herbicide mesotrione[END_REF]Amato et al., 2007) and also because its behaviour during cultivation suggested the synthesis of polysaccharides. The understanding of its metabolism on carbohydrate is important to explain how it can survive in the atmosphere where such substrates might be present, and the identification of metabolites might be of interest for atmospheric chemistry and microphysics. In addition, some metabolites can be of interest for industrial purposes as it is well known that these bacterial species can produce various polysaccharides for food, cosmetic, nutraceutic, and environmental applications. The study of carbohydrates utilisation as a carbon source by this bacterium is

RESULTS

divided into two parts:

•

The first part deals with an extensive study of sucrose metabolism that gave rise to a publication in Carbohydrate Research (see Chapter 4.1.2) focussed on the polysaccharide (PS) production: This study describes a compositional analysis of the incubation medium and individual fractions issued from its separation by GPC.

•

The second part is an extended investigation of the metabolism of carbohydrates focused on their ability to produce exopolymeric substances (EPSs) mainly polysaccharides (PS). The manuscript is planned to be submitted to Carbohydrate Polymers (see Chapter 4.1.3). This study starts with a screening of the ability of this bacterium to metabolise different saccharides. Then the production of EPSs is examined when the bacterium is incubated with low concentration of substrates. Finally, the case study of the incubation with high concentration of glucose is reported.

NMR Structural Study of Fructans Produced by Bacillus sp. 3B6, Bacterium Isolated in Cloud

Water.

90 RESULTS

Introduction

Microorganisms can convert nutriments in exopolymeric substances (EPS) very effectively and thus create a stock of carbon and energy. These

EPSs can be polysaccharides (PS) but also other polymers such as proteic structures or polyhydroxyalkanoates (PHA) for instance.

Many microorganisms are able to synthesise and excrete exopolysaccharides (PS) through rigid cell wall or membrane (Sutherland, 2004;Dedonder, 1966;Han & Clarke, 1990). In some cases it is a feature of all isolates of a given genus and PSs are synthesised virtually under all conditions. Production of PS results from specific nutritional conditions or as a response on the stress. In some prokaryote it may also result from development or morphological changes. In Gram-positive genera of Bacillus and Clostridium it may be due to the sporulation. PS may be firmly attached to a microbial surface or it might be excreted into the incubation medium as an amorphous mass of slime, which can be separated from bacteria by centrifugation. Many of bacterial strain might have the capacity to produce simultaneously more than one type of PS.

The chemical composition of PS depends on the genetics of the microbial cells and the physicochemical environment, in which a biofilm matrix develops (Sutherland, 2001). Generally, monosaccharide composition and structure of PS are independent of the carbon substrate available. Some bacterial genera seldom yield PS producing species. Only few Grampositive Bacillus species produce PSs.

Microbial PSs are multifunctional and can be divided into intracellular polysaccharides, structural and extracellular PS. They are produced by both prokaryotes (eubacteria and archaebacteria) and eukaryotes (phytoplankton, fungi, and algae). The bacterial PSs represent a wide range of chemical structures. Chemically, they may have homo-or heteropolymeric composition of high molecular weight (10 to 30 kDa).

They have newfangled applications due to the unique properties they possess. Owing to this, exopolysaccharides have found various applications in the food, pharmaceutical, and other industries (Kumar et al., 2007). They also play a role in the environmental field as biofloculants for instance (Suh et al., 1997).

Some bacterial strains produce polyhydroxyalkanoates, polyesters of 3-, 4-, 5-, and 6-hydroxyalkanoic acids under nutrient-limiting conditions such as nitrogen, phosphorus, sulphur, magnesium, potassium, or iron with excess of carbon (Anderson & Dawes, 1990;Oeding & Schlegel, 1973;Steinbüchel & Schlegel, 1989). These water insoluble storage polymers are biodegradable, exhibit thermoplastic properties and can be produced from renewable sources. PHAs serve as energy and they are stored as granular inclusion bodies in the cytoplasm of many bacteria and archea (Lemoigne, 1926;Anderson & Dawes, 1990;Steinbüchel, 1991).

Accumulation of intracellular storage polymers has been considered as a strategy used by bacteria to increase survival in a changing environment.

PHA comprises a vast number of structures with repeating subunits of a general formula -[O-CH(R)-(CH 2 ) x -C(O)]-and molecular masses 0.1-30 kDa.

In a recent paper, we had shown that Bacillus sp. 3B6 was able to produce exopolysaccharide levan and FOSs (fructooligosaccharides) of levan and inulin type when incubated at high concentration of sucrose as carbon source [START_REF] Matulová | NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water[END_REF]. These interesting results prompted us to extend our study to a wide range of carbohydrates (alditols, mono-, di-, tetra-, and polysaccharides) to evaluate the potential of this strain to produce EPSs. First, the ability of this bacterium to use these various carbohydrates as carbon sources was investigated using in situ 1 H NMR.

Second, the structure of exopolymeric substances excreted by Bacillus sp.

3B6 was analysed by in situ multidimensional NMR. Finally, a more detailed study concerned the case of EPSs production under high concentration of glucose.

As Bacillus sp. 3B6 has been isolated from cloud water (Amato et al., 2007) a discussion is given about the potential implication of EPS in such atmospheric environment.

Material and methods

Microbial incubations

Bacterial strain

Bacillus sp. 3B6 was isolated from cloud water sampled at Puy de Dôme summit (1465 m) in the Massif Central Region (France) and was identified by 16S rRNA gene sequencing as described by Amato et al. (2007).

General incubation conditions

Liquid pure pre-culture was incubated in 100 mL portion of TSA (Trypcase Soy broth, Biomerieux, Marcy l'Etoile, France) in 500 mL Erlenmeyer flask at 27 °C and 200 rpm. Cells were harvested by centrifugation (8 000 × g and 4 °C for 5 min) after 24 hours of growth and rinsed twice in NaCl 0.8% and thereafter re-suspended in the medium for carbohydrate metabolism study.

Incubation procedure № 1 (Low carbohydrate concentration)

Media for metabolism study were prepared by dissolution of saccharides in Volvic ® mineral water. Each tested saccharide was used as a unique carbon source. The final concentration of saccharides in incubation medium was 20 mM for mono-and disaccharides, and 10 mM for oligo-and polysaccharides. The concentration of used microorganisms was 10 9 cell mL -1 . 24-Hours old inoculum was used to seed the sterile medium.

Bacillus sp. 3B6 was grown in this medium for 6 days under aerobic conditions (27 °C, 200 rpm). Samples were taken in regular intervals (0, 15, 19, 24, 39, 48, 63, 72, 96, 120, 144 h). Extracellular incubation media were separated from cells by centrifugation (3 min, 12 000 × g) and supernatants were kept frozen (-40 °C) until measurement. Inulin, levan, and maltose were ordered from Sigma-Aldrich. All the other tested saccharides were from the production of the Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia.

Incubation procedure № 2 (High glucose concentration)

For EPS production under high glucose concentration a medium proposed by [START_REF] Gandhi | Exopolymer production by Bacillus species[END_REF] was used. Its compositions was as follows: glucose (30 g L -1 ) (Acros), ammonium nitrate (0.4 g L -1 ) (Acros), potassium sulphate (1 g L -1 ) (Acros), sodium chloride (1 g L -1 ) (Acros), potassium dihydrogen phosphate (3 g L -1 ) (Fluka), calcium chloride (0.02 g L -1 ) (Sigma-Aldrich), magnesium sulphate (0.001 g L -1 ) (Sigma-Aldrich).

Medium was autoclaved for 15 min at 121 °C. 24-Hours old inoculum was used to seed the sterile medium. The culture was incubated in a rotary shaker at 200 rpm and 32 °C. After 8 days of incubation, the medium was centrifuged (20 000 × g, 25 °C, 30 min) and Bacillus sp. 3B6 cells were separated from supernatant.

NMR spectroscopy

Structural analyses

NMR spectra were measured on 600 MHz VNMRS Varian equipped with HCN 13 C enhanced salt tolerant cold probe in D 2 O at 25 °C using TSPd 4 as an external standard. Chosen samples containing metabolites were freeze-dried from D 2 O before dissolution in 99.98% D 2 O. Shigemi NMR tubes or micro-capillaries were used in the case of small quantity of metabolites in the samples. Advanced techniques from Varian pulse library of 2D homo-and hetero-correlated spectroscopy (gCOSY, gTOCSY, gHSQCAD, gHMBCAD, gH2BC) including 1D sequences with selective excitations (1DNOESY, 1DTOCSY) were used for the signal assignments.

Measurement of metabolite concentrations

Supernatants from biodegradation test media were prepared Acquisition was performed at 500 MHz on Bruker Avance NMR spectrometer or on 600 MHz VNMRS Varian equipped with HCN 13 C enhanced salt tolerant cold probe. Water signal was eliminated by presaturation and by Pulse field gradient spin echo (GPFSE). The concentration of metabolites was calculated as follows:

[m] = 9 b A 0 A ref [TSPd 4 ]
where [m] is the concentration of compound to be quantify; A 0 is the area of m resonance; A ref is the area of TSPd 4 resonance; 9 and b are the numbers of protons in TSPd 4 and in the metabolite m, respectively.

Calculation of biodegradation rates

The biodegradation rates k c of the compound C have been determined by linear regression fits as follows:

k c = k [C] 0 N cells

Infrared spectroscopy

Fourier-transform infra red (FT-IR) spectra were measured with a Nicolet 6700 (Thermo Fisher Scientific, USA) spectrometer equipped with DTGS detector and Omnic 8.0 software. The spectra were collected in the middle region from 4000 to 400 cm -1 at a resolution of 4 cm -1 , the number of scans was 128. Diamond Smart Orbit ATR accessory was applied for measurement in solid state.

Chemolytic analyses

The carbohydrate content in EPSs-2ns, EPSs-3ns, and EPSs-4ns was determined by the phenol-sulphuric acid assay [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF].

Elemental analysis

Elemental analysis was performed with EA 1108 apparatus (FISONS Instruments, East Grinstead, UK). Protein was calculated from the nitrogen content (%N × 6.25).

Results

3.1 Ability of Bacillus sp.

3B6 to metabolise carbohydrates

The ability of Bacillus sp. 3B6 to metabolise different types of carbohydrates was monitored by in situ 1 H NMR spectroscopy under low concentrations of carbohydrate (incubation procedure № 1). Different saccharides (aldoses, ketoses) were chosen covering penta-and hexasaccharides in acyclic and cyclic forms as well as oligo-and polysaccharides (Table 1). Samples were taken at chosen intervals (0, 24, 39, 48, 63, 72, 96, 120 hours) from the incubation media. Table 1 summarises the results concerning the screening for carbohydrates used as a carbon sources by Bacillus sp. 3B6 as well as their degradation rates.

Alditols

Alditols are acyclic carbohydrates formed by reduction of their corresponding aldoses. In the screening, five chosen alditols were tested:

D-mannitol, D-glucitol, D-galactitol, D-arabitol, and L-arabitol. 1 H NMR Spectra of the incubation media (IM) showed that D-mannitol and Dglucitol were completely metabolised within 24-48 hours. The degradation of L-arabitol started after 48 hours of the incubation and its quantity decreased from 96% at 48 h to 55% at 72 h and it completely disappeared at 96 h. However, D-arabitol and D-galactitol were not degraded. In the case of all alditols no formation of metabolites or intermediates was observed.

Hexoses

A comparison of biodegradation rates of tested aldoses and ketoses (Table 1) showed that the fastest degraded saccharides were D-glucose (Glc)

together with D-galactose (Gal) and D-mannose. Their signals disappeared completely from the 1 H NMR spectra after 48 hours. The degradation of D-fructose (Fru) and L-arabinose was slightly slower and their rates of the degradation were comparable. In both cases, substrate signals disappeared after 48 hours. In the case of arabinose and L-rhamnose, the production of some metabolites was observe. However, their structures were not studied in details.

Pentoses

The intensity of fructose (Fru) signals in the course of the incubation decreased very fast; after 25 hours less than 25% of Fru were present and after 48 hours they disappeared completely. The degradation rate of L-arabinose was only slightly lower than that of Fru followed by those of D-ribose. The biodegradation rate of D-ribose was low; all substrate was degraded after 96 hours of the incubation. The degradation rates of L-ribose and D-xylose were comparable and NMR spectra of their IM showed that after 96 hours almost 60% of substrate was still present there.

Disaccharides

Maltose (αGlc(1-4)Glc) was the fastest degraded disaccharide; it disappeared after 48 hours. The degradation rates of sucrose (αGlc(1-2)βFruf), trehalose (αGlc(1-1)αGlc), turanose (αGlc(1-3)αFruf), and cellobiose (βGlc(1-4)Glc) were similar; in their 1 H NMR spectra all signals were absent after 72 hours. Signals of lactose (αGlc(1-4)Glc) disappeared only after 96 hours indicating slower metabolism of this sugar.

Polysaccharides (PS)

No degradation of levan polymer (2,6-linked Fruf) was observed contrary to inulin (1,2-linked Fruf), which degradation rate was high. After 24 hours already 30% of the polymer was already used and its signals disappeared from the spectrum after 72 hours. The degradation rate of cellotetraose was the same as that of cellobiose. Contrary to cellotetraose, cellulose was not soluble and thus its signals were not present in 1 H NMR spectrum at t = 0 h. The 1 H NMR spectrum of the incubation medium taken at t = 15 h contained signals of amino acids and first signals indicating the presence of some sugar metabolites appeared after 27 hours.

EPSs production on different carbon sources

Analysis of in situ 1 H NMR spectra of samples taken at different times and different substrates revealed similar features of spectral patterns, especially at the end of the incubations when substrate was already depleted. Spectral pattern in all spectra shown in Figure 1 suggest the presence of high molecular mass exopolymeric substances (EPSs). This observation was very interesting due to the fact that different substrates were used as a carbon source (fructose, sucrose, glucose, turanose, cellotetraose, cellulose, and starch; Table 1). However, the complexity of 1 H NMR spectra suggested rich mixtures of metabolites. In most cases the concentration of metabolites was very low due to the conditions used for screening (Incubation procedure № 1) preventing a very detailed analysis of EPS structure. However, in the sample of IM on turanose (t = 72 h) the quantity of individual components was differed to such level that one of them was more or less dominant in the mixture.

This fact allowed its partial structural determination by a combination of selective 1D and 2D NMR techniques. Valuable information in form of characteristic 2D COSY and HSQC spectral patterns due to individual components was also used in the analysis of the complex IM.

3.2.1

EPSs produced on turanose

Comparison of 1 H NMR spectra on different substrates (Figure 1) showed that the spectrum of the medium collected after 72 h of incubation with turanose (Figure 1D) showed the simplest spectral pattern and the quantity of metabolites in this sample was higher than in IM on starch (Figure 1E). Therefore, this sample was chosen for a detailed NMR analysis. Comparison of 1 H NMR spectra suggested that the metabolism of turanose (αGlc(1-3)βFruf) under screening conditions (Incubation procedure № 1) was similar to that one at higher concentration on sucrose (αGlc(1-2)βFruf) (Incubation procedure № 2) described in our previous paper in which Bacillus sp. 3B6 hydrolysed sucrose to Glc and

Fruf. Glc was further used as a source of energy and Fruf for the synthesis of inulin and levan type fructooligosaccharides (FOSs) and levan polysaccharide [START_REF] Matulová | NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water[END_REF]. The same behaviour was observed on turanose, however, here under these screening conditions, the concentration of the substrate was much lower and thus synthesised microbial FOSs were finally used as a source of carbon by the bacterium.

The HSQC spectrum collected after 72 hours of incubation is shown in In the IM spectrum on glucose after 48 h (Figure 4A) traces of glucose were present and between many metabolites present there cross-peaks due to 1,6-linked α-galactan could be identified. In the COSY spectrum of the IM on glucose after t = 63 h (Figure 4B), neither glucose nor low molecular metabolites cross peaks could be detected, only those of EPSs with higher molecular mass were present there. The 1,6-linked α-galactan was one of the main components of the mixture. As cellulose is not soluble, no signal was detected at the beginning of the experiment; first metabolites appeared in the spectrum after 27 hours corresponding to amino acids signals in the region δ 2.4-0.7 ppm. Well resolved H1 anomeric signal at δ 5.179 ppm, indicating the presence of 1,6-linked αgalactan, appeared only after 48 h of the incubation. In the spectrum of IM on cellulose after t = 63 h cross-peaks due to 1,6-linked α-galactan were dominant (Figure 4C).

Polyhydroxyalkanoates (PHA)

In the HSQC spectrum of IM t = 72 h on turanose (Figure 2A) besides cross-peaks due to 1,6-linked α-galactan those due to CH 2 group of high intensity were present: δ 3.97-3.84/67.5, 3.98/65.70, 3.84-3.72/62.14.

Cross-peak of CH 3 group was observed at δ 2.03/22.84 ppm. However, HMBC spectrum did not reveal any cross-peak due to a long range interaction from CH 2 to carboxyl group, which could be present in the molecule. The only cross-peak with carboxyl group was detected from CH 3 group. Moreover, 13 C chemical shifts values of these CH 2 group suggested a close vicinity of oxygen similar to a -CH 2 -O-structure. The complexity of the spectra prevented more precise attribution of signals to some structure. However, on the basis of these data we could deduce the presence of some polyhydroxyalkanoate (PHA) in the incubation medium but its structure remained unknown. Further studies will be necessary for its elucidation.

Analysis of soluble fractions EPS-1, EPSs-2,

EPSs-2s, EPSs-3s

All soluble fractions obtained from the IM after 8 days of incubation on glucose were analysed by NMR; the identified metabolites are listed in Table 3. In the 1 H NMR spectrum of EPSs-2 sample obtained after a second dialysis broad lines indicated the presence of high molecular mass EPSs.

In the HSQC spectrum of the incubation mixture one component was identified as 1,6-linked α-galactan polymer on the basis of its previously characterised spectral pattern. Chemical shifts of its cross-peaks were in accordance with those already assigned for this polymer (see Table 2). In addition, cross-peaks of high intensity due to another polymer present in the mixture could be recognised: CH 3 at δ 2.023/δ 23.00, two CH 2 groups at δ 3.757/δ 61.84 and δ 3.976-3.854/δ 67.74 and CH at δ 4.011/δ 70.61.

Their spectral pattern and chemical shifts were similar to PHA observed in the media obtained at the end of incubations at low concentration of the substrate. They suggested the PHA presence also in this IM.

During the measurement of EPS-2 NMR spectra, a spontaneous precipitation of some non soluble components was observed. The EPSs-2ns precipitate was separated and the supernatant was marked as EPSs-2s. Characteristic broad lines pattern due to low quantity of EPSs were still present in its NMR spectra; however, their quantity was low.

HSQC spectrum indicated a mixture of lower molecular mass compounds.

One of them was identified as 3-hydroxybutyrate. Its structure was confirmed by COSY, HSQC, and HMBC spectra and the chemical shifts data are presented in the Table 2. Another one was glycerol.

During NMR measurements further precipitation occurred in the sample tube in small quantities. New sharp signals, which were not observed before, appeared in the spectra. Analysis of NMR spectra confirmed the presence of butyric acid (Table 2). Moreover, the characteristic spectral pattern due to 1,6-linked α-galactan indicated the presence of its oligomers.

Analysis of water non soluble fractions EPSs-2ns, and EPSs-4ns

Table 3 summarises the results obtained for different non soluble fractions.

EPSs-2ns was subjected to sugar compositional and elemental analyses and infrared spectroscopy. The main carbohydrates identified by sugar compositional analysis were galactose (35%), glucose (33%), and mannose (28%). Only traces of xylose (3%) and arabinose (1%) were found. Elemental analysis confirmed the presence of proteins (~18%). In the FT-IR spectrum (Figure 6) the presence of bands typical for carbohydrate and protein moieties could be identified. In the region 1200-1000 cm -1 characteristic for polysaccharide moiety, significant band maxima were present at 1093, 1072, and 1021 cm -1 derived from hexosans (galactose, glucose, and mannose) based polysaccharide structures. However, due to the EPSs-2ns complex structure and an overlap of vibration bands it was not possible to identify the The precipitated polymer EPSs-2ns and EPSs-3ns was not soluble in DMSO. EPSs-3ns was partially soluble in the mixture of chloroform and methanol (CDCl 3 :MeOH = 7:3). Non soluble material (EPSs-4ns) was removed and dissolved compounds remained in the EPSs-4s.

Carbohydrate compositional analysis did not prove the presence of carbohydrates in EPSs-3ns and EPSs-4ns. NMR analysis of the EPSs-4s fraction confirmed the presence of 3-hydroxybutyrate, glycerol (

Discussion and conclusion

Although Bacillus genus is highly present in the environment, relatively few papers deal with the structural analysis of EPSs by this type of bacterial strains. We show here that the strain Bacillus sp. 3B6, isolated from atmospheric water, can produce many EPSs on various carbohydrates as carbon sources including fructose, glucose, sucrose, turanose, cellotetrarose, cellulose, and starch. In situ NMR allowed identification some of these structures.

Analysis of IM obtained during the incubation of Bacillus sp. 3B6 with different saccharides at low concentration showed that bacterium starts to excrete a mixture of exopolymeric substances (EPS) when this source of the carbon is nearly depleted. First incubation on turanose produced some FOSs. FOSs were previously found in incubation medium of the same strain with sucrose [START_REF] Matulová | NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water[END_REF] The first identified PHA was polyhydroxybutyrate (PHB) from Bacillus megaterium (Lemoigne, 1926). In screening for the best PHA producer, Bacillus cereus was identified as a strain using various carbohydrate substrates (glucose, fructose, sucrose, various fatty acids, and gluconate) for PHA production (Valappil et al., 2007). In this study, Bacillus sp. 3B6

produced PHA when incubated with turanose, glucose, and cellulose.

Interestingly, when the concentration of glucose was increased in the incubation medium other components with a non saccharidic composition, such as 3-hydroxybutyrate, butyric acid, glycerol, and esters of glycerol, have been identified. The presence of 3-hydroxybutyrate is consistent with the synthesis of PHB. Steinbüchel & Valentin (1995) report that a great number of many other different constituents are present in PHB producers and that PHB occurs as an inclusion surrounded by a phospholipid monolayer. In addition, it has been shown that proteins, referred as "granulate-associated proteins", are associated with the membrane and located only at the surface of the PHB granules (Hocking et al., 1994;Huisman et al., 1991;Steinbüchel et al., 1995). The polymer is in fluid state inside the cell and crystallises to form granules when is released from the cells (Kawaguchi & Doi, 1990). In the case of Bacillus sp. 3B6, the detection of glycerol and esters of glycerol, metabolites which are related to phospholipid metabolism, could be involved in the membrane synthesis surrounding PHB granules. Also the presence of proteoglycan identified in the incubation medium could be related to "granulate-associated proteins". These last points are only hypotheses but they are consistent with the actual knowledge about PHB granules in microorganisms.

As already specified, Bacillus genus is highly present in the environment, and thus the production of EPSs has a specific importance for these microorganisms as it is closely related to the ability to form biofilms.

Formation of biofilms seems to be the typical way how bacterial cells grow in nature. These biofilms are important for bacterial cells as i) they serve for the self-defence and survival of cells (chance to resist to physical and chemical external stresses are higher for cells in biofilms),

ii) they allow cells to remain in a favourable niche (bacterial cells are fixed in location where nutrients are more abundant or are constantly replenished), iii) they benefit to bacteria as they can live in close association with other ones increasing their survival chances and their opportunities for genetic exchanges (Madigant et al., 2009). The ability of a microorganism to surround itself with a highly hydrated exopolysaccharide layer may provide it with protection against desiccation and predation by protozoa.

If we now consider the case of the atmospheric environment where Bacillus sp. 3B6 has been isolated, the production of EPSs could be of special interest. First, this structure surrounding the cells could protect them against desiccation, freezing, UV exposure, • OH and NO 3 • radicals, etc. and could help them to survive in this extreme environment (Delort et al., 2010). Second, such functionalised and very hydroscopic structures could influence the formation of Cloud Condensation Nuclei (CCN) and thus the formation of precipitations (Möhler et al., 2007). Finally, EPS could be released in the atmosphere and could be thus considered as Secondary Organic Aerosols (SOAs). This last point is of importance as it

shows that SOAs could be produced by microorganisms as an alternative to radical reactions (Hallquist et al., 2009;[START_REF] Blando | Secondary organic aerosol formation in cloud and fog droplets: a literature evaluation of plausibility[END_REF].

It is also worth noting that some carbohydrates used as carbon sources in this study are present in the atmosphere, it is the case of mannitol, glucitol, arabitol, glucose, sucrose, and trehalose (Jia et al., 2010a(Jia et al., , 2010b;;[START_REF] Wang | Molecular composition and size distribution of sugars, sugar-alcohols and carboxylic acids in airborne particles during severe urban haze event caused by wheat straw burning[END_REF]. We have shown that Bacillus sp. 3B6 could efficiently metabolise them. On the other hand, glycerol, which is also found in airborne particles, can be produced by this strain.

In conclusion, carbohydrate metabolism and the EPS production could be an efficient way for Bacillus sp. 3B6 to survive in the extreme environment of clouds, stay metabolically active and contribute to atmospheric chemistry and nucleation processes.

Conclusion on Carbohydrate Metabolism

The first important result of our study is the fact that the strain Bacillus sp. 3B6 was able to metabolise many carbohydrates. The use of in situ The second important point is that Bacillus sp. 3B6 produced different EPSs on various carbohydrates as carbon sources. This production started when the carbon source was depleted. The 2D NMR spectral patterns of these EPS suggested similar structures.

Incubations with an excess of glucose and sucrose could be analysed in more details thanks to multidimensional NMR experiments performed on fractionated samples. The following metabolites could be evidenced:

• On Sucrose: levan exopolysaccharide (EPS), fructooligosaccharides (FOSs) of levan and inulin types and difructose anhydride DFA IV.

• On Glucose: glycoprotein, polyhydroxyalkanoate polymers (PHA), polysaccharide having 1,6-linked α-galactan backbone, 3-hydroxybutyrate, glycerol, esters of glycerol, and butyric acid.

These types of EPS were also found in the incubation with the other saccharides (fructose, sucrose, glucose, turanose, cellotetrarose, cellulose, and starch) at lower concentrations.

comparison between biocatalysis and radical chemistry showed that microorganisms could be a major sink of organic acids at night [START_REF] Vaïtilingom | Rôle des microorganismes des nuages dans la chimie atmosphérique. Comparaison avec la chimie radicalaire[END_REF](Vaïtilingom et al., , 2011)).

The biotransformation of methanol and formaldehyde, major VOCs in the atmosphere, has not been investigated yet. One of the limitations and difficulties is to measure the concentration of these compounds, especially methanol, and few data on this later substrate are available in the literature.

In this paper we used in situ 1 H and 13 C NMR spectroscopy to monitor specifically the biodegradation kinetics of these compounds and measure precisely the biodegradation rates by various bacteria. Four bacterial strains isolated from cloud water have been chosen, they belong to genera frequently found in the atmosphere (Bacillus, Pseudomonas, and Frigobacterium).

We also used in vivo 13

Biotransformation of Methanol and

Formaldehyde by Bacteria Isolated from Clouds.

Comparison with Radical Chemistry

Conclusion on C1 Compounds Metabolism

The mains conclusions of this paper are the following:

Formaldehyde and methanol metabolic routes

All the bacteria investigated were able to transform formaldehyde and methanol, even at low temperature (5 °C, average temperature in warm clouds).

The metabolic pathways of photo-and biodegradation of methanol and formaldehyde presented some similarities.

As an example, photochemistry, through the production of radicals, is involved in the progressive oxidation of organic compounds to CO 2 in cloud water.

Methanol can be oxidised into formaldehyde by the four strains tested or by reactivity with free radicals. The main difference between radical chemistry and biological activity is the ability of some microbes to catalyse the reduction of formaldehyde into methanol even under very oxidative conditions.

In addition, we showed the ability of Bacillus sp. 3B6 to produce complex compounds such as propane-1,2-diol, propane-1,3-diol and glycerol via the serine pathway. This last point is of importance concerning atmospheric chemistry as it suggests that not only successive oxidations that lead to shorten molecules can occur in cloud water but also reactions that can result in compounds of larger molecular size. In our study, C3

molecules have been built from a C1 compound. It is of major importance to be aware that such functionalisation processes in the atmosphere, identified as determining for atmospheric sciences, can be catalysed by microorganisms.

Relative contribution of microbial activity versus radical chemistry

The relative contributions of microbial and radical activities ( • OH and where we had considered organic acids such as formate, succinate, and acetate [START_REF] Vaïtilingom | Rôle des microorganismes des nuages dans la chimie atmosphérique. Comparaison avec la chimie radicalaire[END_REF].

ii) The origin of air masses modulates the extent of the previous conclusion. Indeed NO 3 • reactivity, which is negligible at night compared to bacteria in remote clouds, becomes of some importance in urban clouds, this is due to the higher concentration of NO 3 • in polluted clouds compared to non polluted ones. However, we are aware that our experimental approach leads to some simplifications where some stressful factors for microorganisms are not considered (very acidic medium, presence of highly toxic chemical species that can characterised polluted clouds).

In conclusion, our results, together with those already obtained with carboxylic acids, suggest that microbial activity could be an alternative route to photochemistry processes in cloud waters. Of course, these experiments were performed in bulk phase while atmospheric chemistry is a multiphase process. So, to go further, cloud chemistry models should integrate both chemical and biological components.

GENERAL CONCLUSION AND PERSPECTIVES

The discovery of the presence of living and active microorganisms in clouds is rather recent. Their survival ability under such hostile environment and their potential role in atmospheric processes are open questions in the scientific community. The objective of this thesis was to bring some answers to these topics.

What have we learned??

1.

For living in the atmosphere

We have shown that Bacillus sp. 3B6 has a very active metabolism towards the compounds tested, many of them are present in the atmosphere (formaldehyde, methanol, mannitol, sorbitol, arabitol, glucose, sucrose, trehalose, and cellulose) providing efficient sources of energy for this bacterium and thus allowing this strain to live in this environment.

The second important feature is the synthesis of ExoPolymericSubstances (EPSs), polymers containing saccharides (FructoOligoSaccharides (FOSs) together of levan and inulin type; 1,6-linked α-galactan) or not PolyHydroxyAlkanoates (PHAs). All these compounds have been shown to protect microorganisms to various environmental stresses, mainly by forming biofilms. In the particular context of the atmospheric environment these EPSs should contribute to help this strain to survive facing dessication events, UV exposure, radical reactivity, acidity, freezing-defreezing etc. EPS synthesis could be a strategy complementary to the unique ability of Bacillus strains to form endospores for survival under extreme condition.

For atmospheric chemistry

Until very recently, only radical chemistry, and particularly photochemistry, was considered in atmospheric chemistry processes.

It has been already shown that the metabolism of microorganisms present in cloud waters can be a possible alternative route of dicarboxylic acid transformations to those of abiotic processes. Here we bring some evidence that microorganisms could also transform methanol and formaldehyde with rates close to those of due to radical reactivity. These biocatalytic reactions could be of major importance during the night.

More, we have also shown that Bacillus sp. 3B6 is able to degrade other types of compounds present in the atmosphere (see above), thus it can also contribute to the transformation of a wide range of organic matter in cloud water.

Finally, the use of NMR as an "objective tool" allowed showing that Bacillus sp. 3B6 was able to synthesise molecules of larger molecular weight than the initial substrate. This was the case of the synthesis of EPS, large polymers from smaller saccharide units and also for C3 compounds synthesised from C1 compounds. These compounds could be released into the cloud water and could be considered as Secondary Organic Aerosols (SOAs). This concept is rather new and of great interest at the moment; even concerning photochemistry processes in cloud water.

For atmospheric microphysics

The production of various metabolites that will be excreted into the medium can have some importance on microphysics. For instance, EPSs which are highly functionalised molecules could have an impact on CCN formation by interacting with water, or due to their ability to change the "wettability" of the cell surface. In this latter case they will act by changing the CCN properties of bio-aerosols.

What do we need in the future??

Our results have brought some new insights in the potential role of microorganisms in atmospheric processes and the ability of microorganisms to survive in clouds. However, they represent only a first step and a lot of experiments are needed to get final answers to these open scientific questions.

For atmospheric microphysics and chemistry

Our results have shown that Bacillus sp. 3B6 was able to metabolise various substrates present in the atmosphere; however, the experimental conditions used were very far from real cloud environment. Therefore new experiments should be conducted using "artificial cloud water" containing most of the metabolites of interest and also carboxylic acids, inorganic ions, etc. with more acidic pH and lower temperatures in order to mimic real cloud waters.

Under these conditions, biodegradation rates should be measured and compared to real photodegradation rates measured under the same conditions. Photo-bioreactors are now available in our laboratories. This will give some indications of the relative contribution of biodegradation processes versus photodegradation ones.

Also the synthesis of EPSs should be tested at lower temperatures close to those in clouds in order to confirm their potential role both in the chemistry ("SOAs hypothesis") and the microphysics ("CCN hypothesis") of clouds.

The further step should be to work with real cloud water containing its endogenous microflora and the complex mixture of organic and inorganic compounds. Now it is possible to collect large amounts of cloud water at the summit of the Puy de Dôme thanks to the rehabilitation of the observatory equipped with chemistry and microbiology laboratories.

However, if one is interested in monitoring the degradation of compounds such as methanol, mannitol, sorbitol, arabitol, glucose, sucrose, and trehalose or other carbohydrates, it is necessary first to develop new tools to show their occurrence and to quantify their concentrations in cloud waters. Our laboratories are fully equipped with sensitive NMR probes and NMR spectrometers as well as Mass Spectrometry instruments, these tools should be used to better characterise the water phase content.

For living in the atmosphere

We have shown that Bacillus sp. 3B6 was able to synthesise numerous EPSs that could potentially protect it against stresses related to atmospheric environment. This hypothesis could be tested under laboratory conditions by exposing the bacterium to UV light, by proceeding to freezing-defreezing cycles, by acting on the composition of the medium to induce acidic, or osmotic stresses. The survival abilities could be evaluated by counting the remaining living cells or by assessing their metabolic changes. This last point could be studied using a metabolomic approach, which is currently under development in our laboratories.

First

  of all, I would like to sincerely thank Dr. Anne-Marie DELORT and Dr. Mária MATULOVÁ for being patient supervisors and for supporting this work with their knowledge, ideas, and criticism. I would like to acknowledge the financial support of the thesis provided by French government (BGF) and Slovak Academy of Science. I would like to thank Martine SANCELME for help with microbiological experiments, Dr. Mounir TRAÏKIA for the help with in vivo NMR experiments, Dr. Laurent DEGUILLAUME for discussions concerning the cloud microphysics, Dr. Peter CAPEK for chemolytical analysis and help with separation techniques, Dr. Vlasta SASINKOVÁ for the help with interpretation of FT-IR spectra. Many thanks to all members of both laboratories, particularly to Dr. Mickaël VAÏTILINGOM, for numerous discussions concerning the atmospheric chemistry and kind help during my first stay at SEESIB laboratory.

Figure 1 .

 1 Figure 1. Life cycle of microorganisms via the atmosphere.

Figure 2 .

 2 Figure 2. AIDA chamber.

Figure 3 .

 3 Figure 3. Simplified scheme of the multiphase cloud chemistry processes taking place in clouds.

Figure 4 .

 4 Figure 4. Similarities between photochemical reactions and microbial metabolism.

Figure 5 .

 5 Figure 5. Estimated relative influence of bacterial activity and free radicals on the degradation of formate, acetate, and succinate in cloud water.

Figure 6 .

 6 Figure 6. Oxidation pathway of methane to methanol and formaldehyde.

Figure 7 .

 7 Figure 7. Pathways of formaldehyde assimilation by the RuMP pathway and path of serine.

Figure 8 .

 8 Figure 8. Disproportionation pathways of formaldehyde to formate and methanol.

Figure 9 .

 9 Figure 9. Different linear pathways of formaldehyde oxidation in Methylobacterium extorquens AM1 and Paracoccus denitrificans.

Figure 10 .

 10 Figure 10. Perfusion system for in vivo NMR experiments.

Figure 11 .

 11 Figure 11. 1 H NMR spectra of the incubation medium of Pseudomonas sp. with formaldehyde and methanol.

Figure 12 .

 12 Figure 12. 13 C NMR spectrum of a suspension of E. coli supplemented with 10 mM 13 C-formaldehyde.

Figure 13 .

 13 Figure 13. 13 C NMR analysis of the kinetics of 13 C-methanol degradation in a cell suspension of B. methanolicus.

Figure 14 .

 14 Figure 14. Example of in vivo 31 P NMR spectrum of the strain E. coli HB101(pUC8).

Figure 15 .

 15 Figure 15. In vivo 2 H NMR spectra of incubations of Staphilococcus aureus, Pseudomonas putida, and E. coli cells with deuteurated formaldehyde.

Figure 1 .

 1 Figure 1. Life cycle of microorganisms via the atmosphere.

Figure 2 .

 2 Figure 2. AIDA chamber: volume of the aerosol vessel is 84 m 3 . Insulated box regulates the temperature in the range -90 °C to +60 °C (temperature control within ±0.3 °C).

Figure 3 .

 3 Figure 3. Simplified scheme of the multiphase cloud chemistry processes taking place in clouds. CCN -cloud condensation nucleus; VOCs -Volatile Organic Carbons; SOAs -Secondary Organic Aerosols.

Figure 4 .

 4 Figure 4. Similarities between photochemical reactions (A) and microbial metabolism (B).

Figure 5 .

 5 Figure 5. Estimated relative influence of bacterial activity (in black) and free radicals (hydroxyl • OH and nitrate NO3 • ) on the degradation of formate, acetate, and succinate in cloud water at 5 °C (A) during the day and (B) during the night and at 17 °C (C) during the day and (D) during the night. During night-time (i.e. in the absence of photochemical reactivity), • OH radicals are not present and were not considered. The following values of concentrations have been used for calculations: [bacteria] = 8.4 × 10 4 cell mL -1 ; [ • OH] = 1.2 × 10 -13 mol L -1 (day-time); [NO3 • ] = 1.5 × 10 -14 mol L -1 ; [formate] = 14.5 × 10 -6 mol L -1 ; [acetate] = 20 × 10 -6 mol L -1 ; [succinate] = 1.5 × 10 -6 mol L -1 . (From

  schemes identify enzymes involved in reactions. Database allows a search according organism or enzymes. Each bacterium has its own pool of enzymes. Although general rules exists (principal reactions are generally conserved), involved enzymes can vary and lateral pathways may not exist. Studies are generally necessary to determine metabolic pathways for a given bacterium or species.

Scheme 4 .

 4 Scheme 4. Metabolic pathways involved in bacterial metabolism. Glycolysis.

Figure 6 .

 6 Figure 6. Oxidation pathway of methane to methanol and formaldehyde. SMMOsoluble methane monooxygenase; pMMO -particulate methane monooxygenase; MDHmethanol dehydrogenase, FADH -formaldehyde dehydrogenase, FDH -formate

Figure 7 .

 7 Figure 7. Pathways of formaldehyde assimilation by (A) the RuMP pathway and (B) path of serine. STHM -serine hydroxymethyl-transferase; HPR -hydroxypyruvate reductase;

Figure 8 .

 8 Figure 8. Disproportionation pathways of formaldehyde to formate and methanol, catalysed by NAD + -dependent oxidoreductase or formaldehyde dismutase.

Figure 9 .

 9 Figure 9. Different linear pathways of formaldehyde oxidation in Methylobacterium extorquens AM1 and Paracoccus denitrificans and their associated cofactors. MtdA -NADP-dependent methylene-H4MPT dehydrogenase; Fch -methenyl-H4F cyclo hydrolase; Fhs -formyl-H4F synthetase; FDH -formate dehydrogenase; Fae -H4MPTdependent formaldehyde activating enzyme; MtdB -NAD(P)-dependent methylene-H4MPT dehydrogenase; Mch -methenyl-H4MPT cyclohydrolase; Ftr -formyltransferase; Fhc -Ftr/hydrolase complex; Gfa -glutathione-dependent formaldehyde activating enzyme; GD-FALDH -NAD + -and glutathione-dependent formaldehyde dehydrogenase;

Figure 10 .

 10 Figure 10. Perfusion system for in vivo NMR experiments described by Chorao et al. (2009). Bacteria are perfused with the incubation medium containing 10% of D2O and

  based on irradiation of water signal by a low energy radiofrequency, are frequently used in the case of samples with higher quantity of metabolites. However, usually in biological samples the energy used for the presaturation cannot be sufficiently strong to suppress efficiently the residual water signal without a partial saturation of signals with the resonance frequency in its neighbourhood and without the baseline distortion of the spectrum. By far, the most often-used water suppression techniques use pulsed field gradients (PFGs) to rapidly attenuate the H 2 O resonance, winding any transverse magnetisation into a tight spatial helix oriented along the PFG axis, and causing the integrated magnetic flux through the NMR receiver coil to nearly vanish for the water magnetisation. A field-gradient pulse is a pulse or a period, during which the magnetic field is made deliberately inhomogeneous. It includes gradient-enhanced coherence transfer pathway selection, which was incorporated into sequences as WATERGATE, excitation sculpting, WET, different modifications of WATERGATE, and PURGE. All these PFG-based methods have their

Figure 11 .

 11 Figure 11. 1 H NMR spectra of the incubation medium of Pseudomonas sp. with formaldehyde and methanol as substrates collected after 2 and 24 hours of incubation.

  C NMR in microbiological studies is a low natural abundance of 13 C isotope (~1%), which does not allow its direct detection due to the low concentration of metabolites. Therefore, utilisation of the 13 C labelled substrates is a widely used technique in microbial metabolism studies. Besides an increased sensitivity, it enables the monitoring of the 13 C label fate in metabolites created by microorganisms during the incubation. Detailed analysis of various isotopomers allows metabolic pathways identification and flux studies.

  studied by in vivo 13 C NMR. Signals at ~60-70 ppm correspond to compounds with longer chain (R-CH 2 -R') formed by the assimilation of formaldehyde. They were assigned to glycerol (propane-1,2,3-triol), propane-1,2-diol, and propane-1,3-diol.

Figure 12 .

 12 Figure 12. 13 C NMR spectrum of a suspension of E. coli supplemented for 4 minutes with 10 mM 13 C-formaldehyde, after 20 minutes (below); after 3 hours (above) (Hunter

Figure 13 .

 13 Figure13.13 C NMR analysis of the kinetics of 13 C-methanol degradation in a cell suspension of B. methanolicus[START_REF] Pluschkell | Dissimilation of[END_REF].

31P

  NMR experiments (Lohmeiner-Vogel et al., 2004) (see Figure14).

Figure 14 .

 14 Figure 14. Example of in vivo 31 P NMR spectrum recorded at 161.8 MHz on the strain E. coli HB101(pUC8). SP -sugar phosphomonoesters; Pi (int), Pi (ext) -intracellular and extracellular pools of inorganic phosphate, respectively; PDE -phosphodiesters; UDPG -uridine diphosphoglucose (Lohmeiner-Vogel et al., 2004).

Figure 15 .

 15 Figure 15. In vivo 2 H NMR spectra of incubations of Staphilococcus aureus, Pseudomonas putida, and E. coli cells with deuteurated formaldehyde[START_REF] Mason | In vivo enzymology: a deuterium NMR study of formaldehyde dismutase in Pseudomonas putida F61a and Staphylococcus aureus[END_REF].

1H

  -1 H homonuclear COSY (COrrelation SpectroscopY) and TOCSY (TOtal Correlation SpectroscopY) experiments reveal covalent correlations (manifested as cross-peaks). COSY and its phase sensitive variant DQF-COSY (Double Quantum Filtered COSY) experiments detect only scalar 1 H-1 H couplings primarily between protons that are 2-4 bonds apart.They use a single step transfer of magnetisation for an identification of neighbouring protons. In the TOCSY experiment, depending on the length of the mixing time, cross-peaks due to the magnetisation transfer to distant protons (showing 3-bond, 4-to 5-bond) are observed. Specific patterns of cross-peaks and their characteristic chemical shifts make thus possible to assign a large number of metabolites directly in crude extracts with high reliability. For metabolite identifications, TOCSY can be superior to COSY, due to its ability to provide a complete covalent network. However, for making individual 1 H-assignments COSY is mostly always needed.For the structure assigning of complex molecules or those ones, which contain fragment(s) with small scalar coupling not detectable by COSY or TOCSY, dipolar correlations may be necessary. Typical example of such molecule is galactose in which the magnetisation transfer from H1 stops at H4 due to a low value of scalar coupling between H4 and H5 protons.Dipole-dipole couplings are observed between spins, which are close to one another in space and which undergo cross-relaxation. The aim of NOESY (Nuclear Overhauser Effect SpectroscopY) experiment is to identify nuclear spins undergoing cross-relaxation and also to measure its rates. Correlations between nuclear spins are established in NOESY during a mixing period. Obtained spectrum is similar to COSY, with diagonal peaks and cross-peaks, however, cross-peaks connect resonances from nuclei that are close in space. Most commonly, due to a sensitivity reason, NOESY is used as a 1 H-1 H homonuclear technique showing cross-peaks for proton nuclei close to each other in space.However, the intensity of build up NOE signals in the spectrum depends on many factors. One of them is a tumbling rate dependence on the molecular mass of the molecule. Thus for certain molecules, whose rotational correlation time falls in range where the NOE effect is too weak or might become zero, it is not detectable in NOESY experiment.

  most cases their structural analysis is based on chemical analyses (sugar compositional analysis and methylation analysis) with simple or no NMR description. The following paragraph is focused on some examples of EPS structure elucidation employing various analytical techniques, including NMR structural analysis. Bacillus circulans belongs to bacteria which yield a number of EPS synthesised isolates: • Fontaine et al. (1991) reported the isolation and structure determination of PS produced by Bacillus circulans with a backbone composed of 1,4-linked galactose (Gal) alternated by two 1,4-linked glucose (Glc) units, all in α-configuration. The lateral chain, linked to galactose unit of the backbone at C3, was composed of β-D-Glcp(1→4)β-D-Man(1→4)β-D-Galp-with pyruvate linked to terminal Glc unit.

•

  Bacillus licheniformis ATCC 9945 was studied by NMR in combination with chemical modification and degradation of the polymer. Results showed that the backbone consists of a pyruvylated pentasaccharide repeating units with the structure {→3)-[(S)Py-(3,4)-β-D-Galp-(1→6)]-α-D-GlcpNAc-(1→3)-β-D-Bacp2N4NAc-(1→3)-[(S)Py-(3,4)-β-D-Galp-(1→6)]-β-D-GalpNAc-(1→}n,

•

  [START_REF] Corsaro | Structure determination of an exopolysaccharide from an alkaliphilic bacterium closely related to Bacillus spp[END_REF] studied a PS produced by an alkaliphilic bacterium closely related to Bacillus sp. by NMR, MS, and chemical analysis. The PS contained D-galacturonic acid (GalA), 2,4diacetamido-2,4,6-trideoxy-D-glucose (QuiNAc4NAc), 2-acetamido-2deoxy-D-mannuronic acid (ManNAcA) and one uncommon unit of D-GalA with the carboxyl group amide-linked to glycine [GalA(Gly)]. The following linear structure of the repeating unit with saccharides in pyranose form was established: [→3)-α-D-GalA(Gly)-(1→4)-β-D-ManNAcA-(1→4)-α-D-GalA-(1→3)-α-D-QuiNAc4NAc-(1→] n .

  considered as a strategy used by bacteria to increase survival in a changing environment. PHA comprises a vast number of structures with repeating subunits of a general formula -[O-CH(R)-(CH 2 ) x -C(O)]-and molecular masses 0.1 to 30 kDa.

  for 1 H NMR by mixing volume of 575 μL of the sample with 25 μL of 20 mM sodium tetra deuterated trimetylsilyl propionate (TSPd 4 , Eurisotop) in D 2 O. D 2 O was used for shimming and locking, while TSPd 4 constituted a reference for chemical shifts (0 ppm) and quantification. Final volume of 600 μL of prepared samples was placed in the 5 mm-diameter NMR tubes.

  with [C] 0 the initial concentration of selected compound C [mol L -1 ]; k the pseudo-first order decays [s -1 ]; N cells the concentration of cells participating to the biodegradation and determined by CFU counts [cells L -1 ].

  Samples were hydrolysed with 2 M trifluoroacetic acid TFA for 1 hour at 120 °C. The quantitative determination of neutral sugars was carried out in the form of their trifluoroacetates by gas chromatography on a Hewlett-Packard Model 5890 Series II chromatograph equipped with a PAS-1701 column (0.32 mm × 25 m), the temperature program of 110-125 (2 °C min -1 ), 125-165 °C (20 °C min -1 ), and a flow rate of hydrogen 2 cm 3 min -1 (Shapira, 1969).

  1 H NMR spectra were measured directly without any further sample treatment. At the beginning of the incubation (t = 0 h) only signals of the substrate and TSPd 4 standard added at known concentration were present in the spectra. Signal intensity changes due to carbohydrate used in relation to that of standard was monitored. Samples, in which the formation of metabolites with carbohydrate structure was observed, were subjected to a detailed structural analysis after freeze-drying and dissolving in D 2 O.

Figure 1 .

 1 Figure 1. 1 H NMR spectra of incubation media (IM) of Bacillus sp. 3B6, in which the carbohydrate substrate was already depleted: A (t = 63 h) on glucose; B (t = 43 h) on cellotetraose; C (t = 63 h) on cellulose; D (t = 72) h on turanose; and E (t = 63 h) on starch. In B (t = 43 h) on cellotetraose still some substrate was present. Similar spectral pattern was observed also in the case of fructose and sucrose at the end of the incubation.

Figure 2 .

 2 Figure 2. EPSs spectral patterns in HSQC spectra of IM media of Bacillus sp. 3B6 on: A -turanose after 72 h and B -cellulose after 63h. Attribution of colours: i) spectrum cross-peak: red -CH2; blue -CH; ii) marks: pink 1,6-linked α-galactan; green and bluenon identified -O-CH2-due to PHA.

Figure 3 .

 3 Figure 3. Selective 1D NMR spectra of the incubation medium of Bacillus sp. 3B6 on turanose: 1 H NMR spectrum of turanose t = 72 h (A); selective 1D TOCSY with mixing time 150 ms spectra with irradiation at δ 5.176 ppm (B) and δ 4.245 ppm (C); selective 1D NOESY spectra with mixing time 200 ms with irradiation of the signal at δ 4.245 (D) and δ 5.176 (E); * unknown.

Figure 2A .

 2A Figure 2A.

Figure 4 .

 4 Figure 4. EPSs spectral patterns in COSY spectra of IM media of Bacillus sp. 3B6 on: A -glucose after 48 h; B -glucose after 63 h and C -cellulose after 63 h. Cross-peaks marked by: pink colour -due to dextran type of polysaccharide with α-1,6-linked galactose in the backbone; green colour -non identified isolated -O-CH2-group.

  Scheme 1. Origin of fractions containing exopolymeric structures EPSs issued by gradual precipitation of non soluble EPSs from the incubation medium obtained after 8 days of the incubation of Bacillus sp. 3B6 on glucose.

Figure 5 .

 5 Figure 5. 1 H NMR spectra of soluble EPSs fractions obtained from IM on glucose (Incubation procedure № 2) by gradual precipitation of non soluble substances: EPSs-1 -IM after first dialysis; EPSs-2 -IM after second dialysis before the precipitation; EPSs-2s -IM after the precipitation; EPSs-3s -after EPSs-2s dialysis; EPSs-4s -soluble part of EPSs-3ns in a mixture of CDCl3:MeOH = 7:3. See also Scheme 1; 3HB -3-hydroxybutyrate.

  heteropolysaccharide backbone. Bands found at 2960, 2935, and 2862 cm -1 could derive from ν(C-H) stretching vibrations of CH groups and that one at 3394 cm -1 from stretching vibrations of OH groups in sugar moiety. The band at 887 cm -1 indicated β-glycosidic linkages of carbohydrates. The presence of carbohydrates linked by α-configuration was deduced on the basis of observed signals at 706 and 775 cm -1 characteristic of α-linkage. Two additional signals characteristic of the α-linkages (expected to appear at 930 and 850 cm -1 ) were hidden by other signals. In addition, IR bands at 1641 and 1566 cm -1 relate to the ν(C=O) and δ(NH 2 ) vibrations of the peptide bonds amide I and amide II, respectively. Bands at 3278 and 3095 cm -1 ν(NH 2 ) originate from the vibrations of primary amide groups. Obtained data suggest that the main component in the precipitate EPS2-ns has a glycoprotein structure.

Figure 6 .

 6 Figure 6. FT-IR spectrum of EPSs-2ns.

  δ 174.64. Identified chemical shifts of glycerol esters were in accordance with published data[START_REF] Lie Ken Jie | 1 H-nuclear magnetic resonance spectroscopic studies of saturated, acetylenic and ethylenic triacylglycerols[END_REF].

  1 H NMR allowed to measure precise biodegradation rates and thus to divide the tested substrates into 3 groups: i) substrates completely degraded within 24-48 hours; (mannitol, glucitol, L-arabinose, glucose, fructose, mannose, rhamnose, sucrose, maltose, trehalose, turanose, cellobiose, cellotetraose, cellulose, glucuronoxylan, and inulin) ii) slowly degraded substrates -their concentration decreased only slowly and at the end of incubation (96-120 hours) only traces of substrates were present (arabitol, ribose, xylose, ribose, lactose, arabinogalactan); iii) not degraded substrates (galactitol, arabitol, glucuronic acid, levan, CMC, α-D-cyclodextrin, β-D-cyclodextrin).

  C NMR to investigate in more details the metabolism of 13 C formaldehyde by Bacillus sp. 3B6, which is the bacterium of major interest in presented PhD work. After the description of the metabolic routes used for formaldehyde and methanol in the various strains, a comparison of the biodegradation rates with those calculated from the reactivity of • OH and NO 3 • radicals has been made. The objective was to quantify the relative contribution of biocatalysis versus photochemistry under various environmental scenarios, including the temperature of the clouds (5 or 17 °C), the origin of the clouds (urban and remote) and the diurnal cycle (day-and nighttime).

NO 3 •

 3 ) have been quantitatively evaluated. Clearly, measured biological and chemical reaction rates are in the same range of magnitude, and their relative contribution varies according to the scenarios we have tested, including the temperature of the clouds (5 or 17 °C), the origin of the clouds (urban and remote) and the diurnal cycle (day-and nighttime). More precisely it was shown that: i) During daytime, • OH are obviously the preponderant actors. However, biotransformation processes could be the main sink for C1 compounds during night-time where the radical chemistry is less efficient than enzymatic reactions. This is explained by the kinetic constant of NO 3 • with these organic compounds that are lower than those with • OH and by the lower NO 3 • concentration during the night compared to those of • OH during the day. The same conclusions were reached in our previous study

Scheme 1 .

 1 Metabolic pathways involved in bacterial metabolism. Citrate cycle (TCA cycle). Metabolic pathways involved in bacterial metabolism. Glyoxalate and dicarboxylate metabolism.

	Scheme 2. Metabolic pathways involved in bacterial metabolism.
	Pyruvate metabolism.
	Scheme 3. Scheme 4. Metabolic pathways involved in bacterial metabolism.
	Glycolysis.

Table 1 .

 1 Concentration of microorganisms in atmospheric water samples.

Table 2 .

 2 Content of most important dicarboxylic acids, cations, and anions in cloud water.

Table 1 .

 1 Concentration of microorganisms in atmospheric water samples.

	Localisation	Type of atmospheric water sample	Concentration of fungi and yeasts [mL -1 ] Concentration of bacteria	References
	Puy de Dôme mountain, France, 1464 m.a.s.l.	Cloud	1.7×10 4 -2.4×10 5 E 8.9×10 2 -2.5×10 4 E	Amato et al., 2007b
	Rax mountain, Austria, 1644 m.a.s.l.	Cloud	2×10 4 C	5.9×10 3 C	Bauer et al., 2002; 2003
	Sonnblick mountain, Austria, 3106 m.a.s.l.	Cloud	1.5×10 3 D		Sattler et al., 2001
	Kleiner Feldberg	Cloud		1.05×10 6 A
	mountain,					Matthias-Maser et
	Germany, 857					al., 2000
	m.a.s.l.	Rain		3.57×10 3 A
	Po valley, Italy	Fog	30-150	B	30-60
	Shizuoka and Tsukuba, Japan	Rain	1.6×10 4 -2.4×10 4 A	Casareto et al., 1996
	Charlottesville, Virginia, USA	Rain	2×10 5 -6×10 5 A	Herlihy et al., 1987

B

[START_REF] Fuzzi | Fog droplets-an atmospheric source of secondary biological aerosol particles[END_REF]

A Total number of Primary Biological Aerosol particles (PBAs) B CFU (colony forming units), i.e. cultivable microorganisms C Average from 3 clouds events D Average from 12 clouds events E Average from 14 clouds events

  . Only two papers reported the description of bacterial population present in clouds using molecular tools. First, Ahern et al. (2007a) used direct analysis of DNA by PCR

(polymerase chain reaction) amplification and cloning the 16S rDNA genes to investigate the microbial population of clouds and rainwaters collected in Scotland. Many strains belonging to the Pseudomonas and Acinetobacter genera have been identified in this way. More recently, Kourtev et al., (2011) used DGGE (Denaturing Gradient Gel Electrophoresis) and sequencing 16S rRNA gene amplicons to analyse cloud bacteria from cumulus clouds in USA. The main bacterial phyla belonged to Cyanobacteria, Proteobacteria, Actinobacteria, and

Table 2 .

 2 Content of most important dicarboxylic acids, cations and anions in cloud water measured by Ionic Chromatography (IC).

	Cloud

water sampled at the Puy de Dôme station Compound min max concentration (μM)

	Acetate	0.6	48.7
	Formate	0.8	71.4
	Succinate	0.1	4.1
	Oxalate	0.1	17.4
	Cl -	0.5	1948.9
	NO3 -	3.4	766.8
	SO4 2-	1.9	369.5
	Na +	0.4	281
	NH4 +	6.3	1801.7
	K +	0.1	124.1
	Mg 2+	0.2	45.6
	Ca 2+	0.3	74.8
	pH	3.9	7.6

Table 3 .

 3 Ambient concentrations of saccharides, sugar-alcohols and vanillin during the

	haze and none-haze days, ng m -3 (haze event caused by wheat straw burning) (Wang et
	al., 2011).
		June 1-5	June 5-17	October 12-24
		Haze (n = 1)	None-haze (n = 3)	None-haze (n = 3)
	Levoglucosan	4030	204 ± 129	689 ± 243
	Fructose	72	32 ± 2.0	32 ± 13
	Glucose	186	49 ± 27	44 ± 8.3
	Sucrose	68	177 ± 24	31 ± 19
	Trehalose	20	21 ± 0.7	17 ± 1.9
	Arabitol	195	16 ± 2.4	21 ± 3.6
	Mannitol	123	26 ± 3.9	24 ± 4.2
	Inositol	19	4.6 ± 0.3	7.0 ± 2.8
	Glycerol	207	41 ± 4.8	44 ± 11
	Vanillin	16	0.5 ± 0.2	2.8 ± 0.9
	Subtotal	4938	571 ± 125	919 ± 280

Formate 5.6% Acetate 26.9% Formate 97.2% Acetate 98.8% Night Day C D Succinate 97.5%

	A	Day
	Formate	Acetate	Succinate
	1.9%	3.0%	14.7%
			Bacteria
			OH•
	B	Night	NO3•	5°C
	Formate	Acetate	Succinate
	90.4%	90.6%	90.3%
			Bacteria
			NO3•
			Succinate
			37.0%
			Bacteria
			OH•
			NO3•	17°C
			Bacteria
			NO3•

Formate 1.9% Acetate 3.0% Formate 90.4% Acetate 90.6% Night Day A B Succinate 90.3%

	Succinate
	14.7%
	Bacteria
	OH•
	NO3•	5°C
	Bacteria
	NO3•

Formate 5.6% Acetate 26.9% Formate 97.2% Acetate 98.8% Night Day C D Succinate 97.5%

	Succinate
	37.0%
	Bacteria
	OH•
	NO3•
	Bacteria
	NO3•

-1 )

	Medium or compound	17 °C	Acetate	Formate	Succinate	Oxalate
		P1	0	7.1 (± 2.2) × 10 -10	0	2.7 (± 1.2) × 10 -10
	Marine	P2	0	2.4 (± 0.1) × 10 -11	0	2.2 (± 0.9) × 10 -11
		Microbial

* 5.3 (± 9.1) × 10 -11

  The natural abundance of 1 H is close to 100% and thus makes the proton

NMR technique the most sensitive: A tagging of the molecules is not necessary. It is a quantitative technique but low range of proton resonance (~15 ppm) may lead to overlapping of peaks. Very important drawback in biological samples studies is the presence of a huge signal of water. Thus, techniques enabling its efficient suppression are of great importance because of the residual water signal adversely affect appearance and subsequent analysis of NMR spectra. A lot of methods allowing the water signal suppression have been developed. The most frequently used techniques are described below.

  The original pulse sequence was composed of an initial 90° pulse bringing all magnetisation into the xy-plane. A gradient is used to encode

the coherences, and refocusing is employed by a non-selective 180° hard pulse surrounded by two 90° shaped pulses of opposite phase. After the refocusing pulses, another identical time period was used containing a final gradient of equal amplitude. Solute was effectively encoded by the first gradient, reversed, and decoded by the final gradient. The type of selective pulses, amplitudes, and durations determined the efficacy and selectivity of the solvent suppression. Optimisation could be achieved by modifying the type of shaped pulse and its duration (McKay, 2009). Nowadays, WATERGATE is often integrated into various pulse sequences as a solvent suppression. For example, Price et al. (2002) developed PGSE-WATERGATE (Pulse Gradient Spin Echo WATERGATE) sequence by integrating the WATERGATE unit into original Hahn spin echo based PGSE sequence. By its application effective solvent signal suppression was echieved. PGSTE-WATERGATE was applied by Zheng et al. (2008) for measuring diffusion coefficients in aqueous solution, where WATERGATE provides excellent solvent suppression without any phase distortions. The progress in water/solvent suppression techniques is clearly visible in many available reviews

  Nevertheless, 2 H NMR spectroscopy of deuterated samples has frequently been used for biosynthetic investigations, in cases where an analyte is available in sufficient amounts. Taking advantage of the strength of NMR for stereochemical analysis, 2 H NMR can be used to determine the steric course of biosynthetic reactions. Chemical shifts of

2

H resonances are similar to its corresponding 1 H signals and thus the assignment can be performed on non labelled compound and further extrapolated to 2 H labelled one. Another advantage, especially for labelling purposes, is the low natural abundance of 2 H (less than 0.012%).

  For example, the one bond coupling constant 1 J CH of the methyl group in alanine and lactate is about 127 Hz, whereas for the methine group it is ca. 145 Hz. Anomeric sugar CH have 1 J CH of around 160-170

	For their assignment correlations to
	heteroatoms (carbon, nitrogen) may be needed. HSQC (Heteronuclear
	Single Quantum Coherence) and HMBC (Heteronuclear Multiple-Bond
	Correlation) are the most frequently used heteronuclear correlated
	experiments. They correlate in most cases 1 H with 13 C via scalar coupling
	interaction between the both nuclei. The efficiency of the magnetisation
	transfer depend on the value of coupling constants, value of which is
	variable.

Table 1 .

 1 Bacillus sp. 3B6 ability to degrade different types of saccharides.

	Tested saccharides	0	24	Incubation time [h] 48	72	96	Biodegradation rate [mol cell -1 s -1 ]
			Alditols
	Mannitol	+++	++	-		-	-	5.4 × -7
	D-Glucitol	+++	+++	+		-	-	5.38 × -7
	D-Galactitol	+++	+++	+++	+++	+++	0
	L-Arabitol	+++	+++	+++		++	-	2.47 × -7
	D-Arabitol	+++	+++	+++	+++	+++	0
			Monosaccharides
	L-Arabinose	+++	++	-		-	-	4.08 × -7
	D-Xylose	+++	+++	+++		++	++	1.04 × -7
	D-Ribose	+++	++	+		+	-	2.33 × -7
	L-Ribose	+++	+++	+++		++	++	1.12 × -7
	D-Fructose	+++	+	EPS		EPS	EPS	6.04 × -7
	D-Glucose	+++	+	EPS		EPS	EPS	7.14 × -7
	D-Galactose	+++	+	-		-	-	7.4 × -7
	D-Mannose	+++	+	-		-	-	6.25 × -7
	L-Rhamnose	+++	++	-		-	-	6.84 × -7
	Glucuronic acid	+++	+++	+++	+++	+++	0
			Disaccharides
	Lactose	+++	++	++		+	-	5.72 × -7
	Sucrose	+++	++	+		-	EPS	5.76 × -7
	Maltose	+++	++	-		-	-	5.14 × -7
	Trehalose	+++	++	+		-	-	5.32 × -7
	Turanose	+++	+++	+		EPS	EPS	5.67 × -7
	Cellobiose	+++	++	+		-	-	5.41 × -7
			Tetrasaccharides
	Cellotetraose	+++	++	+		EPS	EPS	5.21 × -7
			Polysaccharides
	Cellulose	+++	+++	EPS		EPS	EPS	NA
	Arabinogalactan	+++	+++	+++		++	+	1.14 × -7
	Glucuronoxylan	+++	++	+		-	-	5.52 × -7
	Inulin	+++	++	+		-	-	5.73 × -7
	Levan	+++	+++	+++	+++	+++	0
	Starch	+++	++	EPS		EPS	EPS	5.28 × -7
	CMC	+++	+++	+++	+++	+++	0
	α-D-Cyclodextrin	+++	+++	+++	+++	+++	0
	β-D-Cyclodextrin	+++	+++	+++	+++	+++	0
	CMC -carboxymethyl cellulose, EPS -mixture of exopolymeric substances, NA -not
	available +++ 100% of substrate present in IM
	++ cca 60% of substrate present in IM + cca 30% of the substrate present in IM
	-substrate consumed completely

Table 2 .

 2 NMR chemical shifts of various metabolites synthesised by Bacillus sp. 3B6.

	Compound	Solvent	CH	Chemical shift δ H/C [ppm] CH2 CH3	COOH
	PHA	D2O	4.050/70.21	3.908/67.14 3.967;	2.023/22.95	177.52
	3HB	D2O	4.135/66.77	2.297/47.20 2.391;	1.179/22.67	181.28
	3HB	7:3 CDCl3:MeOH=	5.253/67.97	2.492/40,95 2.614;	1.271/19.86	168.95
	acid Butyric	D2O		3.541/63.40 3.63; 3.761/62.25	1.473/17.06	178.5
	Glycerol	CDCl3:MeOH= 7:3	3.877/70.47	3.640, 3.55/63.49
				4.124/65.38
			1,6-linked α-galactan
	Chemical
	shift δ	H1/C1	H2/C2	H3/C3	H4/C4	H5/C5	H6;H6`/C6
	[ppm]
	α-Galactose 5.176/99.47 3.803/69.19 3.917/70.10 4.036/69.76 4.245/70.56	4.026, 3.98/65.21
	PHA -polyhydroxyalkanoate; 3HB -3-hydroxybutyrate

Table 3 .

 3 Identified soluble and insoluble compounds in individual fractions formed by gradual precipitation from the incubation medium (IM) on glucose (Incubation procedure № 2) after 8 days of incubation.

	Identified Compounds	Fractions
	1,6-Linked α-galactan	EPSs-2, EPSs-2, EPSs-3s
	PHA	EPSs-2, EPSs-2s, EPSs-3s
	3HB	EPSs-2s, EPSs-4s
	Butyric acid	EPSs-2s
	Glycerol	EPSs-2s, EPSs-4s
	Esters of glycerol	EPSs-4s
	Proteoglycan	EPSs-2ns

Table 2

 2

	)

some of long range interactions could be detected from CH 3 at δ 0.88 to CH 2 at δ 21.63, 34.86 and from CH 2 at δ 2.34/δ 34.44 to CH 2 at δ 24.19

  FR-0009-07), the French Government Scholarship (fellowship for S.

	Husárová), the Slovak Grant Agency VEGA № 2/0116/10 and the Slovak
	state program 2003SP200280203 and Research & Development
	Operational Program funded by the ERDF for Centre of excellence for
	glycomics. ITMS: 26240120031.

Scheme 2. Metabolic pathways involved in bacterial metabolism. Pyruvate metabolism.

Acknowledgement

This research has been supported by the CNRS LEFE-CHAT program, the French-Slovak collaborative program Štefánik project № 17947UE (SK-

Abstract

The ability of Bacillus sp. 3B6 to metabolise different types of carbohydrates and to produce exopolymeric substances (EPSs) was evaluated by using in situ NMR spectroscopy. Different saccharides (aldoses, ketoses) were chosen covering penta-and hexasaccharides in acyclic and cyclic forms as well as oligo-and polysaccharides. Under low concentration of substrates, it was shown that when carbohydrate carbon source is nearly depleted, this bacterium starts to excrete a mixture of EPSs. One EPS component was identified as 1,6-linked α-galactan. It was detected in incubation media on turanose, glucose, cellotetraose, cellulose, fructose, sucrose, and starch. Other EPSs present in the medium did not contain saccharide molecules. Their structure was not identified precisely. However, NMR data indicate that they belong to polyhydroxyalkanoate polymers (PHA). As 3-hydroxybutyrate was found in the medium it is likely that these PHAs have the structure very close to polyhydroxybutyrate (PHB). When this strain was incubated with high concentration of glucose, non soluble EPSs precipitated gradually form in the incubation medium. Sugar compositional and elemental analyses as well as infrared spectra suggest a glycoprotein structure of the main precipitate. Other components of incubation medium have been also identified: butyric acid, glycerol, and esters of glycerol. Finally, as Bacillus sp. 3B6 has been isolated from cloud water a discussion is given about the potential implication of EPSs in such an atmospheric environment.

Keywords: exopolymeric substances, EPS, structure, NMR, Bacillus,

clouds

RESULTS

Such an extensive study of carbohydrate metabolism by Bacillus species and particularly of EPS structural elucidation by NMR is rather rare in the literature. These data are the first ones reported for a strain isolated from the atmospheric compartment.

Our results show that this strain could be of interest for biotechnological applications in the domain of pharmacy, nutraceutic and food industry.

Mixture of FOSs of levan and inulin type produced by this bacterium on

sucrose could find its application in food industry as a prebiotic. Bacteria only seldom produce inulin FOSs and polysaccharides and inulin FOSs are mostly prepared from chicory inulin. DFA IV, β-D-Fruf-β-D-Fruf-2',6:2,6'-dianhydride, could find its application in food industry as a low calorie sweetener but also as a promising drug for cancer treatment. On glucose, the production of PHA of unknown structure was observed. PHA reveal several properties that lend them to various technical, medical, pharmaceutical and other applications (termoplasticity, biodegradability…). Therefore, PHA have attracted the interest of the chemical industry.

In addition, these results also indicate a new direction of research concerning the potential role of bacteria producing EPS in the atmosphere. Indeed some of the saccharides tested were found in the atmosphere in rather high amounts.

NMR Study of C1 Compounds

Introduction

The role of microoganisms in atmospheric chemistry is still an emerging question. Recent data have shown the potential of microorganisms, as biocatalysts, to transform carboxylic acids present in cloud water. A