Biotransformation of Various Saccharides and Production ofExopolymeric Substances by Cloud-Borne Bacillus sp. 3B6Maria Matulova,† Slavomíra Husarova,†,‡ Peter Capek,† Martine Sancelme,‡,§ and Anne-Marie Delort*,‡,§

†Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dubravska cesta 9, SK-845 38 Bratislava, Slovakia‡Clermont Universite, Universite Blaise Pascal, ICCF, BP 10448, F-63000 Clermont-Ferrand, France§CNRS, UMR 6296, Institut de Chimie de Clermont-Ferrand, F-63177 Aubiere, France

*S Supporting Information

ABSTRACT: The ability of Bacillus sp. 3B6, a bacterial strain isolated fromcloudwaters, to biotransform saccharides present in the atmosphere was evaluatedusing in situ 1D and 2D NMR spectroscopy. Bacillus is one of the genera mostfrequently described in the air and in atmospheric waters. Sugars present in theseenvironments have a biogenic origin; they include alditols, monosaccharides,disaccharides, oligosaccharides, and polysaccharides. Bacillus sp. 3B6 was able toefficiently metabolize sugars, which could thus provide sources of energy for thisbacterium and allow it to live and to be metabolically active in warm clouds. Inaddition, a number of these saccharides (L-arabitol, D-fructose, sucrose, D-glucose,cellotetraose, cellulose, and starch) were transformed to EPSs (exopolymericsubstances). We have clearly identified the structure of two EPSs as 1,6-α-galactanand partially acetylated polyethylene glycol. 1,6-α-Galactan is a newly described polymer. The production of EPSs might protectthis bacterium under hostile cloud environment conditions, including low nutrient availability, cold temperature and freeze−thawprocesses, UV and radical exposure, and evaporation−condensation processes and thus desiccation and osmolarity changes. EPSscould also have a potential role in atmospheric processes because they can be considered as secondary organic aerosols andefficient cloud condensation nuclei.

1. INTRODUCTION

Although air-borne microorganisms have been studied for along time,1−5 the discovery of microorganisms in cloudwaters israther recent.6−16 Only a fraction of atmospheric micro-organisms were cultivable; however, it was shown that most ofthem (notably in clouds) were metabolically active,7,13,14,17−19

thus suggesting that microorganisms are able to survive underthe stressing conditions of the atmospheric environment (UVexposure, freeze−thaw processes, evaporation−condensationcycles, cold stress, etc.) and can use various organic compoundsas carbon sources. This biocatalytic activity raised new scientificquestions about the role of microorganisms in the multiphasechemistry of clouds.20,21 In this context, few papers havereported the effective degradation of organic compounds,including mono and carboxylic acids, as well as methanol andformaldehyde, by atmospheric microorganisms.22−28 Werecently showed that biodegradation of these compounds wascompetitive with radical processes using real cloudwatersamples containing wide microbial biodiversity and a complexchemical composition under realistic atmospheric conditions ina photobioreactor (artificial solar light, low temperature).14 Inaddition to organic acids, methanol, and formaldehyde, theseactive microorganisms could also transform the dissolvedorganic matter present in cloudwaters. The composition of thismatter remains largely unknown; mono and dicarboxylic acids,alcohol, and aldehydes account for less than 20% of the DOC(dissolved organic carbon);29 the rest is composed of

macromolecules including HULIS (humic-like substances)and SOAs (secondary organic aerosols) such as oligomers.Sugars are also likely to be present. Various classes ofsaccharides have been reported as important constituents ofambient atmospheric aerosols (Table S1, Supporting Informa-tion), including sugar alcohols [glycerol, treitol, erytritol,arabitol, xylitol, glucitol (sorbitol), mannitol, inositol, ribitol,and galactitol], monosaccharides (glucose, mannose, galactose,rhamnose, fructose, xylose, arabinose, lyxose, and fucose),disaccharides (sucrose, trehalose, maltose, and cellobiose),trisaccharides (melezitose and rafinose), anhydrosugars (levo-glucosan, mannosan, galactosan, and 1,6-anhydroglucofura-nose), and polysaccharide, such as cellulose. The presence ofthese saccharides in the atmosphere is the signature of biogenicsources of aerosols. Monosaccharides (glucose, fructose, andxylose) and disaccharides (sucrose and trehalose) can bereleased into the atmosphere by microorganisms, plants, andanimals.30 Sugar alcohols, such as glycerol, D- and L-arabitol,mannitol, and D-glucitol, can be produced by fungi, lichens, andbacteria.31−34 Levoglucosan derives from cellulose; galactosansand mannosans derive from hemicelluloses during biomassburning.31−33,35 The nature and concentration of sugars present

Received: March 19, 2014Revised: November 12, 2014Accepted: November 12, 2014

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in aerosols were dependent on the time of year33,36,37 and alsoon the diurnal cycle.31,38 Although no quantitative measure-ment of sugar concentrations was made in cloudwaters, thepresence of sugars in atmospheric waters is likely due to theirhigh solubility in water and their ability to act as cloudcondensation nuclei (CCN).39 A recent work by Santl-Temkivet al.16 reports the presence of carbohydrates in hailstones: 3%of the identified DOC was composed of fatty acids, sulfonicacids, and carbohydrates as measured by FT-ICR-MS.Considering the amount of DOC in cloudwaters,14,29

presumably the carbohydrate concentration should be in themicromolar range.Sugars are well-known substrates for microorganisms; they

represent the main source of carbon and energy for many ofthem. In some cases, microorganisms can convert sugars intoexopolymeric substances (EPSs) very effectively.40 These EPSscan be polysaccharides or other polymers, such as proteicstructures or polyhydroxyalkanoates (PHAs). Microbial poly-saccharides are multifunctional and may have homo- orheteropolymeric compositions of high molecular weight (10−30 kDa).The aim of this paper was to investigate the biotransforma-

tion of sugars usually encountered in the atmosphere by thecloud microorganism Bacillus sp. 3B6. This bacterium wasisolated in cloudwaters at the Puy de Dome station.10 There aretwo reasons for the choice of this strain: first, Bacillus is one ofmajor genera described in the free troposphere, includingclouds6,13 and the air,4 and second, the metabolism of thisstrain was shown to be active on some compounds present inthe atmosphere, including methanol and formaldehyde,28 aswell as sucrose.41 We report here the ability of Bacillus sp. 3B6to use various carbohydrates present in the atmosphere and toexcrete EPSs. The kinetics of sugar transformations wasmonitored by in situ 1H nuclear magnetic resonance (NMR)spectroscopy performed directly on the samples of crudeincubation media. The EPSs structures were elucidated usingmultidimensional NMR techniques. A discussion is given aboutthe potential implication of these results for the atmosphericenvironment. First, EPSs could protect bacteria and help themto survive under the stressing conditions encountered in clouds.Second, these highly functionalized molecules could beconsidered as SOAs and favor CCN formation.

2. MATERIALS AND METHODS2.1. Bacterial Strain and Culture Conditions. Bacillus sp.

3B6 was isolated from cloudwater sampled at the Puy de Domesummit (1465 m) in the Massif Central Region of France andwas identified by 16S rRNA gene sequencing as described byAmato et al.10

Liquid, pure preculture was incubated in 100 mL portions ofTSA (trypcase soy broth, Biomerieux, Marcy l’Etoile, France)in 500 mL Erlenmeyer flasks at 27 °C and 200 rpm. Cells wereharvested by centrifugation (8000g and 4 °C for 5 min) after 24h of growth and rinsed twice in 0.8% NaCl and resuspended inthe medium for biodegradation studies.2.2. Incubations of Bacillus sp. 3B6 with Low Sugar

Concentrations. Arabinogalactan and glucuronoxylan (In-stitute of Chemistry, Slovak Academy of Sciences) and all theother tested saccharides (Sigma-Aldrich) were used as received.Incubation media were prepared by the dissolution of

saccharides in Volvic mineral water. Each tested saccharide wasused as a unique carbon source. The final concentration ofsaccharides in the incubation medium was 20 mM for mono-

and disaccharides, 10 mM for oligosaccharides, and 4 g L−1 forpolysaccharides. The microbial concentration was 109 cellsmL−1. The incubations of Bacillus sp. 3B6 with sugars wereconducted under aerobic conditions (200 rpm) at 27 °C. Theexperimental conditions used here were similar to thoseconducted with this strain incubated with methanol andformaldehyde in a previous paper;28 therefore, the resultsobtained with sugars and VOCs can be directly compared. Dueto the limited sensitivity of NMR experiments, it was inevitableto scale up the concentration of the tested substrates togetherwith the bacterial concentration while the substrate-to-cell ratioidentified in the natural environment of clouds was maintained.In a previous work26 it was shown that the biodegradation ratesare only substrate-to-cell ratio dependent, regardless of theconcentration chosen.Samples of 1.5 mL were taken at regular intervals (0, 15, 19,

24, 39, 63, 72, 96, and 120 h), these samples were centrifuged(3 min, 12 000g), the cell pellet was discarded, and thesupernatants were kept frozen (−20 °C) until NMR measure-ments were done.

2.3. Incubation of Bacillus sp. 3B6 with a HighConcentration of D-Glucose. The objective of the incubationwith a high concentration of glucose was to get a maximumyield of EPSs necessary for their structural study by low-sensitivity 2D 1H−13C heterocorrelated NMR sequences; thus,the conditions, including the temperature, had to be differentfrom those described when a low concentration of glucose wasused. For EPS production with a high D-glucose concentration,a medium proposed by Gandhi et al.42 was used. Itscomposition was as follows: D-glucose (30 g L−1) (Acros),ammonium nitrate (0.4 g L−1) (Acros), potassium sulfate (1 gL−1) (Acros), sodium chloride (1 g L−1) (Acros), potassiumdihydrogen phosphate (3 g L−1) (Fluka), calcium chloride(0.02 g L−1) (Sigma-Aldrich), and magnesium sulfate (0.001 gL−1) (Sigma-Aldrich). The medium was autoclaved for 15 minat 121 °C, and 24 h old inoculum was used to seed the sterilemedium. The culture was incubated in a rotary shaker at 200rpm and 32 °C. After 8 days of incubation, the medium wascentrifuged (20 000g, 25 °C, 30 min), and Bacillus sp. 3B6 cellswere separated from the supernatant. Low molecular weightresidues were removed from the crude incubation medium bydialysis against distilled water (two times) until the 1H NMRspectra confirmed the absence of D-glucose, salts, and aminoacids in the incubation medium. After a volume reduction, thesample was freeze-dried and redissolved in D2O for NMRmeasurements. The soluble fraction containing EPSs wasfurther analyzed by 1D and 2D NMR.

2.4. NMR Spectroscopy. Monitoring Sugar Biodegrada-tion by 1D in Situ 1H NMR. Supernatants from thebiodegradation test media were prepared for 1H NMR bymixing 575 μL of the sample with 25 μL of 20 mM thetetradeuterated sodium salt of trimethylsilyl propionic acid(TSP-d4, Eurisotop) dissolved in D2O. D2O was used forshimming and locking, whereas TSP-d4 constituted a referencefor chemical shifts (0 ppm) and quantification. A final volumeof 600 μL of prepared sample was added to the 5 mm diameterNMR tubes. Acquisition was performed at 500 MHz on aBruker Avance NMR spectrometer or on a 600 MHz VNMRSVarian spectrometer equipped with an HCN 13C-enhanced salt-tolerant cold probe. The water signal was eliminated bypresaturation and by pulse field gradient spin echo (GPFSE).The concentrations of metabolites were calculated as follows

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= ‐b

AA

d[m]9

[TSP ]0

ref4

(1)

where [m] is the concentration of compound to be quantified,A0 is the area under the m resonance, Aref is the area under theTSP-d4 resonance, and 9 and b are the numbers of protons inTSP-d4 and in the metabolite m, respectively.Structural Analyses of EPSs. Chosen samples were freeze-

dried, dissolved in 99.98% D2O, and further measured at 25 °Cin 3 mm sample tubes on a 600 MHz VNMRS Varian equippedwith an HCN 13C-enhanced salt-tolerant cold probe using TSP-d4 as an external standard. Advanced techniques from theVarian pulse library of two-dimensional homo- and hetero-correlated spectroscopy (COSY, 1H−1H homocorrelatedspectroscopy; TOCSY, 1H−1H total correlation spectroscopy;HSQC, 1H−13C Heteronuclear single quantum correlationspectroscopy; HMBC, 1H−13C heteronuclear multiple quan-tum correlation spectroscopy; and H2BC, heteronuclear two-bond correlation spectroscopy) including one-dimensionalsequences with selective excitations (1D NOESY, NOEspectroscopy and 1D TOCSY) were used for the signalassignments.

3. RESULTS3.1. Biodegradation of Different Sugars. The ability of

Bacillus sp. 3B6 to metabolize different types of carbohydrateswas monitored by in situ 1H NMR spectroscopy. The different

mono- and disaccharides (aldoses and ketoses) were chosen tocover pentoses and hexoses in acyclic and cyclic forms (Table1), the presence of which was determined in aerosol samples(Table S1, Supporting Information). We also investigated thedegradation of polysaccharides, constituents of plant debris,which can be potentially aerosolized, including cellulose (andits products of degradation cellobiose and cellotetraose),arabinogalactan, glucuronoxylan, inuline, and starch. Sampleswere taken from the incubation media at regular intervals. Aftercentrifugation, used for cell elimination, the supernatant wasdirectly analyzed by 1H NMR without any further sampletreatment. The sugar concentrations at different intervals wereestimated as the ratio of the anomeric signal intensity (ifpresent; otherwise, another not-overlapped signal was chosen)and the signal intensity of the TSP-d4 standard added at aknown concentration (as described above).Experimental results showed that Bacillus sp. 3B6 metabo-

lized all the tested carbohydrates that can be potentially presentin the atmosphere (Table S1, Supporting Information).Although the exact rates of degradation were difficult todetermine using these complex spectra, the percent degradationwas roughly quantified (100%, 60%, and 30%) at measuredintervals of 0, 24, 48, 72, and 96 h of incubation (Table 1). Thisrough estimation allowed for comparing the efficiency of thebiodegradation of the different saccharides. After 48 h ofincubation, mannitol, L-arabinose, D-fructose, D-glucose, D-galactose, L-mannose, L-rhamnose, sucrose, and maltose were

Table 1. Ability of Bacillus sp. 3B6 To Degrade Different Types of Saccharides and Produce EPS

products detected over time

tested saccharides 0 h 24 h 48 h 72 h 96 h

Alditolsmannitol +++ ++ − − −D-glucitol +++ +++ + − −L-arabitol (arabinitol) +++ +++ +++ ++/EPS EPSD-arabitol +++ +++ +++ +++ +++

MonosaccharidesL-arabinose +++ ++ − − −D-xylose +++ +++ +++ ++ ++D-ribose +++ ++ + + −L-ribose +++ +++ +++ ++ ++D-fructose +++ + − EPS EPSD-glucose +++ ± EPS EPS EPSD-galactose +++ ++ − − −L-mannose +++ ++ − − −L-rhamnose +++ + − − −

Disaccharideslactose +++ ++ + ± −sucrose +++ ++ − EPS EPSmaltose +++ ++ − − −trehalose +++ ++ + − −cellobiose +++ ++ ± − −

Tetrasaccharidescellotetraose +++ ++ ++/EPS +/EPS −/EPS

Polysaccharidescellulose ND ND ND/EPS ND/EPS ND/EPSarabinogalactan +++ +++ +++ ++ +glucuronoxylan +++ ++ + ± −inulin +++ ++ + − −starch ND ND/EPS ND/EPS ND/EPS ND/EPS

EPS, mixture of exopolymeric substances. +++ = 100% of substrate, ++ = cca 60%, + = cca 30%, ± = traces of the sugar substrate, − = no sugarpresent in the incubation medium. ND, not determined.

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completely degraded. D-Glucitol, D-ribose, lactose, andtrehalose were degraded up to 30%. After 72 h, approximately60% of L-arabitol, D-xylose, and L-ribose and 30% of D-riboseand D-cellotetraose still remained in the media. Only D-arabitolwas not degraded by this strain. The slowest degradations wereobserved for D-xylose and L-ribose; 60% was still present in themedium after 96 h. The degradation of water-solublepolysaccharides (inulin, arabinogalactan, and glucuronoxylan)was monitored as described above: glucuronoxylan and inulinwere completely degraded within 72 h, whereas 30% ofarabinogalactan remained after 96 h. The limited solubility ofcellulose and starch due to their higher molar masses made thedirect quantification of these substrates from the 1H NMRspectra of the incubation media impossible. However, thesignals of the newly formed EPSs were detectable (see section3.2).

Although the biodegradation rates for sugars could not bemeasured very precisely, they were evaluated as follows:considering that 109 cells in 1 mL incubation volume degrade2 × 10−7 mol of sugars within 48−96 h, the biodegradationrates were estimated to be in the range from 1.16 × 10−19 to5.78 × 10−20 mol cell−1 s−1. These values are in the same rangeas those measured with the same strain when degradingmethanol (2.9 × 10−21 mol cell−1 s−1) and formaldehyde (2.0 ×10−20 mol cell−1 s−1).28 Therefore, Bacillus sp. 3B6 degradessugars with the same efficiency as it degrades formaldehyde andmethanol.

3.2. Exopolymeric Substances. In several cases (L-arabitol, D-glucose, D-fructose, sucrose, cellotetraose, cellulose,and starch), the formation of EPSs was observed. Theirproduction was identified from the 1H NMR spectra by thepresence of characteristic broad, complex spectral patterns of

Figure 1. 1H NMR spectra of incubation media samples of Bacillus sp. 3B6 incubated on L-arabitol (t = 72 h), D-fructose (t = 72 h), sucrose (t = 72h), D-glucose (t = 63 h), cellotetraose (t = 48 h), cellulose (t = 63 h), and starch (t = 63 h).

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EPSs (Figure 1). The excretion of EPSs, synthesized intra-cellularly, to the extracellular incubation medium was observedafter the depletion of the initial saccharide substrate. Becausewe only examined this extracellular medium by NMR, wecannot detect the presence of EPSs before their excretion. Inaddition, due to the relatively low sensitivity of the NMRtechnique, EPS signals are only detected when they start toaccumulate in the medium. Other 1H NMR resonances weredetected notably in the range of 1−3 ppm (not shown), but thestructure of the corresponding metabolites was not identified.The present paper will focus only on the identification of EPSs,as these polymers could be relevant for the atmosphericenvironment.For fast-metabolized saccharides (D-fructose and D-glucose)

and for oligo- and polysaccharides (cellotetraose, cellulose, andstarch), the formation of EPSs was revealed after 48 h ofincubation (characteristic triplet signal appeared at δ 4.245; seea detailed description in the Supporting Information); for moreslowly metabolized sucrose and L-arabitol, the formation ofEPSs was observed after 72 h.The EPS concentrations were too low to perform all

necessary analyses to elucidate their detailed structures.Therefore, an up-scaled incubation with a higher concentrationof D-glucose (30 g L−1) was performed to obtain enoughmaterial for further analysis. The 1H NMR fingerprints of thewater-soluble EPSs formed with high concentrations of D-glucose (not shown) were similar to the signal patternsobserved in previous experiments. This indicates that the sametypes of EPSs are produced under both incubation conditions.Details of the NMR structural analysis of these EPSs are fullydescribed in the Supporting Information (Figures S1 and S2and Table S2). The analysis of the NMR spectra of theincubation media led to the identification of two distinct EPSs.The first one is an exopolysaccharide with a 1,6-linked α-galactan (1,6-α-galactan) backbone (Scheme 1A; Table S2,

Supporting Information); this structure has never beendescribed before for a Bacillus strain. The second one is apolymer, polyethylene glycol (PEG), with −CH2−O− groupsrandomly interleaved by −CH−O−-bearing O-acetyl groups(Scheme 1B; Table S2, Supporting Information).Both EPSs identified in the incubation medium obtained

with a high concentration of D-glucose showed characteristicspectral patterns in the 2D NMR spectra. To confirm that thepolymers formed in the incubation medium of the experimentswith less concentrated substrates have the same structure as the

identified 1,6-α-galactan and AcPEG, the 2D NMR spectrafrom previous incubations with different saccharides werereinvestigated (for more details, see the Supporting Informa-tion). The COSY spectrum of the sample taken after 63 h ofincubation with a low concentration of D-glucose is shown inFigure 2A. The signals of 1,6-α-galactan are highlighted in pink.The signals of the CH2 group of the partially acetylated PEGare highlighted in green. The same specific NMR spectralfeatures could be identified in the COSY spectra of theincubation media on cellotetraose (Figure 2B), cellulose(Figure 2C), sucrose (Figure 2D), and starch (Figure 2E).Due to the low concentration of metabolites in most of theincubation media, the HSQC spectra were measured only for D-glucose, cellulose, and sucrose (Figure 3). These spectra alsoshowed characteristic 1H−13C correlation spectral patterns dueto 1,6-α-galactan (pink) and AcPEG (green), which attestedtheir presence in the studied incubation media.In conclusion, the presence of EPSs including AcPEG and

1,6-α-galactan was clearly detected under different experimentalconditions. Many arguments show that these EPSs weresynthesized from the sugars added to the incubation mediumby Bacillus 3B6. First EPSs were only found with some sugars,including arabitol, fructose, glucose, sucrose, cellotetraose,cellulose, and starch (see Table 1); for all the other saccharides,no EPS was produced, even in the case where they wereconsumed by the bacteria (see mannitol, arabinose, lactose,maltose, trehalose, cellobiose, inuline) within 48 or 72 h.Second EPSs appeared at different times depending on thesugars used: from 24 h in the case of starch, 42 h for glucose(Figure S3, Supporting Information), to 72 h for some others(arabitol, fructose, sucrose). In some cases, EPSs appearedwhen the saccharide was completed consumed (fructose,sucrose), while they appeared before the end of theirconsumption in the case of arabitol and cellotetraose. Anotherargument is given by the comparison of the degradation of thetwo enantiomers of arabitol. Bacillus 3B6 was able to fullydegrade L-arabitol and produced EPSs under these conditions,while no EPS was produced in the presence of D-arbitol, whichwas not degraded. Also, in the absence of sugar in theincubation medium, no EPS was detected in the supernatant.So it means that these EPSs (AcPEG and 1,6-α-galactan) werenot produced before the addition of saccharide, previouslystored in the cells, and then released after a cell lysis. If it wasthe case, EPSs should be found in all the incubations, whateverthe sugar used and just after the end of their consumption, asthe cells were prepared and incubated in the same way (culturein TSA, centrifugation, and then incubation with the saccharideof interest in Volvic mineral water).

4. DISCUSSIONS

The presented work focuses on the metabolism of the strainBacillus sp. 3B6 isolated from cloudwaters and its ability tobiotransform saccharides present in the atmosphere. Thesesugars have a biogenic origin; they include alditols, mono-saccharides, disaccharides, oligosaccharides, and polysacchar-ides (Table S1, Supporting Information). This bacterial strainbelongs to the genus Bacillus, which is one of the mostfrequently described genera in the air4 and in free troposphericclouds,6,13 which are considered “warm clouds”. In such warmclouds, Bacillus is likely to be active under its vegetative form.We are not considering here clouds present in the highstratosphere, in which this bacterium should form endospores

Scheme 1. Structures of the Identified ExopolymericSubstances Produced by Bacillus sp. 3B6 on Glucose: (A)1,6-Linked α-Galactan and (B) EPS Containing a BackboneClose to Polyethylene Glycol (PEG) with −CH2−O−Groups Randomly Interleaved by −CH−O−-Bearing O-Acetyl Groups (R = H or OAc, m ≥ 1, n ≥ 1)

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to resist to very cold temperature and exposure to UV−C andconsequently should no longer transform saccharides.We have shown that Bacillus sp. 3B6 has a very active

metabolism toward the tested saccharides; all of them weredegraded except D-arabitol. It is worth noting that sugar (as anyother substrate) metabolism takes place within the cells wherethe pH and metabolite concentrations are highly regulated bythe bacteria. As a consequence, the transformation of sugars isnot greatly affected by the evaporation/condensation processes;microbial metabolism continues inside the cell along thedynamic processes of cloud formation and life. In addition, it islikely that a constant water layer is maintained around the cells.It was shown that, whatever the hydrophilicity/hydrophobicitybalance of the cell surface, bacteria are very efficient atactivating cloud droplets and thus at condensing water at theirsurface.43 Santl-Temkiv et al.15 also showed that bacteriaisolated from hailstones (seven strains of Methylobacterium andone strain of Bradyrhizobacterium) were able to grow on variouscarbohydrates. These strains belong to completely differentgenera, showing that sugar metabolism could be a rathergeneral process for atmospheric microorganisms.In addition, some of these saccharides (L-arabitol, D-fructose,

sucrose, D-glucose, cellotetraose, cellulose, and starch) can beefficiently transformed to EPSs by Bacillus sp. 3B6. Weidentified the structure of two EPSs as 1,6-α-galactan andpartially acetylated polyethylene glycol (AcPEG). 1,6-α-Galactan is a newly described polymer. In a previous study,we showed that in the presence of sucrose, Bacillus sp. 3B6 isable to produce the 2,6-linked fructose polymer levan andfructooligosaccharides of levan and inulin type in which thefructose units are 2,6- and 1,2-linked.41 Although the Bacillus

genus is highly present in the environment, relatively fewpapers address the structural analysis of EPSs produced by thistype of bacterial strain.44 Neutral levan polysaccharides havebeen described in structural EPSs of Bacillus subtilis.44

Additionally, Bacillus strains have been reported to synthesizepolyhydroxyalkanoate (PHA) structures. This was the case for aBacillus cereus strain using various carbohydrates as substrates(glucose, fructose, sucrose, various fatty acids, and gluconate).45

Our experiments did not prove their formation by Bacillus sp.3B6.These results could have implications regarding the

survivability of this strain under hostile cloud environmentconditions and its potential role in atmospheric processes.First, sugars provide efficient sources of energy for this

bacterium, and thus, they allow this strain to live and to bemetabolically active in warm clouds. We have shown thatBacillus sp. 3B6 could metabolize sugars as efficiently asmethanol and formaldehyde. The present data suggest that itcould also biotransform other types of substrates present inwarm clouds, such as amino acids and proteins for instance.In the future, experiments should be conducted under more

complex conditions mimicking cloud systems to quantify thereal impact of sugar degradation by microorganisms in warmclouds. We have recently published the use of real water cloudsamples in specifically designed photobioreactors to assess theimpact of microbial activity on the oxidant capacity and organiccarbon budget in clouds. We showed that formaldehyde wasbiodegraded by the endogenous and biodiverse microflora ofclouds. This result was consistent with the recent description ofthe presence of methylobacteria in the atmosphere.5,16 Thesame type of experiments could be conducted to monitor sugar

Figure 2. Parts of the COSY spectra of the incubation media of Bacillus sp. 3B6 with D-glucose (t = 63 h) (A), cellotetraose (t = 48 h) (B), cellulose(t = 63 h) (C), sucrose (t = 96 h) (D), and starch (t = 63 h) (E). Pink, H2 to H6, H6′ signals of 1,6-α-galactan; green, EPS containing a backboneclose to polyethylene glycol (PEG) with −CH2−O− groups randomly interleaved by −CH−O−-bearing O-acetyl groups; pink with green border,overlapped signals of 1,6-α-galactan and AcPEG.

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degradation. It will be necessary to develop new analytical toolsto measure sugar concentrations in cloudwaters. Massspectrometry could be an alternative to NMR as a moresensitive technique.46 Because sugars are major carbon sourcesfor microorganisms, they should be degraded by most of themicroorganisms present in warm clouds, as suggested by Santl-Temkiv et al.,16 who showed the presence of carbohydrates inatmospheric waters.The second important point is related to the production of

EPSs by Bacillus sp. 3B6. EPSs are known to surround thebacterial cells and to form biofilms. The formation of biofilms

seems to be the typical way that bacterial cells grow in naturebecause it confers many ecological advantages.44,47−50 Thesecompounds have been shown to protect microorganisms fromvarious environmental stresses, mainly by forming biofilmswhen a solid surface is available or by forming aggregates. Thishighly hydrated layer surrounding the cells can protect themagainst desiccation and UV exposure.47 Biofilms were alsoshown to decrease the diffusion rate of metabolites and thus toadjust their availability for microorganisms in the environ-ment.51 EPSs have also been found in extreme marinehabitats;52 for instance, they play a major role in Antarcticmarine environments, such as sea ice and ocean particles, toprotect microorganisms against extreme cold temperature, highsalinity, and the lack of nutrients.53 Consequently, EPSs couldthus contribute to the survival of microorganisms in theatmosphere, particularly in clouds, where they are subjected tolow nutrient availability, to cold temperature and freeze−thawprocesses, to UV and radical exposure, to evaporation−condensation processes, and thus to desiccation and osmolaritychanges.21 In the case of the Bacillus genus, EPS synthesis couldbe a complementary strategy to its unique ability to formendospores to survive under extreme conditions.44

EPS production might also have an importance concerningatmospheric processes. Bacillus sp. 3B6 was able to synthesizemolecules with a higher molecular mass (EPSs) than the initialsubstrate (mono-, di-, and tetrasaccharides). EPSs could bereleased in cloudwater and could be considered as SOAs. Theunderstanding of SOAs formation in atmospheric waters is ofgreat interest at the moment in the scientific community;54−57

microbial activity could contribute to these processes inaddition to radical chemistry.Because EPSs are highly functionalized molecules able to

interact with water and are potentially able to form aggregatesand gels, EPSs can also act as efficient CCN. Concerningsugars, Rosenorn et al.39 have shown that many saccharidespresent in the atmosphere (glucose, fructose, mannose, lactose,maltose, sucrose, and levoglucosan) are CCN-active. Also PEGhas been shown to activate cloud droplets.58 Therefore, it isvery likely that 1,6-α-galactan, which is polymer of sugars, andAcPEG, which is a derivative of PEG, also have CCNproperties. In the high arctic, Orellana et al.59 have shownthat marine microgels issued from EPS, synthesized by marinephytoplankton, were a source of CCN. They coulddemonstrate that these EPSs were present both in the ArcticOcean surface (source) but also in airborne aerosols, in fog, andin cloudwaters. Russel et al.60 found carbohydrate derivatives(EPSs) on submicrometer particles and showed that they wereissued from the ocean by bubble bursting.In addition, it should be stressed that the formation of EPSs

by this strain could also take place before the bacteria areaerosolized from the sugars present in terrestrial and aquaticenvironments, including the ocean.61 EPSs could pass into theatmosphere directly as pure compounds by mechanisms ofbubble bursting from aquatic water bodies (as observed forphytoplankton EPSs) or by wind-driven resuspension fromterrestrial environments (dust, dry leaves, etc.). This phenom-enon could be general for many bacteria forming EPSs on theearth, even though they are not further aerosolized.Alternatively, Bacillus could be aerosolized embedded in itsEPSs in the form of aggregates or as a biofilm on a solidsupport; we can suppose than many other microorganismscould be also aerosolized under this form. In this later case, the

Figure 3. Parts of the HSQC spectra of the incubation media ofBacillus sp. 3B6 with D-glucose (t = 63 h) (A), cellulose (t = 63 h) (B),and sucrose (t = 96 h) (C). Pink, 1,6-α-galactan; green, EPScontaining a backbone close to polyethylene glycol (PEG) with−CH2−O− groups randomly interleaved by −CH−O−-bearing O-acetyl groups (AcPEG); pink with green border, overlapped signals of1,6-α-galactan and AcPEG.

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EPS could also protect bacteria during their airborne transportand be further integrated in cloudwaters.In conclusion, to our knowledge, this is the first study

reporting the biotransformation of sugars of atmosphericinterest and the production of EPSs by a cloud bacterium.This study focused on a Bacillus strain that can be considered asa case study; the same approach could be valid for many otherstrains present in cloudwaters. This topic is new and innovative;these results are a first step in the exploration of sugarmetabolism in clouds, and as such our conclusions are stillspeculative, but they point out the potential key role of thisclass of compounds for cloud microorganisms. Although ratherpreliminary, this work gives some new insight into survivalstrategies of microorganisms in such extreme environmentalconditions, as well as their potential contribution toatmospheric processes. It opens new directions of researchconcerning the potential impact of microorganisms in clouds.

■ ASSOCIATED CONTENT*S Supporting InformationStructure of EPSs obtained during the incubation of Bacillus sp3B6 with high glucose concentration, interpretation of 2DNMR experiments performed on incubation media with lowsugar concentration, Tables S1 and S2, and Figures S1−S3.This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: Anne-Marie.DELORT@univ-bpclermont.fr. Tel: 33473 40 77 14.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research has been supported by the CNRS LEFE-CHATprogram, the French−Slovak collaborative program Stefanikproject No. 17947UE (SK-FR-0009-07), the French Govern-ment Scholarship (fellowship for S.H.), the Scientific GrantAgency of the Ministry of Education of the Slovak Republic andof the Slovak Academy of Sciences (VEGA No 2/0007/13), theSlovak Research and Development (APVV) (No. 0125-11), theSlovak state program 2003SP200280203, and Research &Development Operational Program funded by the ERDF forthe Centre of Excellence for Glycomics (ITMS: 26240120031).

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