Biotransformation of Various Saccharides and Production of

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Biotransformation of Various Saccharides and Production of Exopolymeric Substances by Cloud-Borne Bacillus sp. 3B6 Mária Matulová,† Slavomíra Husárová,†,‡ Peter Capek,† Martine Sancelme,‡,§ and Anne-Marie Delort*,‡,§ †

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

S Supporting Information *

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

1. INTRODUCTION Although air-borne microorganisms have been studied for a long time,1−5 the discovery of microorganisms in cloudwaters is rather recent.6−16 Only a fraction of atmospheric microorganisms were cultivable; however, it was shown that most of them (notably in clouds) were metabolically active,7,13,14,17−19 thus suggesting that microorganisms are able to survive under the stressing conditions of the atmospheric environment (UV exposure, freeze−thaw processes, evaporation−condensation cycles, cold stress, etc.) and can use various organic compounds as carbon sources. This biocatalytic activity raised new scientific questions about the role of microorganisms in the multiphase chemistry of clouds.20,21 In this context, few papers have reported the effective degradation of organic compounds, including mono and carboxylic acids, as well as methanol and formaldehyde, by atmospheric microorganisms.22−28 We recently showed that biodegradation of these compounds was competitive with radical processes using real cloudwater samples containing wide microbial biodiversity and a complex chemical composition under realistic atmospheric conditions in a photobioreactor (artificial solar light, low temperature).14 In addition to organic acids, methanol, and formaldehyde, these active microorganisms could also transform the dissolved organic matter present in cloudwaters. The composition of this matter 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 © 2014 American Chemical Society

macromolecules including HULIS (humic-like substances) and SOAs (secondary organic aerosols) such as oligomers. Sugars are also likely to be present. Various classes of saccharides have been reported as important constituents of ambient atmospheric aerosols (Table S1, Supporting Information), 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 (levoglucosan, mannosan, galactosan, and 1,6-anhydroglucofuranose), and polysaccharide, such as cellulose. The presence of these saccharides in the atmosphere is the signature of biogenic sources of aerosols. Monosaccharides (glucose, fructose, and xylose) and disaccharides (sucrose and trehalose) can be released into the atmosphere by microorganisms, plants, and animals.30 Sugar alcohols, such as glycerol, D- and L-arabitol, mannitol, and D-glucitol, can be produced by fungi, lichens, and bacteria.31−34 Levoglucosan derives from cellulose; galactosans and mannosans derive from hemicelluloses during biomass burning.31−33,35 The nature and concentration of sugars present Received: Revised: Accepted: Published: 14238

March 19, 2014 November 12, 2014 November 12, 2014 November 12, 2014 dx.doi.org/10.1021/es501350s | Environ. Sci. Technol. 2014, 48, 14238−14247

Environmental Science & Technology

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and disaccharides, 10 mM for oligosaccharides, and 4 g L−1 for polysaccharides. The microbial concentration was 109 cells mL−1. The incubations of Bacillus sp. 3B6 with sugars were conducted under aerobic conditions (200 rpm) at 27 °C. The experimental conditions used here were similar to those conducted with this strain incubated with methanol and formaldehyde in a previous paper;28 therefore, the results obtained with sugars and VOCs can be directly compared. Due to the limited sensitivity of NMR experiments, it was inevitable to scale up the concentration of the tested substrates together with the bacterial concentration while the substrate-to-cell ratio identified in the natural environment of clouds was maintained. In a previous work26 it was shown that the biodegradation rates are only substrate-to-cell ratio dependent, regardless of the concentration 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 the supernatants were kept frozen (−20 °C) until NMR measurements were done. 2.3. Incubation of Bacillus sp. 3B6 with a High Concentration of D-Glucose. The objective of the incubation with a high concentration of glucose was to get a maximum yield of EPSs necessary for their structural study by lowsensitivity 2D 1H−13C heterocorrelated NMR sequences; thus, the conditions, including the temperature, had to be different from those described when a low concentration of glucose was used. For EPS production with a high D-glucose concentration, a medium proposed by Gandhi et al.42 was used. Its composition was as follows: D-glucose (30 g L−1) (Acros), ammonium nitrate (0.4 g L−1) (Acros), potassium sulfate (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), and magnesium sulfate (0.001 g L−1) (Sigma-Aldrich). The medium was autoclaved for 15 min at 121 °C, and 24 h 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 000g, 25 °C, 30 min), and Bacillus sp. 3B6 cells were separated from the supernatant. Low molecular weight residues were removed from the crude incubation medium by dialysis against distilled water (two times) until the 1H NMR spectra confirmed the absence of D-glucose, salts, and amino acids in the incubation medium. After a volume reduction, the sample was freeze-dried and redissolved in D2O for NMR measurements. The soluble fraction containing EPSs was further analyzed by 1D and 2D NMR. 2.4. NMR Spectroscopy. Monitoring Sugar Biodegradation by 1D in Situ 1H NMR. Supernatants from the biodegradation test media were prepared for 1H NMR by mixing 575 μL of the sample with 25 μL of 20 mM the tetradeuterated sodium salt of trimethylsilyl propionic acid (TSP-d4, Eurisotop) dissolved in D2O. D2O was used for shimming and locking, whereas TSP-d4 constituted a reference for chemical shifts (0 ppm) and quantification. A final volume of 600 μL of prepared sample was added to the 5 mm diameter NMR tubes. Acquisition was performed at 500 MHz on a Bruker Avance NMR spectrometer or on a 600 MHz VNMRS Varian spectrometer equipped with an HCN 13C-enhanced salttolerant cold probe. The water signal was eliminated by presaturation and by pulse field gradient spin echo (GPFSE). The concentrations of metabolites were calculated as follows

in aerosols were dependent on the time of year33,36,37 and also on the diurnal cycle.31,38 Although no quantitative measurement of sugar concentrations was made in cloudwaters, the presence of sugars in atmospheric waters is likely due to their high solubility in water and their ability to act as cloud condensation nuclei (CCN).39 A recent work by Šantl-Temkiv et al.16 reports the presence of carbohydrates in hailstones: 3% of the identified DOC was composed of fatty acids, sulfonic acids, and carbohydrates as measured by FT-ICR-MS. Considering the amount of DOC in cloudwaters,14,29 presumably the carbohydrate concentration should be in the micromolar range. Sugars are well-known substrates for microorganisms; they represent the main source of carbon and energy for many of them. In some cases, microorganisms can convert sugars into exopolymeric substances (EPSs) very effectively.40 These EPSs can be polysaccharides or other polymers, such as proteic structures or polyhydroxyalkanoates (PHAs). Microbial polysaccharides are multifunctional and may have homo- or heteropolymeric compositions of high molecular weight (10− 30 kDa). The aim of this paper was to investigate the biotransformation of sugars usually encountered in the atmosphere by the cloud microorganism Bacillus sp. 3B6. This bacterium was isolated in cloudwaters at the Puy de Dôme station.10 There are two reasons for the choice of this strain: first, Bacillus is one of major genera described in the free troposphere, including clouds6,13 and the air,4 and second, the metabolism of this strain was shown to be active on some compounds present in the atmosphere, including methanol and formaldehyde,28 as well as sucrose.41 We report here the ability of Bacillus sp. 3B6 to use various carbohydrates present in the atmosphere and to excrete EPSs. The kinetics of sugar transformations was monitored by in situ 1H nuclear magnetic resonance (NMR) spectroscopy performed directly on the samples of crude incubation media. The EPSs structures were elucidated using multidimensional NMR techniques. A discussion is given about the potential implication of these results for the atmospheric environment. First, EPSs could protect bacteria and help them to survive under the stressing conditions encountered in clouds. Second, these highly functionalized molecules could be considered as SOAs and favor CCN formation.

2. MATERIALS AND METHODS 2.1. Bacterial Strain and Culture Conditions. Bacillus sp. 3B6 was isolated from cloudwater sampled at the Puy de Dôme summit (1465 m) in the Massif Central Region of France and was identified by 16S rRNA gene sequencing as described by Amato et al.10 Liquid, pure preculture was incubated in 100 mL portions of TSA (trypcase soy broth, Biomerieux, Marcy l’Etoile, France) in 500 mL Erlenmeyer flasks at 27 °C and 200 rpm. Cells were harvested by centrifugation (8000g and 4 °C for 5 min) after 24 h of growth and rinsed twice in 0.8% NaCl and resuspended in the medium for biodegradation studies. 2.2. Incubations of Bacillus sp. 3B6 with Low Sugar Concentrations. Arabinogalactan and glucuronoxylan (Institute of Chemistry, Slovak Academy of Sciences) and all the other tested saccharides (Sigma-Aldrich) were used as received. Incubation media were prepared by the dissolution of saccharides in Volvic mineral water. Each tested saccharide was used as a unique carbon source. The final concentration of saccharides in the incubation medium was 20 mM for mono14239

dx.doi.org/10.1021/es501350s | Environ. Sci. Technol. 2014, 48, 14238−14247


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Table 1. Ability of Bacillus sp. 3B6 To Degrade Different Types of Saccharides and Produce EPS products detected over time tested saccharides

0h

mannitol D-glucitol L-arabitol (arabinitol) D-arabitol

+++ +++ +++ +++

L-arabinose

L-rhamnose

+++ +++ +++ +++ +++ +++ +++ +++ +++

lactose sucrose maltose trehalose cellobiose

+++ +++ +++ +++ +++

cellotetraose

+++

cellulose arabinogalactan glucuronoxylan inulin starch

ND +++ +++ +++ ND

D-xylose D-ribose L-ribose D-fructose D-glucose D-galactose L-mannose

24 h

48 h

Alditols ++ +++ +++ +++ Monosaccharides ++ +++ ++ +++ + ± ++ ++ + Disaccharides ++ ++ ++ ++ ++ Tetrasaccharides ++ Polysaccharides ND +++ ++ ++ ND/EPS

72 h

96 h

− + +++ +++

− − ++/EPS +++

− − EPS +++

− +++ + +++ − EPS − − −

− ++ + ++ EPS EPS − − −

− ++ − ++ EPS EPS − − −

+ − − + ±

± EPS − − −

− EPS − − −

++/EPS

+/EPS

−/EPS

ND/EPS +++ + + ND/EPS

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 sugar present in the incubation medium. ND, not determined.

[m] =

9 A0 [TSP‐d4] b A ref

mono- and disaccharides (aldoses and ketoses) were chosen to cover pentoses and hexoses in acyclic and cyclic forms (Table 1), the presence of which was determined in aerosol samples (Table S1, Supporting Information). We also investigated the degradation of polysaccharides, constituents of plant debris, which can be potentially aerosolized, including cellulose (and its products of degradation cellobiose and cellotetraose), arabinogalactan, glucuronoxylan, inuline, and starch. Samples were taken from the incubation media at regular intervals. After centrifugation, used for cell elimination, the supernatant was directly analyzed by 1H NMR without any further sample treatment. The sugar concentrations at different intervals were estimated as the ratio of the anomeric signal intensity (if present; otherwise, another not-overlapped signal was chosen) and the signal intensity of the TSP-d4 standard added at a known concentration (as described above). Experimental results showed that Bacillus sp. 3B6 metabolized all the tested carbohydrates that can be potentially present in the atmosphere (Table S1, Supporting Information). Although the exact rates of degradation were difficult to determine using these complex spectra, the percent degradation was roughly quantified (100%, 60%, and 30%) at measured intervals of 0, 24, 48, 72, and 96 h of incubation (Table 1). This rough estimation allowed for comparing the efficiency of the biodegradation of the different saccharides. After 48 h of incubation, mannitol, L-arabinose, D-fructose, D-glucose, Dgalactose, L-mannose, L-rhamnose, sucrose, and maltose were

(1)

where [m] is the concentration of compound to be quantified, A0 is the area under the m resonance, Aref is the area under the TSP-d4 resonance, and 9 and b are the numbers of protons in TSP-d4 and in the metabolite m, respectively. Structural Analyses of EPSs. Chosen samples were freezedried, dissolved in 99.98% D2O, and further measured at 25 °C in 3 mm sample tubes on a 600 MHz VNMRS Varian equipped with an HCN 13C-enhanced salt-tolerant cold probe using TSPd4 as an external standard. Advanced techniques from the Varian pulse library of two-dimensional homo- and heterocorrelated spectroscopy (COSY, 1H−1H homocorrelated spectroscopy; TOCSY, 1H−1H total correlation spectroscopy; HSQC, 1H−13C Heteronuclear single quantum correlation spectroscopy; HMBC, 1H−13C heteronuclear multiple quantum correlation spectroscopy; and H2BC, heteronuclear twobond correlation spectroscopy) including one-dimensional sequences with selective excitations (1D NOESY, NOE spectroscopy and 1D TOCSY) were used for the signal assignments.

3. RESULTS 3.1. Biodegradation of Different Sugars. The ability of Bacillus sp. 3B6 to metabolize different types of carbohydrates was monitored by in situ 1H NMR spectroscopy. The different 14240



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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 = 72 h), D-glucose (t = 63 h), cellotetraose (t = 48 h), cellulose (t = 63 h), and starch (t = 63 h).

Although the biodegradation rates for sugars could not be measured very precisely, they were evaluated as follows: considering that 109 cells in 1 mL incubation volume degrade 2 × 10−7 mol of sugars within 48−96 h, the biodegradation rates were estimated to be in the range from 1.16 × 10−19 to 5.78 × 10−20 mol cell−1 s−1. These values are in the same range as those measured with the same strain when degrading methanol (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 degrades sugars with the same efficiency as it degrades formaldehyde and methanol. 3.2. Exopolymeric Substances. In several cases (Larabitol, D-glucose, D-fructose, sucrose, cellotetraose, cellulose, and starch), the formation of EPSs was observed. Their production was identified from the 1H NMR spectra by the presence of characteristic broad, complex spectral patterns of

completely degraded. D-Glucitol, D-ribose, lactose, and trehalose were degraded up to 30%. After 72 h, approximately 60% of L-arabitol, D-xylose, and L-ribose and 30% of D-ribose and D-cellotetraose still remained in the media. Only D-arabitol was not degraded by this strain. The slowest degradations were observed for D-xylose and L-ribose; 60% was still present in the medium after 96 h. The degradation of water-soluble polysaccharides (inulin, arabinogalactan, and glucuronoxylan) was monitored as described above: glucuronoxylan and inulin were completely degraded within 72 h, whereas 30% of arabinogalactan remained after 96 h. The limited solubility of cellulose and starch due to their higher molar masses made the direct quantification of these substrates from the 1H NMR spectra of the incubation media impossible. However, the signals of the newly formed EPSs were detectable (see section 3.2). 14241



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identified 1,6-α-galactan and AcPEG, the 2D NMR spectra from previous incubations with different saccharides were reinvestigated (for more details, see the Supporting Information). The COSY spectrum of the sample taken after 63 h of incubation with a low concentration of D-glucose is shown in Figure 2A. The signals of 1,6-α-galactan are highlighted in pink. The signals of the CH2 group of the partially acetylated PEG are highlighted in green. The same specific NMR spectral features could be identified in the COSY spectra of the incubation 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 the incubation media, the HSQC spectra were measured only for Dglucose, cellulose, and sucrose (Figure 3). These spectra also showed characteristic 1H−13C correlation spectral patterns due to 1,6-α-galactan (pink) and AcPEG (green), which attested their presence in the studied incubation media. In conclusion, the presence of EPSs including AcPEG and 1,6-α-galactan was clearly detected under different experimental conditions. Many arguments show that these EPSs were synthesized from the sugars added to the incubation medium by 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 were consumed by the bacteria (see mannitol, arabinose, lactose, maltose, trehalose, cellobiose, inuline) within 48 or 72 h. Second EPSs appeared at different times depending on the sugars 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 appeared when the saccharide was completed consumed (fructose, sucrose), while they appeared before the end of their consumption in the case of arabitol and cellotetraose. Another argument is given by the comparison of the degradation of the two enantiomers of arabitol. Bacillus 3B6 was able to fully degrade L-arabitol and produced EPSs under these conditions, while no EPS was produced in the presence of D-arbitol, which was not degraded. Also, in the absence of sugar in the incubation medium, no EPS was detected in the supernatant. So it means that these EPSs (AcPEG and 1,6-α-galactan) were not produced before the addition of saccharide, previously stored in the cells, and then released after a cell lysis. If it was the case, EPSs should be found in all the incubations, whatever the sugar used and just after the end of their consumption, as the cells were prepared and incubated in the same way (culture in TSA, centrifugation, and then incubation with the saccharide of interest in Volvic mineral water).

EPSs (Figure 1). The excretion of EPSs, synthesized intracellularly, to the extracellular incubation medium was observed after the depletion of the initial saccharide substrate. Because we only examined this extracellular medium by NMR, we cannot detect the presence of EPSs before their excretion. In addition, due to the relatively low sensitivity of the NMR technique, EPS signals are only detected when they start to accumulate in the medium. Other 1H NMR resonances were detected notably in the range of 1−3 ppm (not shown), but the structure 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 atmospheric environment. For fast-metabolized saccharides (D-fructose and D-glucose) and for oligo- and polysaccharides (cellotetraose, cellulose, and starch), the formation of EPSs was revealed after 48 h of incubation (characteristic triplet signal appeared at δ 4.245; see a detailed description in the Supporting Information); for more slowly metabolized sucrose and L-arabitol, the formation of EPSs 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 concentration of D-glucose (30 g L−1) was performed to obtain enough material for further analysis. The 1H NMR fingerprints of the water-soluble EPSs formed with high concentrations of Dglucose (not shown) were similar to the signal patterns observed in previous experiments. This indicates that the same types of EPSs are produced under both incubation conditions. Details of the NMR structural analysis of these EPSs are fully described in the Supporting Information (Figures S1 and S2 and Table S2). The analysis of the NMR spectra of the incubation media led to the identification of two distinct EPSs. The first one is an exopolysaccharide with a 1,6-linked αgalactan (1,6-α-galactan) backbone (Scheme 1A; Table S2, Scheme 1. Structures of the Identified Exopolymeric Substances Produced by Bacillus sp. 3B6 on Glucose: (A) 1,6-Linked α-Galactan and (B) EPS Containing a Backbone Close to Polyethylene Glycol (PEG) with −CH2−O− Groups Randomly Interleaved by −CH−O−-Bearing OAcetyl Groups (R = H or OAc, m ≥ 1, n ≥ 1)

4. DISCUSSIONS The presented work focuses on the metabolism of the strain Bacillus sp. 3B6 isolated from cloudwaters and its ability to biotransform saccharides present in the atmosphere. These sugars have a biogenic origin; they include alditols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides (Table S1, Supporting Information). This bacterial strain belongs to the genus Bacillus, which is one of the most frequently described genera in the air4 and in free tropospheric clouds,6,13 which are considered “warm clouds”. In such warm clouds, Bacillus is likely to be active under its vegetative form. We are not considering here clouds present in the high stratosphere, in which this bacterium should form endospores

Supporting Information); this structure has never been described before for a Bacillus strain. The second one is a polymer, polyethylene glycol (PEG), with −CH2−O− groups randomly 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 characteristic spectral patterns in the 2D NMR spectra. To confirm that the polymers formed in the incubation medium of the experiments with less concentrated substrates have the same structure as the 14242



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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 backbone close 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.

genus is highly present in the environment, relatively few papers address the structural analysis of EPSs produced by this type of bacterial strain.44 Neutral levan polysaccharides have been described in structural EPSs of Bacillus subtilis.44 Additionally, Bacillus strains have been reported to synthesize polyhydroxyalkanoate (PHA) structures. This was the case for a Bacillus 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 environment conditions 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 be metabolically active in warm clouds. We have shown that Bacillus sp. 3B6 could metabolize sugars as efficiently as methanol and formaldehyde. The present data suggest that it could also biotransform other types of substrates present in warm 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 the real impact of sugar degradation by microorganisms in warm clouds. We have recently published the use of real water cloud samples in specifically designed photobioreactors to assess the impact of microbial activity on the oxidant capacity and organic carbon budget in clouds. We showed that formaldehyde was biodegraded by the endogenous and biodiverse microflora of clouds. This result was consistent with the recent description of the presence of methylobacteria in the atmosphere.5,16 The same type of experiments could be conducted to monitor sugar

to resist to very cold temperature and exposure to UV−C and consequently should no longer transform saccharides. We have shown that Bacillus sp. 3B6 has a very active metabolism toward the tested saccharides; all of them were degraded except D-arabitol. It is worth noting that sugar (as any other substrate) metabolism takes place within the cells where the pH and metabolite concentrations are highly regulated by the bacteria. As a consequence, the transformation of sugars is not greatly affected by the evaporation/condensation processes; microbial metabolism continues inside the cell along the dynamic processes of cloud formation and life. In addition, it is likely that a constant water layer is maintained around the cells. It was shown that, whatever the hydrophilicity/hydrophobicity balance of the cell surface, bacteria are very efficient at activating cloud droplets and thus at condensing water at their surface.43 Šantl-Temkiv et al.15 also showed that bacteria isolated from hailstones (seven strains of Methylobacterium and one strain of Bradyrhizobacterium) were able to grow on various carbohydrates. These strains belong to completely different genera, showing that sugar metabolism could be a rather general process for atmospheric microorganisms. In addition, some of these saccharides (L-arabitol, D-fructose, sucrose, D-glucose, cellotetraose, cellulose, and starch) can be efficiently transformed to EPSs by Bacillus sp. 3B6. We identified the structure of two EPSs as 1,6-α-galactan and partially acetylated polyethylene glycol (AcPEG). 1,6-αGalactan is a newly described polymer. In a previous study, we showed that in the presence of sucrose, Bacillus sp. 3B6 is able to produce the 2,6-linked fructose polymer levan and fructooligosaccharides of levan and inulin type in which the fructose units are 2,6- and 1,2-linked.41 Although the Bacillus 14243



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seems to be the typical way that bacterial cells grow in nature because it confers many ecological advantages.44,47−50 These compounds have been shown to protect microorganisms from various environmental stresses, mainly by forming biofilms when a solid surface is available or by forming aggregates. This highly hydrated layer surrounding the cells can protect them against desiccation and UV exposure.47 Biofilms were also shown to decrease the diffusion rate of metabolites and thus to adjust their availability for microorganisms in the environment.51 EPSs have also been found in extreme marine habitats;52 for instance, they play a major role in Antarctic marine environments, such as sea ice and ocean particles, to protect microorganisms against extreme cold temperature, high salinity, and the lack of nutrients.53 Consequently, EPSs could thus contribute to the survival of microorganisms in the atmosphere, particularly in clouds, where they are subjected to low nutrient availability, to cold temperature and freeze−thaw processes, to UV and radical exposure, to evaporation− condensation processes, and thus to desiccation and osmolarity changes.21 In the case of the Bacillus genus, EPS synthesis could be a complementary strategy to its unique ability to form endospores to survive under extreme conditions.44 EPS production might also have an importance concerning atmospheric processes. Bacillus sp. 3B6 was able to synthesize molecules with a higher molecular mass (EPSs) than the initial substrate (mono-, di-, and tetrasaccharides). EPSs could be released in cloudwater and could be considered as SOAs. The understanding of SOAs formation in atmospheric waters is of great interest at the moment in the scientific community;54−57 microbial activity could contribute to these processes in addition to radical chemistry. Because EPSs are highly functionalized molecules able to interact with water and are potentially able to form aggregates and gels, EPSs can also act as efficient CCN. Concerning sugars, Rosenorn et al.39 have shown that many saccharides present in the atmosphere (glucose, fructose, mannose, lactose, maltose, sucrose, and levoglucosan) are CCN-active. Also PEG has been shown to activate cloud droplets.58 Therefore, it is very likely that 1,6-α-galactan, which is polymer of sugars, and AcPEG, which is a derivative of PEG, also have CCN properties. In the high arctic, Orellana et al.59 have shown that marine microgels issued from EPS, synthesized by marine phytoplankton, were a source of CCN. They could demonstrate that these EPSs were present both in the Arctic Ocean surface (source) but also in airborne aerosols, in fog, and in cloudwaters. Russel et al.60 found carbohydrate derivatives (EPSs) on submicrometer particles and showed that they were issued 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 are aerosolized from the sugars present in terrestrial and aquatic environments, including the ocean.61 EPSs could pass into the atmosphere directly as pure compounds by mechanisms of bubble bursting from aquatic water bodies (as observed for phytoplankton EPSs) or by wind-driven resuspension from terrestrial environments (dust, dry leaves, etc.). This phenomenon could be general for many bacteria forming EPSs on the earth, even though they are not further aerosolized. Alternatively, Bacillus could be aerosolized embedded in its EPSs in the form of aggregates or as a biofilm on a solid support; we can suppose than many other microorganisms could be also aerosolized under this form. In this later case, the

Figure 3. Parts of the HSQC spectra of the incubation media of Bacillus 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, EPS containing a backbone close to polyethylene glycol (PEG) with −CH2−O− groups randomly interleaved by −CH−O−-bearing Oacetyl groups (AcPEG); pink with green border, overlapped signals of 1,6-α-galactan and AcPEG.

degradation. It will be necessary to develop new analytical tools to measure sugar concentrations in cloudwaters. Mass spectrometry could be an alternative to NMR as a more sensitive technique.46 Because sugars are major carbon sources for microorganisms, they should be degraded by most of the microorganisms present in warm clouds, as suggested by ŠantlTemkiv et al.,16 who showed the presence of carbohydrates in atmospheric waters. The second important point is related to the production of EPSs by Bacillus sp. 3B6. EPSs are known to surround the bacterial cells and to form biofilms. The formation of biofilms 14244



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EPS could also protect bacteria during their airborne transport and be further integrated in cloudwaters. In conclusion, to our knowledge, this is the first study reporting the biotransformation of sugars of atmospheric interest and the production of EPSs by a cloud bacterium. This study focused on a Bacillus strain that can be considered as a case study; the same approach could be valid for many other strains present in cloudwaters. This topic is new and innovative; these results are a first step in the exploration of sugar metabolism in clouds, and as such our conclusions are still speculative, but they point out the potential key role of this class of compounds for cloud microorganisms. Although rather preliminary, this work gives some new insight into survival strategies of microorganisms in such extreme environmental conditions, as well as their potential contribution to atmospheric processes. It opens new directions of research concerning the potential impact of microorganisms in clouds.

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ASSOCIATED CONTENT

S Supporting Information *

Structure of EPSs obtained during the incubation of Bacillus sp 3B6 with high glucose concentration, interpretation of 2D NMR experiments performed on incubation media with low sugar concentration, Tables S1 and S2, and Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 33 473 40 77 14. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research has been supported by the CNRS LEFE-CHAT program, the French−Slovak collaborative program Štefánik project No. 17947UE (SK-FR-0009-07), the French Government Scholarship (fellowship for S.H.), the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and of the Slovak Academy of Sciences (VEGA No 2/0007/13), the Slovak Research and Development (APVV) (No. 0125-11), the Slovak state program 2003SP200280203, and Research & Development Operational Program funded by the ERDF for the Centre of Excellence for Glycomics (ITMS: 26240120031).

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