# Phototrophy improves the aerial fitness in a photoheterotrophic Methylobacterium isolated from clouds

Frédéric Mathonat, Federico Mazzei, Marie Prévot, Virgina Vernocchi, Elena Gatta, Muriel Joly, Mariline Theveniot, Anne-Catherine Lehours, François Enault, Barbara Ervens, et al.

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## To cite this version:

Frédéric Mathonat, Federico Mazzei, Marie Prévot, Virgina Vernocchi, Elena Gatta, et al. Phototrophy improves the aerial fitness in a photoheterotrophic Methylobacterium isolated from clouds. 2025. hal-05344310

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Phototrophy improves the aerial fitness in a photoheterotrophic

*Methylobacterium* isolated from clouds

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Frédéric Mathonat¹,², Federico Mazzei³,⁴, Marie Prévot¹, Virginia Vernocchi⁴, Elena Gatta³, Muriel

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Joly¹, Mariline Théveniot¹, Anne-Catherine Lehours², François Enault², Barbara Ervens¹, Pierre Amato²

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¹ Université Clermont Auvergne, CNRS, Clermont Auvergne INP, ICCF, 63000, Clermont-Ferrand, France

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² Université Clermont Auvergne, CNRS, LMGE, 63000, Clermont-Ferrand, France

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³ University of Genoa, Department of Physics, 16146, Genova, Italy

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⁴ National Institute of Nuclear Physics, 16146, Genova, Italy

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E-mail addresses:

12 frederic.mathonat@uca.fr

13 federico.mazzei@ge.infn.it

14 marie.prevot@uca.fr

15 virginia.vernocchi@ge.infn.it

16 elena.gatta@unige.it

17 muriel.joly@uca.fr

18 mariline.theveniot@uca.fr

19 a-catherine.lehours@uca.fr

20 francois.enault@uca.fr

21 barbara.ervens@uca.fr

22 pierre.amato@uca.fr

23

24 Short running title: Phototrophy and aerial fitness in bacteria

25 Keywords: Photoheterotrophy – Atmosphere – Anoxygenic photosynthesis – Bacteria – Survival –

26 *Methylobacterium* sp. – Aerial fitness – Atmospheric simulation chamber

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29 **Type of article:** Letter

30 **The number of words in the abstract:** 150

31 **The number of words in the main text:** 4972

32 **The number of references:** 73

33 **The number of figures and tables:** 6

34 **The number of supplementary figures and tables:** 8

35 **Corresponding author:**

36 Frédéric Mathonat ; +33 6 89 41 43 05 ; frederic.mathonat@uca.fr

37

38 **Authors contributions:**

39 FrM, PA, MJ and MT designed the experiments. FrM, PA, MJ, MT and MP performed the experiments.

40 FeM, VV and EG helped with the use of on-site infrastructures, and in particular the use of the

41 atmospheric simulation chamber. FrM, PA, MJ, MT and MP collected samples and data. FrM and FE

42 conducted the bioinformatics analysis. FrM and PA wrote the paper. All authors have contributed to

43 the editing and formatting of figures and text. PA supervised the research, coordination and strategy.

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53 Abstract

54
Surface ecosystems such as the phyllosphere host diverse microbial communities, a fraction of
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which may detach and remain aloft for up to several days. As airborne, bacteria are challenged by high
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exposure to oxidants and limited nutrients, and we postulate that specific biological trait such as
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phototrophy may support the maintenance of metabolic functions. Omics data reanalysis indicates the
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presence of the genetic biomarker of anoxygenic photosynthesis in the atmosphere and clouds. Using
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an atmospheric simulation chamber, we monitored proxies for viability and metabolic activity in
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airborne photoheterotrophic aerobic *Methylobacterium* sp. cells expressing or not photosynthetic
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capabilities (bacteriochlorophyll). We observe shorter airborne residence time in [Bchl+] cells than in
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[Bchl-] cells, and higher survival rate and potential for metabolic activity, in particular under light
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exposure. These results imply that aerial transport is not neutral, and can favor the maintenance and
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spread of biological traits often considered elsewhere as poorly competitive.
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75 **Introduction**

76 The phyllosphere, i.e. the aerial parts of plants, hosts abundant and diverse bacteria with
77 multiple biological functions and trophic modes, which play pivotal ecological roles by contributing to
78 the recruitment and processing of major nutrients in plants e.g., (Lehours et al. 2016; Vergne et al.
79 2021; Vorholt 2012). These are subjected to aerial dispersion, and vegetated surfaces were indeed
80 reported to emit viable bacteria to the air at high rate (Lindemann et al. 1982). The soil and leaf
81 surfaces are often considered the main contributors of airborne bacteria (Bowers et al. 2011), with
82 other identified sources including, for instance, water bodies (de Leeuw et al. 2011), humans and
83 animals (Fujimura et al. 2010). Once aerosolized, airborne bacteria can be transported upwards by
84 convective air movements and remain aloft for an estimated average time of 3 to 4 days (Burrows et
85 al. 2009), during which they can travel over large scales (Smets et al. 2016). Meanwhile, their survival,
86 and so their chance of successful aerial dispersal, is compromised by conditions including nutrient and
87 water limitations, and potentially high exposure to UV-light and reactive oxygen species (Joly et al.
88 2015; Šantl-Temkiv et al. 2022; Smith et al. 2011; Tong & Lighthart 1998). Various biological traits have
89 been identified in bacteria as contributing to their survival during air transport, including the capacity
90 to form spore, or to synthesize carotenoid pigments (Lighthart & Shaffer 1997; Tong & Lighthart 1998),
91 but no specific trophic mode has yet been associated with such a role.

92 Among the bacterial taxa frequently reported as viable and metabolically active in the
93 atmosphere, including natural clouds above vegetated regions, is the genus *Methylobacterium*, of the
94 order *Rhizobiales* within the phylum *Pseudomonadota* (Amato *et al.* 2017; Klein *et al.* 2016; Šantl-
95 Temkiv *et al.* 2018). Members of this genus have a wide variety of features that make them well-fitted
96 for survival in the atmosphere (desiccation tolerance, nitrogen-fixing activity, biofilm formation,
97 facultative methylotrophy (Cáceres *et al.* 2014; Dourado *et al.* 2015). *Methylobacterium* also include
98 aerobic anoxygenic photosynthetic bacteria (AAPB). These photoheterotrophic microorganisms use
99 bacteriochlorophyll (Bchl), a pigment containing reaction centres and light harvesting complexes, in
the presence of oxygen to generate biological energy *via* phototrophy, while they lack carbon fixation
genes and are incapable of synthetizing organic components from inorganic sources (Koblížek 2015).
Surprisingly, the role of photoheterotrophy in the maintenance of survival and metabolic activity in
airborne bacteria has never been investigated, although the ability to use light may be particularly
beneficial (Soora & Cypionka 2013). In the present study, using an atmospheric simulation chamber
(ASC) dedicated to bioaerosol studies, we evaluated the possibility that phototrophy could support
survival and metabolic activity in a photoheterotrophic *Methylobacterium* strain isolated from clouds.
Total airborne cell abundance, particle size, viability, cultivability and ATP content were monitored
over time using a combination of on-line and off-line methods. In addition, metagenomes and
metatranscriptomes of natural aerosols and clouds were reanalysed for bioindicators of anoxygenic
photosynthesis. We demonstrate that phototrophy provides selective advantages in airborne
photoheterotrophic bacteria, notably increased viability and shorter residence time, and that this
function is prevalent in the atmosphere and clouds. This could be key in the evolutionary persistence
and distribution of this biological function in nature.

114

115 **2 Materials and Methods**

116 **2.1 Bacterial strain**

117 *Methylobacterium* sp. R17b-9, the model strain investigated here, was originally isolated from
118 cloud water at Reunion island (Indian Ocean) (Charpentier et al. 2024). We confirmed the
119 phototrophic potential of *Methylobacterium* sp. R17b-9 by PCR targeting the *pufM* gene
120 **(Supplementary Material)**, which encodes the M sub-unit of the reaction center of AAPB commonly
121 used as a biomarker for anoxygenic photosynthesis, following the conditions described in (Vergne et
122 al. 2021). The production of bacteriochlorophyll *a* (Bchl *a*) in cultures was confirmed by fluorescence
123 using an infrared kinetic fluorometer (IRF, model FL6000, Photon Systems Instruments Ltd.) according
124 to the protocol described by (Koblížek et al. 2005, 2006). The kinetics of synthesis and degradation of
the pigment were measured in resting cells (suspensions in 0.8% NaCl w/v) under agitation, from cells
harvested from their mid-log phase of growth (Supplementary Material, and Supplementary Figure
S1).

For ASC experiments, cells were grown aerobically on R2A agar medium (Reasoner & Geldreich
1985), at room temperature (~22°C), under constant light exposure (COKOLILA, light spectrum from
410 to 700 nm, maximum light intensity: 60 µW/cm²) or dark condition leading to distinct phenotypes:
[Bchl-], devoid of the photoreceptor pigment bacteriochlorophyll, and therefore uncapable of
photosynthesis, and [Bchl+], respectively. Strain growth kinetics were obtained by monitoring the
optical density of liquid cultures at 600 nm (OD₆₀₀nm).

## 2.2 Chamber experiments

The atmospheric simulation chamber (ASC) ChAMBRe (Chamber for Aerosol Modelling and
Bio-aerosol Research) at the National Institute of Nuclear Physics in Genoa (INFN) is mainly dedicated
to bioaerosol studies. Its characteristics have been described previously (Massabò et al. 2018;
Vernocchi et al. 2023). Neutral density optical filters can be installed to attenuate the irradiance
spectrum of 29%, 50%, 66% and 81% of the full light intensity. A waveband-integrated bioaerosol
sensor (WIBS-NEO; Droplet Measurement Technologies®) sampling air at a flow rate of 0.3 L min⁻¹ was
been integrated in the particle monitoring system to measure the bioaerosol particle concentration
and size in real time.

Experiments in ChAMBRe (Figure 1) consisted *Methylobacterium* sp. R17b-9 cell suspensions
injected and exposed, as aerosols, to 3 conditions of constant light intensities (dark, 50% or full light
exposure, the 105% condition) for 2 hours, with samples collected periodically. Independent triplicates
were performed for each light condition and phenotype, leading to a total of 18 experiments. Cells
were initially grown either under constant dark or light conditions, to allow for the expression of the
[Bchl+] phenotype, respectively. For each experiment, a fresh cell suspension was prepared from agar
plates (to limit the formation of aggregates that occur in liquid culture) incubated for 5 days at ambient
temperature (~22°C), by scrapping off the surface using sterile plastic loops, and suspended in sterile NaCl (0.8%) at a concentration adjusted to ~10⁹ cells mL<sub>liq</sub><sup>-1</sup> based on OD<sub>600nm</sub>. Aliquots of the suspension were collected for analyses (T<sub>-1</sub>), and the suspension was then nebulized in ChAMBRe using the Sparging Liquid Aerosol Generator (SLAG, CH Technologies) with a 0.75" diameter porous disc and nominal pore size of 2 µm. Four milliliters of the bacterial suspension were dripped onto the SLAG porous stainless-steel disk with a syringe pump at a flow rate of 0.4 mL min<sup>-1</sup> and nebulized inside ChAMBRe in order to achieve a concentration of airborne cells of ~10⁶ cells m<sub>air</sub><sup>-3</sup>, as monitored by WIBS.

Aerosol samples were collected 3 minutes after the end of injection of the cell suspension (T₀), and every 30 min for 2 hours (T₁ - T₄). The three minutes after the injection was selected as T₀ because this is the necessary time for the injected liquid solution to mix and achieve homogeneity inside the ChAMBRe's volume (Massabò et al. 2018). Sampling was performed at the air flow rate of 12.5 L min⁻¹ for 10 min, or 20 min for the final time T₄, using impingers (SKC Biosampler; (Lin et al. 1999)) sterilized by autoclave and filled with 20 mL of sterile 0.8% NaCl solution (w/v). The precise volume of liquid contained in the sampler before and after sampling was determined by weighting, in order to adjust possible loss due to evaporation. The concentrations of total and cultivable cells and ATP, and the fraction of viable cells were determined for the sprayed suspension and from the sampling liquid for each sampling time, as detailed below. Since the atmospheric pressure inside ChAMBRe is maintained constant, the data at each sampling time were corrected by dilution factor considering the flowrate of the WIBS (constant at 0.3 L min⁻¹) and sampling for analyses (10 min at 12.5 L min⁻¹). The correction factors F<sub>dil</sub> due to dilution of airborne material in the chamber at each sampling time are: T₀ = 1.00; T₁ = 1.06; T₂ = 1.13; T₃ = 1.20; T₄ = 1.27. For clarity where possibly ambiguous, units are specified as referring to liquid or air volumes in the form of subscripts.

The airborne concentration of a variable of interest x<sub>air</sub> (in #.m<sub>air</sub><sup>-3</sup>) was estimated from measurements in the sampling liquid, as:
175

$$x_{air} = \frac{x_{liq} \times F_{dil} \times V_{imp}}{V_{air}}$$

176 Where $x_{liq}$ is the concentration measured in the liquid (# mL$_{liquid}^{-1}$), $F_{dil}$ is the dilution factor (see above),
177 $V_{imp}$ the volume of collection liquid in the impinger (20 mL), and $V_{air}$ is the quantity of air drawn off by
178 the impinger during sampling, in $m_{air}^{-3}$.

179

## 2.3 Assessment of viability

180 The viable cell fractions both in the initial cell suspensions, and in the air from the collection
181 liquid of the impinger at T0, T2 and T4 after injection was quantified by flow cytometry, using a LSR
182 Fortessa flow cytometer (Becton Dickinson, Franklin Lakes, NJ) on samples stained using the
183 “Live/dead™ fixable far red” kit (Invitrogen; ref L34974) (Supplementary Material).

184

## 2.4 Quantification of culturable cells

185 The concentration of cultivable cells in initial cell suspensions and in the impinger's collection
186 liquid at each sampling time was estimated by plate-counting. 100 µL of the appropriate 10-fold serial
187 dilutions of the cell suspension ($10^{-2}$; $10^{-3}$; $10^{-4}$ dilutions) or collection liquid (no dilution; $10^{-1}$, $10^{-2}$) were
188 spread on R2A medium, and incubated at 22–25°C for 5-7 days in the dark before counting the colonies
189 formed. Cultivability was expressed as the ratio between the concentration of colonies and that of
190 total airborne particles in the chamber quantified by WIBS.

191

## 2.5 Quantification of ATP

192 Adenosine-5'-triphosphate (ATP) content was quantified by bioluminescence (ATP Biomass Kit
193 HS; BioThema; Handen, Sweden), using luminometer (Lumac Biocounter M2500), from triplicate
194 volumes of 50 µL of the initial cell suspensions and of the impinger's collection liquid, as in (Vaïtilingom
195 et al. 2013), and normalised to the total particle number quantified by WIBS.

196

## 2.6 Reanalysis of metagenomes and metatranscriptomes for anoxygenic photosynthesis biomarkers
The metagenomes and metatranscriptomes of nine cloud water and five aerosol samples acquired previously in the natural atmosphere were re-examined here for *pufM*, the biomarker gene of anoxygenic photosynthesis in bacteria. The datasets were originally presented in (Péguilhan *et al.* 2025). For each sample, the number of reads with a hit was normalized to the total number of reads, and to gene length (334 pb). For each read with a hit, the taxonomy of the most similar Uniprot protein sequence was retrieved (Supplementary Material).

## 2.7 Statistical analyses, particle loss rate coefficient and survival rates

The concentration of airborne cells in the chamber decreased following exponential decay, which can be expressed as:

$$y = a \times e^{(-bt)}$$

Where *a* is the initial cell concentration, *b* is the particle loss rate coefficient, and *t* is time in minutes.

With this equation, increasing trends, i.e., positive exponentials, result in negative values for *b*.

The half-life time $t_{1/2}$, during which the initial concentration is changed by a factor of 2 (halves or doubles in the case of negative *b*), can be calculated as follows:

$$t_{\frac{1}{2}} = \frac{\ln 2}{b}$$

The same principle applies to the survival rate, using the concentration of cultivable cells as the input variable.

All statistical analyses were performed using PAST version 4.03 (Hammer *et al.* 2001) and RStudio. Non-parametric Mann-Whitney, Kruskal-Wallis or Wilcoxon statistical and parametric Student tests were used depending on data distribution (Shapiro's normality test), as specified.
219 **3 Results**

220 **3.1. Kinetics of growth and Bchl synthesis in *Methylobacterium* sp. R17b-9**

221 In liquid culture, *Methylobacterium* sp. R17b-9 has a generation time of ~7 hours. In resting
222 [Bchl-] cells of *Methylobacterium* sp. R17b-9, initially grown in the light then deprived of dissolved
223 nutrients and light, the amount of bacteriochlorophyll reached a maximum after 4 days, then a plateau.
224 No bacteriochlorophyll production could be observed under exposure to light (**Supplementary Figure
225 S1A; Data S1**). In [Bchl+] exposed to light, initially grown under dark conditions, then exposed to light
226 in the absence of nutrient, a slow temporal decrease of fluorescence intensity was observed, indicating
227 some extent of degradation of bacteriochlorophyll by light, as shown by (Iba & Takamiya 1989)
228 (**Supplementary Figure S1B**). There was no difference in the absorption at wavelengths typical of
229 carotenoids (~400 to ~550 nm; (Yurkov & Beatty 1998) between [Bchl+] and [Bchl-] cells
230 (**Supplementary Figure S2**).

231 **3.2 ChAMBRe: Initial conditions**

232 A total of 18 experiments were carried out in the atmospheric simulation chamber (ChAMBRe),
233 from *Methylobacterium* sp. R17b-9 cell suspensions at concentrations ranging from 7.87x10⁸ to
234 2.20x10⁹ cells mL⁻¹ as estimated from OD₆₀₀nm. The proportion of viable cells estimated by flow
235 cytometry was 98 to 100 ±1%, regardless of the phenotype (**Figure 2B**). Just after injection (T₀), WIBS
236 data indicated total airborne cell particle concentrations from 4.12x10⁶ to 7.59x10⁶ cells m⁻³ in the
237 chamber. This corresponds to a high but realistic concentration of airborne bacteria in the natural
238 atmosphere (Bowers *et al.* 2009). All data are presented in **Data S1**.
239

239 **3.3 Temporal evolution of the number and size of airborne cells in the ASC**

240 During the course of the 2-hour experiments, bacteria particles were physically removed from
241 air, i.e., deposited, at rates depending on the phenotype (p<0.05, Mann-Whitney test; **Table 1**): on
242 average, [Bchl+] cells deposited about 1.71 times faster than their counterparts without the pigment
243

[Bchl-], with half-residence times of ~89 min vs ~152 min, respectively, as quantified by WIBS. Light intensity had no influence (p>0.05, Mann-Whitney test, **Table 1**).

244

245

WIBS measurements indicate larger average airborne particle size for [Bchl+] cells than for [Bchl-] cells (p<0.05, Mann-Whitney test; **Figure 2A**). Immediately after injection into the ASC, the mean particle diameter for [Bchl+] was 1.20 µm, compared with 1.11 µm for [Bchl-], i.e. ~8% larger. Assuming spherical particles, the corresponding average biovolume represented by [Bchl+] airborne cell particles (0.90 µm³) was ~30% times bigger than that of [Bchl-] cells (0.72 µm³). Over the course of the experiment, the mean particle diameter decreased by around 14% independently from the phenotype and light condition, to 1.02 µm (~0.56 µm³) for [Bchl+] and 0.96 µm (~0.46 µm³) for [Bchl-] at the end of the experiments (**Figure 2A**). Assuming that the latter correspond to single cells we can estimate an average of 1.6 cells per particle at the beginning of the experiment, independently from the phenotype. Much bigger cell aggregates also rarely occurred (~0.05% of the total particles), with a maximum aerodynamic diameter of 4.5 µm, i.e., ~50 µm³, corresponding to ~100 cells.

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**3.4 Temporal evolution of viability and cultivability in airborne [Bchl+] and [Bchl-] cells exposed to different conditions of light**

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The aerosolization process by nebulization affected viability, as estimated by flow cytometry, by ~10% to 20%: just after injection (T₀), the fraction of viable cells was on average 88 ±12% for [Bchl+] and 83 ±23% for [Bchl-] cells (**Figure 2B**). The highest and lowest viability were observed in [Bchl+] and [Bchl-] cells exposed to light, independently from its intensity, with 90 ±6% and 81 ±21% viable cells, respectively. Under exposure to moderate or high light intensity, [Bchl+] cells maintained higher viability than [Bchl-] cells (p<0.05, Mann-Whitney test), with 89 ±11% and 78 ±12% of viable cells, respectively, after 120 min airborne.

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The temporal evolution of cell cultivability after injection (i.e. the proportion of cultivable cells to that of total airborne particles as quantified by WIBS) as well depended on phenotype, with higher cultivability in [Bchl+] cells than in [Bchl-] cells at every time point independently from light exposure

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267
and intensity (p<0.05; Mann-Whitney test) (Figure 2C). Just after injection (T₀), there were between 0.72 ±0.15 and 0.95 ±0.16 CFU particle⁻¹ for [Bchl+] cells exposed to moderate and high light intensities, respectively, and between 0.05 ±0.04 and 0.20 ±0.07 CFU particle⁻¹ for [Bchl-] exposed to moderate light and dark conditions, respectively. At the end point, after 2 hours airborne, cultivability ranged between 0.90 ±0.19 to 1.71 ±0.20 CFU particle⁻¹ for [Bchl+] cells, and between 0.02 ±0.01 and 0.14 ±0.04 CFU particle⁻¹ for [Bchl-] cells under high light intensity and dark conditions, respectively.

Exposure to light caused differences in the temporal evolution of cultivability depending on the phenotype (Figure 2D). In [Bchl-] bacteria exposed to moderate or high light intensity, cultivability decreased to 48 ±28% and 38 ±13% of the initial values after 2 hours airborne, respectively, corresponding to t₁/₂ of 121 and 100 min, respectively (Table 2). In airborne [Bchl-] cells under dark conditions, as well as in [Bchl+] cells exposed to high light intensity, there was no significant change of cultivability over time (p>0.05; Wilcoxon test), with 94 ±20% and 71 ±19% of the initial values at the end of the experiments, respectively. Finally, in [Bchl+] in the dark or exposed to moderate light intensity, cultivability increased over time (p<0.05; Wilcoxon test), following very similar trends, to 187% ±23% and 187% ±32% of their initial values, respectively.

## 3.5 Temporal evolution of ATP content

ATP concentration (i.e. ATP concentration normalized to particle concentration as quantified by WIBS) was phenotype-dependent, with higher (up to twice) ATP concentrations in [Bchl+] than in [Bchl-] cells at every time point, independently from light intensity (p<0.05; Mann-Whitney test) (Figure 4). Just after injection in the ASC, this ranged from (2.2 ±0.6) x10⁻¹⁶ mol ATP particle⁻¹ for [Bchl-] cells to (8.4 ±2.8) x10⁻¹⁶ mol ATP particle⁻¹ for [Bchl+] cells under dark conditions. After 2 hours airborne, there were between (2.1 ±0.5) x10⁻¹⁶ mol ATP particle⁻¹ for [Bchl-] cells under high light intensity, to (1.2 ±0.2) x10⁻¹⁵ mol ATP particle⁻¹ for [Bchl+] cells in the dark. When normalized to the biovolumes estimated from WIBS measurements (see section 3.3), [Bchl+] cell particles always had higher ATP contents than [Bchl-] ones, with averages of (1.1 ±0.4) x10⁻¹⁶ mol ATP µm⁻³ and (5.1 ±2.6)
293 x10⁻¹⁷ mol ATP µm⁻³, respectively, just after the injection and (1.3 ±0.5) x10⁻¹⁶ and (4.5 ±1.7) x10⁻¹⁷ mol
294 ATP µm⁻³, respectively, after 2 hours.

295 In all experiments, the ATP content in airborne cell particles did not significantly vary over time
296 after injection (p>0.05, Wilcoxon test). However, trends differed depending on phenotype and light
297 exposure: in [Bchl-] cells, the ATP concentration remained at low and constant regardless of light
298 exposure. In [Bchl+] cells in the dark, this tended to increase for 90 min after injection, before levelling
299 off or decreasing. Under moderate light exposure, we observed a trend of increasing ATP
300 concentration after 60 min airborne. Finally, at high light exposure, ATP content rather tended to
301 decrease over time. At the end of the experiments, higher ATP concentrations were observed in [Bchl+]
302 cells exposed to moderate light or dark conditions than in cells exposed to high light (p<0.05, Mann-
303 Whitney test) (Figure 4).

304 **3.6 Proportion and activity of anoxygenic photosynthetic bacteria in atmospheric metagenomes and**
305 **metatranscriptomes**

306 Metagenomes (MG) and metatranscriptomes (MT) of cloud water and aerosol samples from
307 natural atmosphere at a high mountain site (puy de Dôme, France, 1465 m asl) previously acquired
308 (Péguilhan et al. 2025) were re-examined here for genes and transcripts of *pufM*, the biomarker of
309 anoxygenic photosynthesis. Sequences of *pufM* were detected in all the metagenomes, with an
310 average proportion of 3.2 ppb (part per billion base-pairs) (Supplementary Table S1 ; Data S2). The
311 taxonomic affiliations point to the bacteria orders *Rhodospirillales*, *Burkholderiales*,
312 *Sphingomonadales* and *Hyphomicrobiales* (including numerous *Methylobacterium*). Transcripts were
313 detected in all the 5 cloud-free air samples, at an average relative abundance of 6.4 ±7.2 ppb, and in
314 33% of the 9 cloud samples, at an average of 1.7 ±4.1 ppb (Supplementary Table S1). The gene
315 expression level (i.e. the average relative representation of *pufM* transcripts in MTs respect to genes
316 in MGs, referred to as RNA:DNA ratio) was significantly higher in clear atmosphere (1.61 ±0.78) than
317 in clouds (0.52 ±1.13; p < 0.05, Mann-Whitney test (Supplementary Table S1)), suggesting higher
318 photosynthetic activity in these bacteria taxa.

319

320 **4 Discussion**

321 For the first time this study examines the influence of a particular phenotype on bacteria
322 exposed to aerial transport on a single clone (as opposed to studies investigating genetically modified
323 organisms). By growing cells under dark or light conditions, we could control the production of
324 photoheterotrophic pigment, bacteriochlorophyll, in a facultative anoxygenic strain of
325 *Methylobacterium* sp. isolated from clouds. Such control of bacteriochlorophyll synthesis by light in
326 facultative anoxygenic phototrophic bacteria was reported in other members of Alphaproteobacteria
327 and Hyphomicrobiales, including *Porphyrobacter*, *Erythrobacter*, *Rhodospirillum*, *Dinoroseobacter*,
328 *Hoeflea*, *Stappia* and others (Biebl & Wagner-Döbler 2006; Breznak *et al.* 1978; Okamura *et al.* 1986;
329 Tinguely *et al.* 2023), and also occurs in natural aquatic systems (Fecskeová *et al.* 2019).

330 **4.1 Phototrophy supports survival and metabolic activity in airborne cells**

331 The survival of airborne [Bchl-] *Methylobacterium* sp. R17b-9 cells decreased over time, in
332 particular when exposed to light, with half-life survival rates ranging from ~1.5 h under light exposure
333 to ~12 h in the dark. Although the experimental conditions (temperature, humidity, etc) may vary
334 between studies, this is in the upper range of airborne temporal survival rates reported for bacteria in
335 ASC, with half-life times of ~30 min for *E. coli* (Vernocchi *et al.* 2023) and ~4 h in *P. syringae* and *P.
336 fluorescens* (Amato *et al.* 2015). This may be related to biological trait(s) other than bacteriochlorophyll
337 and responsible for a high propensity to airborne dispersal in this bacterial strain. In turn, we observed
338 no loss of viability or cultivability in airborne [Bchl+] cells over the course of the experiments, lasting 2
339 hours, either under dark or light conditions. This evidences that the [Bchl+] phenotype is linked with a
340 significant selective advantage to airborne cells.
Under bulk conditions (Supplementary Material and Supplementary Figure S3), in the absence of dissolved nutrient, the [Bchl] phenotype did not influence the survival of *Methylobacterium* sp. R17-b9 over the time frame considered of a few hours (5 hours) (Supplementary Figure S4). Previous studies indicated a half-life time of 3 to 14 days in an AAPB (*Rhodospirillum rubrum*) placed under such starvation conditions in the presence of light (Breznak *et al.* 1978).

We observed higher ATP concentrations in airborne [Bchl+] cells as compared to [Bchl-] cells, for all the conditions assessed, and under moderate light exposure, the cellular ATP concentration tended to increase over time. This suggests active phototrophy, i.e., the direct synthesis of ATP from light through the translocation of protons, as observed in the bulk phase experiments (Supplementary Figure S5). AAPB can use both organic carbon sources and light to generate biochemical energy, in the presence of oxygen. This is not associated with the uptake of CO₂, and the energy from light is directly converted into ATP molecules, without the release of oxygen. The energy absorbed by the carotenoids is transferred to the Bchl *a* molecule within picoseconds (Šlouf *et al.* 2013) and used for primary charge separation in the reaction center. It has been estimated that phototrophy in AAPB generates up to ~15 ATP molecules per reaction center per second (Koblížek *et al.* 2003), which may account for up to 20% of the cellular needs (Kolber *et al.* 2001). Surprisingly, high ATP levels also occurred in [Bchl+] cells in the dark, where photosynthetic processes are not possible. Short exposure to light, on the order of minutes, during sample preparation and injection in the chamber likely was involved. [Bchl-] cells were grown under constant light conditions, which may have generated photo-oxidative stress during culture (Gourmelon *et al.* 1994; Ziegelhoffer & Donohue 2009) and solicited energy resources and led to a decrease of ATP content, as opposed to [Bchl+] cells, which rather took benefit of exposure to light.

We observed no difference in the carotenoid content of [Bchl+] and [Bchl-] cells in our study so this is not involved in the differential survival rates between the phenotypes. AAPB species have high levels of carotenoid pigments, which typically act as photoprotectors and against phototoxic
molecules and photo-oxidation (Takaichi et al. 1991; Tuveson et al. 1988; Yurkov et al. 1993). 366

Carotenoid synthesis is strain-dependent, either constitutive or modulated (promoted or inhibited) by 367 light exposure (Iba & Takamiya 1989; Soora & Cypionka 2013; Takano et al. 2006). 368

## 4.2 Trophic conditions during atmospheric transport 369

Aerial transport is thought to exert strong selective pressure on microorganisms, due to high 370 exposure to UV light and oxidants, and low water and nutrient availability (Ervens et al. 2025; Joly et 371 al. 2015; Šantl-Temkiv et al. 2022; Smith et al. 2011; Tong & Lighthart 1998). Metabolic flexibility 372 provides a selective advantage in such environments where the conditions fluctuate (Chen et al. 2021). 373

Anoxygenic photosynthesis enhances survival and fitness in oligotrophic environments (Soora & 374 Cypionka 2013). Diverse potential AAPB are frequently recovered viable from atmospheric samples, 375 including *Sphingomonas*, *Burkholderia* and *Methylobacterium* (Amato et al. 2017; Vaïtilingom et al. 376 2012; Woo & Yamamoto 2020). The reanalysis of metagenomes and metatranscriptomes of natural 377 atmospheric samples indicates that anoxygenic photosynthesis functions are indeed prevalent and 378 may be particularly active outside of clouds, at low levels of dissolved organic nutrients. 379

In cloud and precipitation, typically micromolars of dissolved bioavailable organic carbon and 380 inorganic ions can support microbial heterotrophic activity and some extent of growth, as shown in 381 laboratory studies (Amato et al. 2007; Bianco et al. 2019; Deguillaume et al. 2014; Šantl-Temkiv et al. 382 2013; Sattler et al. 2001). However, the distribution of the aqueous solution in cloud droplets, as 383 opposed to bulk solutions in flasks, creates microenvironments where dissolved substrates remain 384 limited (Ervens et al. 2025; Khaled et al. 2021; Nuñez López et al. 2024; Péguilhan et al. 2025). Under 385 these conditions, photoheterotrophy, the alternative capacity to use light besides organic carbon as a 386 source of energy to fuel metabolic processes, including responses to stress and nutrient processing, 387 appears as a powerful trophic mode to support microbial survival and metabolic activity in the 388 atmosphere. 389
390 Methylotrophs such as *Methylobacterium* are heterotrophic bacteria that can utilize $C_1$ compounds as carbon sources. Methanol or methylamine is oxidized to formaldehyde and formate 391 and eventually to $CO_2$ for energy metabolism, and carbon is assimilated via the serine cycle 392 (Chistoserdova *et al.* 2003; Quayle 1963). Many dissolved $C_1$ compounds are present in cloud water at 393 micromolar concentrations (Amato *et al.* 2007; Deguillaume *et al.* 2014; Vaïtilingom *et al.* 2013). 394 Recent atmospheric model studies suggest that biodegradation in the atmosphere may present a 395 significant loss of formic acid/formate whereas its role of less soluble compounds, such as methanol 396 and formaldehyde, is less important (Khaled *et al.* 2021; Nuñez López *et al.* 2024). Facultative 397 photoheterotrophic and methylotrophic bacteria such as *Methylobacterium* could play a major role in 398 the biodegradation of these compounds in clouds. 399

400 **4.3 Bchl phenotype influences transport range**

401 The concentration of airborne cultivable cells decreased over time due to combined removal 402 by deposition and by loss of viability (Supplementary Figure S6). As observed with WIBS, the 403 phenotype influenced airborne particle size, and also their residence time: [Bchl+] cells were 8% larger, 404 and had 1.7 times shorter atmospheric residence time than [Bchl-] cells. The increasing cultivability in 405 airborne [Bchl+] cells exposed to moderate light over time implies that they maintained viability longer 406 they were removed by physical processes, i.e., they survived longer than they remained airborne. The 407 possibility that cells multiplied in the chamber while airborne can reasonably be excluded based on 408 the generation time of cell cultures under our conditions (~7h gen⁻¹). Rather, it is likely that larger cell 409 aggregates disrupted over time, as attested by the temporal decrease of the average particle size 410 (Figure 3 and Supplementary Figure S7), leading to an increasing number of colonies contributed by 411 each airborne particle. As each particle can only carry at the maximum one CFU independently of its 412 size, the cultivability, as CFU particle⁻¹, is likely an underestimation of the actual fraction of cultivable 413 cells, in particular at the beginning of the experiments. 414
We could observe a rapid and significant production of bacteriochlorophyll, within a few hours, in starved cells under dark conditions, and no production under exposure to light. Bacteria cells, as spherical particles of 1 µm of diameter, have an estimated average residence time of 3.4 days in the natural atmosphere (Burrows et al. 2009). It is therefore conceivable that cells synthesize pigments once airborne during night time and gain phototrophic capacities and compensate for the degradation and damages accumulated to Bchl by photolysis or photobleaching during daytime (Bopp et al. 1997). It is also notable that bacteriochlorophyll synthesis follows a rhythmic diurnal cycle in many AAPB (Tinguely et al. 2023). One could see here some extent of physiological synchronization with the atmospheric life cycle of these bacteria, by concurring with increased aerosolization rates from vegetated surfaces at sunrise (Lighthart 1997). Such link between microbial traits and their atmospheric life cycle was suggested earlier regarding ice nucleation active bacteria and their possible influence on clouds and the water cycle (bioprecipitation) (Morris et al. 2008).

## 4.4 Ecological implications

The auxiliary metabolism of anoxygenic photosynthesis in AAPB is often associated with other, energy-intensive, metabolism: biological nitrogen fixation, i.e., the ability to utilize atmospheric nitrogen ($N_2$) (Galloway et al. 2003). Nitrogen fixers are often classified as mutualists with leguminous plants through nodules in the rhizosphere and phyllosphere (Giraud et al. 2000). Some nitrogen fixers adopted anoxygenic photosynthesis to support biochemical energy needs. A large number of anoxygenic photosynthetic bacteria are also nitrogen fixers (Madigan 1995), found in specialized structures of the phyllosphere such as phytotelms, which collect rain water and promote the development of nitrogen fixers (Lehours et al. 2016; Vergne et al. 2021, 2023). This mechanism of recovering gaseous nitrogen in association with anoxygenic photosynthesis could support the maintenance of metabolic functions during atmospheric transport.

The question arises as to why photoheterotrophic type of metabolism is not more frequent and abundant in ecosystems. Anoxygenic photosynthesis provides a selective advantage in nutrient-
limited environments such as the atmosphere but falls short of offering a competitive advantage in interspecies interactions. For instance, in aquatic ecosystems, grazing pressure by protists may explain their relatively low abundance (Strom 2000), by targeting preferentially larger cells and therefore disproportionately affecting anoxygenic photosynthetic bacteria due to their relatively large size (Fauteux et al. 2015; Sieracki et al. 2006). In the atmosphere, the selective advantage provided by facultative phototrophy could have contributed to the maintenance of this ancestral metabolism during evolution (Hamilton 2019), and to its worldwide distribution in the environment.

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<figure><img src="image_3.png" /></figure>

462

463 **Figure 1:** Schematic of the experimental strategy.

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<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

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<caption>**Figure 2:** Temporal evolution of airborne [Bchl+] and [Bchl-] *Methylobacterium* sp. exposed to dark, moderate light or high light intensity. (A) Average airborne particle size (B) viability, (C) absolute and (D) relative cultivability. Each experiment was realized as independent triplicates (n=3). The letters (a and b) on the right of the symbols indicate statistical differences between phenotypes. Asterisks (*) indicate significant differences with the initial value, (p < 0.05, Mann-Whitney or Wilcoxon test). For A and B, standard deviations are not shown for clarity reasons, and were on the order of 40% and 10% of the mean, respectively. For A, all sampling times are statistically different from one another (p < 0.05, Mann-Whitney).</caption>
479

<table><caption>**Table 1:** Half-residence time of bacteria aerosol particles in the chamber, based on WIBS data.</caption><thead><tr><th rowspan="3">Half-residence time</th><th colspan="3">[Bchl+]</th><th colspan="3">[Bchl-]</th></tr><tr><th>Dark</th><th>Light50%</th><th>Light105%</th><th>Dark</th><th>Light50%</th><th>Light105%</th></tr><tr><th>t<sub>1/2</sub> (min)</th><th>t<sub>1/2</sub> (min)</th><th>t<sub>1/2</sub> (min)</th><th>t<sub>1/2</sub> (min)</th><th>t<sub>1/2</sub> (min)</th><th>t<sub>1/2</sub> (min)</th></tr></thead><tbody><tr><td>Experiment #1</td><td>n.a.</td><td>56</td><td>57</td><td>141</td><td>133</td><td>154</td></tr><tr><td>Experiment #2</td><td>51</td><td>57</td><td>76</td><td>198</td><td>154</td><td>105</td></tr><tr><td>Experiment #3</td><td>126</td><td>83</td><td>204</td><td>231</td><td>133</td><td>114</td></tr><tr><td>Average ± standard error of the mean</td><td>88 ±53</td><td>65 ±15</td><td>112 ±80</td><td>190 ±45</td><td>140 ±12</td><td>124 ±26</td></tr><tr><td>Phenotype average ± standard error of the mean</td><td>89 ±53</td><td></td><td></td><td>152 ±40</td><td></td><td></td></tr></tbody></table>

480

*n.a. : data not available

481

<figure><img src="image_8.png" /></figure>

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<caption>**Figure 3:** Aerosols particle size (aerodynamic diameter) distribution, as measured by WIBS, in the atmospheric simulation chamber after injection of cell suspensions ("Initial time"; plain filling) and at the last sampling time ("Final time", dashed filling) for [Bchl+] and [Bchl-] cells, all conditions of light exposure included. Asterisks (*) indicate significant differences between [Bchl+] and [Bchl-] cells at a given time (p < 0.05, Mann-Whitney or Student test). Triangles (Δ) indicate significant differences between initial and final times, for [Bchl+] (green) or [Bchl-] (brown) phenotypes (p < 0.05, Student paired or Wilcoxon test).</caption>

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**Table 2:** Half-life time for cultivability of bacteria as aerosol in the chamber, estimated for each individual experiment based on the temporal evolution of CFU particle⁻¹ concentration. The plus symbol (+) indicates an increase in cultivability over time, while the minus symbol (-) denotes a loss of cultivability. Asterisks (*) indicate statistical differences between initial and final times (p<0.05, Mann-Whitney or Student test).

<table><thead><tr><th rowspan="3">Half-cultivability time</th><th colspan="3">[Bchl+]</th><th colspan="3">[Bchl-]</th></tr><tr><th>Dark</th><th>Light50%</th><th>Light105%</th><th>Dark</th><th>Light50%</th><th>Light105%</th></tr><tr><th>t<sub>1/2 life</sub> (min)</th><th>t<sub>1/2 life</sub> (min)</th><th>t<sub>1/2 life</sub> (min)</th><th>t<sub>1/2 life</sub> (min)</th><th>t<sub>1/2 life</sub> (min)</th><th>t<sub>1/2 life</sub> (min)</th></tr></thead><tbody><tr><td>Experiment #1</td><td>n.a.</td><td>99 (+)</td><td>231 (-)</td><td>1733 (-)</td><td>63 (-)</td><td>173 (-)</td></tr><tr><td>Experiment #2</td><td>108 (+)</td><td>131 (+)</td><td>533 (+)</td><td>231 (-)</td><td>69 (-)</td><td>63 (-)</td></tr><tr><td>Experiment #3</td><td>187 (+)</td><td>365 (+)</td><td>533 (+)</td><td>231 (-)</td><td>231 (-)</td><td>63 (-)</td></tr><tr><td>Average ± standard error of the mean</td><td>148 ±56 (*) (+)</td><td>198 ±145 (*) (+)</td><td>278 ±441 (+)</td><td>732 ±867 (-)</td><td>121 ±95 (*) (-)</td><td>100 ±64 (*) (-)</td></tr></tbody></table>

*n.a.: data not available

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<figure><img src="image_9.png" /></figure>

497

**Figure 4:** Average temporal evolution of ATP concentration (mol particle⁻¹) in the [Bchl+] and [Bchl-] airborne *Methylobacterium* sp. under dark conditions or exposed to moderate or high light. Each experiment was realized as independent triplicate (n=3) Asterisks (*) indicate significant differences between phenotypes at given time after injection and light condition (p<0.05, Mann-Whitney test).

501

502
503 Acknowledgments:

504 We are grateful for the support and facilities provided by the National Institute of Nuclear Physics in

505 Genoa (INFN), in particular to Dario Massabò, Elena Abd El and Paolo Prati. We would also like to thank

506 Jonathan Colombet and Hermine Billard for their help with the cytometry platform at the Laboratoire

507 Microorganismes: Genome Environnement (LMGE). F.M. was supported by a PhD fellowship from the

508 French Ministry of Education and Research.

509

510 Study funding:

511 This work has received funding from the European Union's Horizon 2020 research and innovation

512 program through the ATMO-ACCESS Integrating Activity under grant agreement No 101008004.

513 ATMO-ACCESS TNA (Trans-national access): TNA-5G--0000000021, *Advantage of light utilization in*

514 *bacterial survival in the atmosphere*.

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524
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