Maxime Deforet
email: maxime.deforet@sorbonne-universite.fr.

Long-range alteration of the physical environment mediates cooperation between Pseudomonas aeruginosa swarming colonies

Keywords: Pseudomonas aeruginosa, swarming, agar gel, surfactants, rhamnolipids

Pseudomonas aeruginosa makes and secretes massive amounts of rhamnolipid surfactants that enable swarming motility over biogel surfaces. But how this rhamnolipids interact with biogels to assist swarming remains unclear. Here I use a combination of optical techniques across scales and genetically-engineered strains to demonstrate that rhamnolipids can induce agar gel swelling over distances >10,000x the body size of an individual cell. The swelling front is on the micrometric scale, and is easily visible using shadowgraphy. Rhamnolipid transport is not restricted to the surface of the gel, but occurs through the whole thickness of the plate and, consequently, the spreading dynamics depends on the local thickness. Surprisingly, rhamnolipids can cross the whole gel and induce swelling on the opposite side of a two-face Petri dish. The swelling front delimits an area where the mechanical properties of the surface properties are modified: water wets the surface more easily, which increases the motility of individual bacteria and enables collective motility. A genetically-engineered mutant unable to secrete rhamnolipids (rhlA -), and therefore unable to swarm, is rescued from afar with rhamnolipids produced by a remote colony.

These results exemplify the remarkable capacity of bacteria to change the physical environment around them and its ecological consequences.

Introduction

Bacteria have a remarkable ability to change the world around them through their collective behaviors [START_REF] Papenfort | Quorum sensing signal-response systems in gramnegative bacteria[END_REF][START_REF] Despoina | Bacteria use collective behavior to generate diverse combat strategies[END_REF][START_REF] Ratzke | Modifying and reacting to the environmental ph can drive bacterial interactions[END_REF]. Some of these changes are physical : bacteria interact mechanically with their environment [START_REF] Persat | The mechanical world of bacteria[END_REF] and they are also able to feedback on the physical environment [START_REF] Veysel | Molecular architecture and assembly principles of vibrio cholerae biofilms[END_REF][START_REF] Su Chuen Chew | Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides[END_REF][START_REF] Tropini | How the physical environment shapes the microbiota[END_REF].

Petri dishes with an agar biogel are widely used to investigate collective bacterial motility and its interplay with other biological processes [START_REF] Wadhwa | Bacterial motility: machinery and mechanisms[END_REF]. This approach was used to uncover chemotactic genes [START_REF] Greenfield | Self-organization of the escherichia coli chemotaxis network imaged with super-resolution light microscopy[END_REF]10], mechanisms of action of pharmaceutical molecules [START_REF] Ok Mirzoeva | Antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and motility of bacteria[END_REF], evolutionary dynamics [START_REF] Baym | Spatiotemporal microbial evolution on antibiotic landscapes[END_REF], quorum sensing [START_REF] Daniels | Quorum sensing and swarming migration in bacteria[END_REF][START_REF] Nachiket | Surface hardness impairment of quorum sensing and swarming for pseudomonas aeruginosa[END_REF], and other social behaviors of bacteria [START_REF] Jeckel | Learning the space-time phase diagram of bacterial swarm expansion[END_REF][START_REF] Badal | Foraging signals promote swarming in starving pseudomonas aeruginosa[END_REF][START_REF] Monaco | Spatial-temporal dynamics of a microbial cooperative behavior resistant to cheating[END_REF].

Agar gel serves as a physical and chemical substrate: cells are inoculated at the surface of the gel, they proliferate by consuming essential nutrients supplemented to the gel. In that regard, an 2 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 2, 2022. ; https://doi.org/10.1101/2022.06. [START_REF] Daniel | A field guide to bacterial swarming motility[END_REF].498166 doi: bioRxiv preprint agar plate is generally viewed as a passive element merely providing mechanical support, water, and nutrients. During colony growth, bacteria secrete osmolytes (exopolysaccharides), which draw water from the gel to equilibrate osmotic imbalance and contribute to colony swelling and expansion, in non-motile [START_REF] Gabriel E Dilanji | Entropy-driven motility of sinorhizobium meliloti on a semi-solid surface[END_REF]19] as well as in motile colonies [START_REF] Yang | Influence of physical effects on the swarming motility of pseudomonas aeruginosa[END_REF][START_REF] Ma | An expanding bacterial colony forms a depletion zone with growing droplets[END_REF][START_REF] Rhodeland | Bacterial surface motility is modulated by colony-scale flow and granular jamming[END_REF]. While colony morphogenesis modeling attempts sometimes include nutrient and water depletion [START_REF] Srinivasan | A multiphase theory for spreading microbial swarms and films[END_REF][START_REF] Yan | Extracellular-matrix-mediated osmotic pressure drives vibrio cholerae biofilm expansion and cheater exclusion[END_REF], structural changes of agar gel are rarely considered.

In the case of the bacterium Pseudomonas aeruginosa, a common opportunistic pathogen, another gel modification needs to be considered: P. aeruginosa secretes rhamnolipids, a family of glycolipid surfactants of strong interest in medicine [START_REF] Thakur | Rhamnolipid the glycolipid biosurfactant: Emerging trends and promising strategies in the field of biotechnology and biomedicine[END_REF] and industry [START_REF] Varvaresou | Biosurfactants in cosmetics and biopharmaceuticals[END_REF][START_REF] Ña | Bioconversion of agro-industrial by-products in rhamnolipids toward applications in enhanced oil recovery and bioremediation[END_REF], in particular for their antimicrobial properties, their capacity to emulsify oil and participate in bioremediation, and their good environmental compatibility and biodegradability. Secretion of rhamnolipids allows the colony to rapidly and collectively spread into a branched shape, a phenotype called swarming [START_REF] Matthew | Bacterial swarming: a model system for studying dynamic self-assembly[END_REF][START_REF] Daniel | A field guide to bacterial swarming motility[END_REF]. Mutants that do not secrete rhamnolipids are unable to swarm [START_REF] Caiazza | Rhamnolipids modulate swarming motility patterns of ¡i¿pseudomonas aeruginosa¡/i¿[END_REF][START_REF] Xavier | A molecular mechanism that stabilizes cooperative secretions in pseudomonas aeruginosa[END_REF]. Due to their surfactant nature [START_REF] Yang | Evidence for biosurfactant-induced flow in corners and bacterial spreading in unsaturated porous media[END_REF], rhamnolipids are often said to lubricate the surface [START_REF] Boyle | Integration of metabolic and quorum sensing signals governing the decision to cooperate in a bacterial social trait[END_REF], decrease friction against the gel [START_REF] Theresa | Sliding on the surface: bacterial spreading without an active motor[END_REF], act as a wetting agent [START_REF] Tremblay | Self-produced extracellular stimuli modulate the pseudomonas aeruginosa swarming motility behaviour[END_REF], or lower surface tension [START_REF] Yang | Influence of physical effects on the swarming motility of pseudomonas aeruginosa[END_REF][START_REF] Sina R Ütschlin | Inhibitors of bacterial swarming behavior[END_REF]. But how the rhamnolipids interact with the agar biogel to enable swarming motility remains unclear.

Previous work used biophysical approaches to study how surfactant-assisted spreading of liquid film can yield to branching. This has been explored on liquid and solid substrates [START_REF] Omar | The development of transient fingering patterns during the spreading of surfactant coated films[END_REF][START_REF] Omar | Dynamics of surfactant-assisted spreading[END_REF],

and was extended to the case of P. aeruginosa swarming colonies [START_REF] Fauvart | Surface tension gradient control of bacterial swarming in colonies of pseudomonas aeruginosa[END_REF][START_REF] Trinschek | Modelling of surfactant-driven front instabilities in spreading bacterial colonies[END_REF]. In those works, rhamnolipids were modeled as a thin film of insoluble surfactants secreted by the colony and diffusing at the surface of a gel. Differences of surfactant concentration inside and outside the colony yield to gradients of surface tension and induced a branching instability (Marangoni effect). These modeling efforts ignored what was happening on the colony side, where a precursor film was sometimes observed. In particular, rhamnolipids are soluble in water [START_REF] Mohammad | Characterization of rhamnolipid produced by pseudomonas aeruginosa isolate bs20[END_REF] and these molecules are substantially smaller than the agar gel pores. Therefore, they are expected to diffuse though the entire agar gel, a hydrogel, whose structure can be modified through swelling [START_REF] Bibi | Effect of surfactants on swelling capacity and kinetics of alginate-chitosan/cnts hydrogel[END_REF][START_REF] Li | Delayed swelling and dissolution of hydrophobically associated hydrogel coatings by dilute aqueous surfactants[END_REF]. Bulk effects might complement surface effects and alter colony morphogenesis.

Here, I use a combination of imaging methods across scales to demonstrate that rhamnolipids secreted by the colony alter the physical properties of the agar gel, not only locally where they are secreted, but also all more widely around the bacteria. Moreover, their transport is not restricted to the surface. I find, instead, that the rhamnolipids imbibe the whole gel, which yields to gel swelling with a sharp swelling front. Bulk transport of rhamnolipids directly affects the expansion rate of the region imbibed by rhamnolipids. Gel imbibition by rhamnolipids cover so large distances that rhamnolipid-deficient colonies can be remotely rescued. More generally, this is an example of the remarkable ability of bacteria to change the mechanical properties of the world surrounding them.

Methods

Bacterial strains and growth conditions

The bacterial strains used in this study are described in Table S1. Bacterial cells were routinely grown in LB at 37ºC with aeration. Swarming plates were prepared as previously described [START_REF] Xavier | A molecular mechanism that stabilizes cooperative secretions in pseudomonas aeruginosa[END_REF].

Agar concentration, unless specified otherwise, is 0.5 % (w/v). Overnight culture of bacteria were washed twice in PBS and 2 µL of the washed suspension were used to inoculate a swarming plate in the center. The plates were then flipped and placed inside a 37ºC microbiological incubator.

rhlA -pBAD-RhlAB colonies were grown on 1% L-arabinose swarming plates to induce expression of the rhlA gene and robust secretion of rhamnolipids.

Whole swarming colony

An automated device was built inside a microbiological incubator (Heratherm IGS 100, Ther-moFisher Scientific), for fluorescence, shadowgraphy, and brighfield imaging of a 10-cm diameter Petri dish. A LED light source (pE-4000, Coolled, UK) was used for fluorescence excitation. A set of lenses and mirrors were used to bring an even illumination pattern onto the plate. A single LED light source (Thorlabs, USA) was positioned on one side of the chamber for shadowgraphy imaging. Generic white LED strings (Mouser, France) were positioned above the plate for brightfield imaging. Images were acquired with a CMOS camera (Cellcam Centro, Cairn Research, UK) with a macro lens (Navitar MVL7000, Thorlabs, USA). A dual-band emission filter (59010m, Chroma, USA) was mounted between the camera and the lens, for green and red fluorescence imaging. All devices were controlled with µManager (http://www.micro-manager.org).

Sessile droplets

Plates and swarming colonies were prepared as explained above. Plates were taken out of the incubator and placed under a Axiozoom V16 macroscope (Zeiss), set at the lowest magnification (field of view is 25.7x21.5mm), equipped with a CMOS camera (BlackFly S, FLIR, USA), and illuminated through a transilluminator (Zeiss) with a mirror position that mimics DIC illumination. 1 µL droplets of swarming media (recipe identical to that of the swarming plate, but without agar) were deposited on the surface of the gel, either inside or outside the swelling front generated by the colony. A movie was recorded as tens of droplets were deposited. Footprint diameters were measured, immediately after deposition, using ImageJ.

Fluorescent bead tracking

For quantifying gel swelling, 1 µm fluorescent polysterene beads (F13083, ThermoFisher Scientific) were added during the swarming plate preparation (final concentration of 10 5 beads/mL).

Colony were grown inside a microscopy incubator (Oko-Lab, Italy), mounted an a IX-81 Olympus inverted microscope, equipped with a LED light source (pE-4000, Coolled, UK). Images were taken with a 20x objective and a CMOS camera (BlackFly S, FLIR, USA), every minute, for 30 minutes, while the swelling front was passing through the field of view (confirmed by phasecontrast imaging). Diffraction pattern detection, beads detection, tracking, and calculation of the vertical displacement were performed with custom-made routines in MATLAB (MathWorks, USA). Calibration between diffraction pattern radius and vertical position was done by taking a vertical stack of images.

Profilometry

Height profiles were measured with a Zegage Pro interferometry profilometer (Zygo) equipped with a 5x Michelson objective (Nikon). An acquisition took 5 seconds (for one field of view 2x2.7 mm), and 20 positions were stitched together to reconstruct the whole height profile (Figure 1E).

For Figure 5C, the profiles were measured when the diameter of the rhamnolipids-imbibed area was approximately 3 cm in diameter. Four positions were recorded per plate (the 4th roots of unity). Each profile was then measured perpendicularly to the swelling front. The X = 0 location was identified as the inflection point of the profile. The reference height Z = 0 was imposed at location X = -500 µm and X = -400 µm, which tilt-corrected the whole profile. Heights in Figure 5C were measured at X = 1000 µm.

Stepwise substrate A 1.5 mm thick sheet of polydimethylsiloxane (PDMS) was made following standard protocol (RTV615A+B, 10:1, Momentive Performance Materials). A 1x2 cm slab was cut out of the PDMS sheet and was placed in a Petri dish. 20 mL of agar gel was then poured on top of the substrate, following usual swarming plate protocol.

Two-face Petri dish

The bottom of a Petri dish was drilled with a 10-mm drill bit. Holes were deburred with a 13-mm drill bit. Dishes were sterilized in 70% ethanol. The bottom side of each hole was taped with a piece of Parafilm, which was peeled off after the agar gel was poured and solidified.

Single-cell motility

A suspension of rhlA -cells growing in liquid swarming media was spun down. A 20 µL plastic pipette tip was used to sample cells from the pellet and to transfer cells onto the gel surface (approximately 2 mm from the swelling front identified in shadowgraphy). One second videos (46 frames-per-second) were acquired every 5 minutes, at 10x magnification, in phase contrast, 6 with an IX-81 Olympus inverted microscope. Auto-focus was performed before each acquisition.

Image analysis was performed independently for each video: Local density was measured on the first frame of the video, by thresholding the phase-contrast image, followed by local averaging.

The Density Index, defined to be between 0 (no cell) and 1 (cells form a uniformly dark population) is therefore the proportion of neighboring pixels that contains cells. To evaluate motility, the difference between the maximum projection and the minimum projection across the timestack yields to a map where pixels were bright if their grey values changed during the video.

Then, a threshold was applied and the result was locally average to produce a map of Speed Index between 0 (no pixel change) and 1 (all pixels have change values). This coarse-grained quantification is valid as long as cells are not too dense and cells do not move too much during one video (therefore, the analysis was limited to the region of the field of view where cells did not pile up and only one-second long videos were considered). To check the robustness of the method, various thresholding values have been tried, without significantly affecting the results.

Results

Rhamnolipids cause biogel swelling

Wild-type P. aeruginosa colony spreads on soft agar by forming a branched colony, a behavior called swarming [START_REF] Daniel | A field guide to bacterial swarming motility[END_REF]. rhlA, a gene involved in production of rhamnolipids, is required for swarming: an rhlA -colony, which does not produce rhamnolipids, is unable to swarm (Figure 1A). Shadowgraphy, an optical method that reveals non-uniformities in transparent media by casting a shadow onto a white background [START_REF] Gary | Schlieren and shadowgraph techniques: visualizing phenomena in transparent media[END_REF], revealed that an area of the gel around the rhamnolipid-producing colony was modified: a thin darker line surrounded the colony (Fig- rhamnose groups, called monorhamnolipids (mono-RLs) or dirhamnolipids (di-RLs) [START_REF] Gutierrez | Simultaneous inhibition of rhamnolipid and poly-25 hydroxyalkanoic acid synthesis and biofilm formation in pseudomonas aeruginosa by 2bromoalkanoic acids: Effect of inhibitor alkyl-chain-length[END_REF]. These molecules are lipids, therefore they could be localized using a lipid dye. Nile Red, whose emission spectrum depends on the polarity of the solvant [START_REF] Teo | Nile red fluorescence spectroscopy reports early physicochemical changes in myelin with high sensitivity[END_REF], was mixed to the agar gel during gel preparation. Fluorescence microscopy confirmed a sharp drop of fluorescence intensity coincides with the location of the darker line visualized in shadowgraphy (Figure 1C). rhlA -colony does not produce HAA but still produces fatty acid chain precursors, whose lipid nature can be captured by Nile Red imaging. In this case, fluorescence intensity gradually varied with the distance to the colony, but no sharp transition was observed. I also measured the wetting property of the gel surface by performing sessile droplets experiments [START_REF] Banaha | Spreading of liquid drops on agar gels[END_REF]. Water droplets were deposited on each side of the darker line localized by shadowgraphy. On average, the droplet footprint radius was 3.78 ± 0.27 (SD) mm on the colony side, and 2.13 ± 0.09 (SD) mm on the other side (Figure 1D). Neglecting gravity and therefore assuming a spherical cap shape allow the calculation of contact angles of 22.7º and 35.6º, respectively. The wetting footprint size was found to be uniform on the colony side, independent of the distance to the colony or the distance to the darker line. Similarly, the wetting footprint size was found to be uniform also outside the darker line.

This confirms that the darker line marks a sharp limit of the rhamnolipid range, with surface properties that correlates with the presence of rhamnolipids. (Of note, the resolution of the sessile droplet experiment does not enable to measure a gradient of surfactant concentration; the results merely demonstrate a contrast of concentration inside/outside the darker line.)

To understand the origin of the darker line visible in shadowgraphy, I had to first evaluate its 3D profile. Using an optical profilometer (a device capable to measure 3D profile of a reflective surface with a nanometric resolution), I measured a jump of 8 µm, with a maximal slope of 3º

(Figure 1E). The jump could emerge from two possible mechanisms: either it corresponds to a thick layer of rhamnolipid molecules covering the surface of the agar gel, as assumed by previous works [START_REF] Fauvart | Surface tension gradient control of bacterial swarming in colonies of pseudomonas aeruginosa[END_REF][START_REF] Trinschek | Modelling of surfactant-driven front instabilities in spreading bacterial colonies[END_REF], or alternatively rhamnolipids infiltrate the gel and the jump corresponds to a gel found to propagate faster on top of obstacles (Figure 2A), where the gel section was smaller, and slower on top of thicker sections of the gel, which is compatible with a transport of rhamnolipids through the whole gel. This effect was also found in gels of various thicknesses. Once again, thinner gels induced faster traveling speed of the swelling front (Figure 2B), in agreement with transport through the gel. Intriguingly, while thinner gels induce faster spreading of the swelling front, they also induce smaller swarming colonies (Figure S2). Finally, I tested the swelling speed in a gel with gradual change of thickness. Pouring liquid agar on a tilted dish resulted in a gel with a spatial gradient of thickness: on this substrate, the swelling front advanced faster on 10 the thinning side, slower on the thickening side, and at intermediate speed on the transverse directions (Figure 2B). Colony did not swarm at all on plates lower than 7 mL. They gradually were larger and larger on thicker gels and the colony size was maximal on plates of the nominal volume (20 mL).

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To further demonstrate transport of rhamnolipids through gels, I designed a "two-face" Petri dish. I inoculated bacteria on the lower side of the dish and a swelling front was observed on the upper side. The upper surface was characterized with shadowgraphy, profilometry, and sessile droplets (Figure 2D). These measurements confirmed that a rhamnolipid-induced swelling front propagated to the upper surface, even though rhamnolipids were secreted from the lower surface.

Rhamnolipids enable surface motility at the single-cell level

Rhamnolipids, transported within the gel, are required for the colony to spread at the surface of the gel. To better understand the interplay between bulk transport of rhamnolipids and surface motility, I used rhlA -mutant, which does not secrete rhamnolipids but is motile, as a sensor of ability to move on the gel as a function of presence of rhamnolipids. A wild-type colony was grown on agar gel and a small amount of planktonic rhlA -cells were seeded on the naked gel. Individual cells were found to be initially mostly non-motile. Motility was detected only in denser areas. The passage of the swelling front dramatically perturbed spatial organization of the cells (Figure 3A and Movie S3), and hampered direct comparison before/after. In particular, following individual cells was impossible. Instead, motility was assessed by coarse-grained image analysis (see Methods). The motility-density dependence was quantified and plotted against time (Figure 3B-C). Even though cells gradually became more motile with time, incoming rhamnolipids substantially increased cell motility, at all densities (Movie S4). Therefore, rhamnolipids are used by bacteria to alter their physical environment over large distances and enable surface motility.

Swarming rescue experiments confirm rhamnolipids diffuse across the biogel

This microscopic-scale rescue of motility rhlA -mutants inspired me to test if such distant rescue could occur at a macroscopic scale. A plate was inoculated with wild-type cells on one side, and with rhlA -cells on the other side. As already showed in Figure 1, the wild-type colony secretes rhamnolipids (inducing a swelling front) and swarms into a branched shape. The rhlA -colony does not secrete rhamnolipids and does not swarm. However, wild-type colony spreading closely follows the swelling front. To avoid the rhlA -colony from being reached by the expanding wildtype colony before it started swarming, I turned into a mutant over-producer of rhamnolipids:

rhlA -pBAD-RhlAB, whose rhamnolipid promoter expression is induced by a chemical cue, Larabinose. The rhlA -pBAD-RhlAB colony dedicates a large part of its metabolism to rhamnolipid secretion rather than cell proliferation, which yields to a faster swelling front and a slower colony spreading. As shown on Figure 4A (and Movie S5), rhlA -colonies started swarming a few hours after the swelling front reached it. Interestingly, the rhlA -colonies did not spread radially, but formed tendrils, whose size is comparable to wild-type colony branches. These tendrils swarmed outward, following the direction of rhamnolipids propagation. These observations are in agreement with the surface tension induced spreading mechanism, where a gradient of surfactants concentration at the colony surface induce a Marangoni stress [START_REF] Trinschek | Modelling of surfactant-driven front instabilities in spreading bacterial colonies[END_REF].

I reproduced the rescue experiment on a two-face Petri dish, with the over-producing colony (rhlA -pBAD-RhlAB) on one side and rhlA -on the other side (Figure 4B, Movie S5). Here again, the rhlA -colonies did not spread radially, but formed a tendril spreading in the direction of rhamnolipid propagation. This confirms rhamnolipids imbibe the whole gel, physically alter the gel over large distance, and generate a gradient of concentration on the opposite surface, which yield to colony branching following Marangoni effect.

Physical alteration of the gel by rhamnolipids correlates with colony shape

Since P. aeruginosa is capable to alter physical properties of its environment over long distances, I wanted to verify whether this would hold true for a range of environments. I used agar gels of various agar concentrations (0.4%, 0.5%, 0.6%, and 0.7%) to simulate environments of different physical properties. Agar gel elastic modulus and pore size were previously characterized and they were found to be highly dependent on agar concentration. From the literature, pore size varies from 400 nm at 0.4% to 100 nm at 0.7% [START_REF] Narayanan | Determination of agarose gel pore size: Absorbance measurements vis a vis other techniques[END_REF][START_REF] Nicholas L Cuccia | Pore-size dependence and slow relaxation of hydrogel friction on smooth surfaces[END_REF]; elastic modulus varies from 4 kPa at 0.4% to 18 kPa at 0.7% [START_REF] Guenet | Agarose sols and gels revisited[END_REF][START_REF] Mao | Normal force controlled rheology applied to agar gelation[END_REF]. (Most studies focused on agarose, a polysaccharide isolated from agar, but with comparable mechanical properties; rheological measurements are difficult to perform on these soft gels, and those figures must be taken with a grain of salt.) I grew P. aeruginosa on plates at the four agar concentrations and confirmed a result already described: swarming colony morphogenesis is highly dependent on the gel properties (Figure 5A). At low concentration (0.4%), the colony was smaller and more circular than the branch colony formed at 0.5%. At 0.6%, the colony was still branched but smaller than 0.5% colony.

The 0.7% colony did not spread at all. Following the same trend, macroscopic swarming rescue on two-face Petri dished occurred similarly at 0.4% and 0.5% (once again forming tendrils, in agreement with Marangoni mechanisms for surface tension induced spreading), little spreading was visible at 0.6%, and no spreading at all at 0.7% (Figure 5B). In parallel, I characterized the gel and its alteration by secreted rhamnolipids, for the four agar concentrations.

First, I reproduced the sessile droplet experiment as in Figure 1D. Footprint diameters increased with gel concentration when droplets were deposited on the raw gel surface (outside the rhamnolipid imbibed area), from 1.77 ± 0.13 mm for 0.4% to 2.41 ± 0.10 mm for 0.7% (Figure 5C). In contrast, when droplets were deposited inside the rhamnolipids-imbibed area, footprint diameters were overall much greater and independent of agar concentrations. They were about 3.86 (±0.27 mm) for all conditions, as if rhamnolipids equalized the wetting properties of the imbibed gels.

Second, I measured the swelling front height profiles generated by rhamnolipid imbibition (Figure 5D). I observed a strong influence of agar concentration: the swelling front jump (measured 1 mm away from the front, see details in Methods) was 27.4 ± 4.2 µm for 0.4% and gradually decreased for larger agar concentrations (3.9 ± 1.8 µm for 0.7%). This trend was confirmed by shadowgraphy, a contrast imaging method that relies on non-uniformities deviating light rays: the front on a 0.4% gel yielded to a strongly contrasted black line, whereas the front on a 0.7% gel was nearly not visible.

Third, I located the position of the swelling front as a function of time, on two-face Petri dishes. An rhlA -pBAD-RhlAB colony was seeded on the small aperture on the opposite side, 16 in order to limit the spatial expansion of the rhamnolipid source and to allow for comparison of swelling front advancing speeds across agar concentrations without having to control for the source colony size. The position of the swelling front followed a power law r = t α with α ranging between 0.4 and and 0.6 (Figure 5D, inset), in agreement with imbibition processes [START_REF] Azevedo | Fluid imbibition in paper fibers: Precursor front[END_REF]. However, a reproducible estimation of the exponent was difficult, considering the day-to-day variability of agar gel preparation previously described [START_REF] Tremblay | Improving the reproducibility of pseudomonas aeruginosa swarming motility assays[END_REF][START_REF] Ha | Plate-based assay for swarming motility in pseudomonas aeruginosa[END_REF][START_REF] Pearson | Methods for studying swarming and swimming motility[END_REF]. I turned to a simpler and more robust quantification: the position of the front 15 hours after inoculation. Using this measure, I found that the swelling front advanced significantly faster in low concentration agar gels compared to high concentration agar gels (Figure 5E).

Taken together, these results demonstrate that swarming colony morphogenesis correlates with the long distance physical alteration of the gel, across a range of agar concentrations.

Discussion

Self-produced surfactants play an critical role in a many bacterial genera. For example, the swarming of Rhizobium etli [START_REF] Daniels | Quorum signal molecules as biosurfactants affecting swarming in rhizobium etli[END_REF], Serratia marcescens [START_REF] Ang | The role of rsma in the regulation of swarming motility in serratia marcescens[END_REF], Bacillus subtilis [START_REF] Thomas E Angelini | Bacillus subtilis spreads by surfing on waves of surfactant[END_REF], and P. aeroginosa [START_REF] Caiazza | Rhamnolipids modulate swarming motility patterns of ¡i¿pseudomonas aeruginosa¡/i¿[END_REF] were shown to be linked to the presence of biosurfactants. Here, I documented that rhamnolipids, secrected by P. aeruginosa, physically modify the agar gel by inducing a gel swelling. The swelling front displays a maximum angle of 3º that is sufficient to make the edge visible to the naked eye and by shadowgraphy. Previous work had already noted a change of the surface visual aspect.

Those studies used terms like "zone of fluid", "zone of liquid" [START_REF] Caiazza | Rhamnolipids modulate swarming motility patterns of ¡i¿pseudomonas aeruginosa¡/i¿[END_REF], or "ring of biosurfactants" [START_REF] Xavier | A molecular mechanism that stabilizes cooperative secretions in pseudomonas aeruginosa[END_REF] to describe this modified surface state, but my findings show those views were incomplete.

Further investigation will be necessary to understand the swelling mechanism, which might involve complex interplay between gel pores, osmolarity, hydrophobic interactions, etc., and goes beyond the scope of this study.

Additionally, I demonstrated that transport of rhamnolipids is not restricted to the surface of the agar gel. On the contrary, rhamnolipids imbibe the whole thickness of the gel. A direct consequence of the transport in volume is that spreading speed depends on the local section of the gel, with faster spreading on thinner gels. The modulation of the swelling front traveling speed is likely to be conflated with resource availability: a thinner gel contains less nutrient, yields to a smaller colony, and is expected to produce to a slower swelling front. Experimental data goes in the opposite direction: thinner gels yields to faster, not slower, swelling front. Interestingly, colonies do not spread faster on thinner gel. This highlights the complex interplay between resources availability, rhamnolipids spatial distribution, and swarming motility [START_REF] Xavier | A molecular mechanism that stabilizes cooperative secretions in pseudomonas aeruginosa[END_REF][START_REF] Boyle | Integration of metabolic and quorum sensing signals governing the decision to cooperate in a bacterial social trait[END_REF].

The swelling front advancing speed, typically 1 mm/h, is substantially lower than the spreading dynamics of surfactants on liquid films (typically 1 mm/s [START_REF] Omar | The development of transient fingering patterns during the spreading of surfactant coated films[END_REF]). It resembles precursor front in fluid imbibition dynamics of porous materials, which can be interpreted as non-Fickian transport in a Darcy flow [START_REF] Azevedo | Fluid imbibition in paper fibers: Precursor front[END_REF]. Moreover, the deformation of the agar gel is likely to be coupled to the effective diffusivity of rhamnolipids through the gel. Since rhamnolipids are capable to reach the opposite surface, one could assume, in first approximation, a uniform concentration profile across the gel thickness. Yet, agar gel is a physically-crosslinked hydrogel, and the physicochemistry of its interaction with surfactants could lead to a non-uniform profile of concentration [START_REF] Banaha | Spreading of liquid drops on agar gels[END_REF][START_REF] Boral | Syneresis in agar hydrogels[END_REF]. Colony branching in two-face rescue experiments confirms that bulk gel imbibition generates a lateral gradient of surfactant concentration that could potentially interact with the spreading colony itself.

Alteration of the biotic or abiotic environment by living organisms have the potential to modulate motility and dispersal mechanics. Secreted metabolites can act as chemoattractants [START_REF] Evanthia T Roussos | Chemotaxis in cancer[END_REF][START_REF] Colin | Emergent properties of bacterial chemotaxis pathway[END_REF], trigger quorum sensing [START_REF] Christopher M Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF], participate in self-organization in stigmergic behavior [START_REF] Theraulaz | A brief history of stigmergy[END_REF][START_REF] Joseph | Cell migration guided by long-lived spatial memory[END_REF],

and control single-cell motility [START_REF] Wershof | Matrix feedback enables diverse higher-order patterning of the extracellular matrix[END_REF]. These mechanisms rely on release of diffusible molecules, or local deposition of non-diffusible signals. Here, I evidenced a mechanism where bacteria alter their physical world on a lengthscale substantially larger that their own size. I confirmed this effect holds true across a range of agar concentration. It would be interesting to test other biogel materials [START_REF] Charles | Use of alternative gelling agents reveals the role of rhamnolipids in pseudomonas aeruginosa surface motility[END_REF], especially those with biomedical implications, such as mucus [START_REF] Amy | Mucin promotes rapid surface motility in pseudomonas aeruginosa[END_REF][START_REF] Rossy | Pseudomonas aeruginosa contracts mucus to rapidly form biofilms in tissue-engineered human airways[END_REF]. Moreover, rhamnolipids are shown to be involved in many killing processes (towards other bacteria [START_REF] Bharali | Rhamnolipid (rl) from pseudomonas aeruginosa obp1: a novel chemotaxis and antibacterial agent[END_REF],

fungus [START_REF] Soltani Dashtbozorg | Rhamnolipids as environmentally friendly biopesticide against plant pathogen phytophthora sojae[END_REF], immune cells [START_REF] Peter Ø Jensen | Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorumsensing-controlled production of rhamnolipid by pseudomonas aeruginosa[END_REF], or higher organisms [START_REF] Zaborin | Red death in caenorhabditis elegans caused by pseudomonas aeruginosa pao1[END_REF][START_REF] Vinicius | Rhamnolipids: solution against aedes aegypti?[END_REF]): understanding how this killing agent is transported through complex media is of critical importance.

This complex interplay between surfactants secretion, biogel properties alteration and bacterial motility is a unique example of the capacity of bacteria to change the mechanical properties of the world around them, and ultimately, to interact with their distant peers [START_REF] Estrela | Environmentally mediated social dilemmas[END_REF].

7 (Figure 1 :

 71 Figure 1: Rhamnolipids induce agar gel swelling. A-B-C: Wild-type P. aeruginosa and rhlA - colonies were grown on an agar gel. The former swarmed out and formed a branched colony, whereas the latter did not swarm. A: Brightfield image. B: Shadowgraphy image reveals a darker line (depicted by a black arrowhead) that corresponds to a swelling front. C: Fluorescence image, for Nile Red imaging, confirms the darker line correponds to a boundary of an area imbibed by rhamnolipids. D: An illustrative micrograph showing the sessile droplets experiments. Dashed line depicts the swelling front. The source colony is on the right side. Inset: comparison of the wetting footprint radius in the two conditions. Error bars are standard deviation. E: Top: a height profile of a gel, obtained from optical interferometry. Bottom: The local slope of the height profile. F: Vertical positions of fluorescent microbeads reveals gel swelling. The time axis for each trajectory is shifted using time synchronization from lateral displacement data (See Figure S1). Colors code for initial depth of each bead.

  swelling front. I embedded micrometric fluorescent beads during preparation of the gel and performed 3D-tracking to follow each bead while the line passed through the field of view. If rhamnolipids cover the gel surface, beads should not move. If rhamnolipids diffuse through the gel and make it swell, beads should move up. Data shown in Figure1Fconfirms a vertical movement of all the beads, synchronized with the passing line (see also Movie S2). The swelling amplitude was uniform across the first 50 µm (optical limitations of fluorescence microscopy hamper deeper observations). Of note, beads also moved laterally with a similar amplitude: they first moved outwards and then inwards (this corresponds to a dilatation wave, see FigureS1for more details). This confirms rhamnolipids make the agar gel swell over large distances, and the darker line corresponds to a swelling front that is so steep that it can be seen by a naked eye and by shadowgraphy.Rhamnolipids imbibe the bulk of the gel, not the surfaceIf transport of rhamnolipids is restricted to the gel surface, or if rhamnolipids diffuse through the gel following normal diffusion, their spreading rate should not depend on the local thickness of the gel. On the contrary, if Darcy's law governs rhamnolipids' transport, their spreading dynamics should depend on the local section of the gel. I used gels with varying thicknesses to test this hypothesis and check whether rhamnolipids imbibed the whole gel or only the upper part.First, I used gels with stepwise thickness. I placed a thick inert obstacle at the bottom surface of the Petri dish and poured the agar gel on top of them. The thickness of obstacles (1.5 mm) was chosen to be smaller than the gel thickness (3.5 mm) to make sure the upper surface remained flat. To simplify the geometry of the system and keep a local source of rhamnolipids, I inoculated a non-motile mutant (flgK -) along a line. The swelling front coming out of the colony was

Figure 2 :

 2 Figure 2: A: Snapshot of the swelling front on a plate with an obstacle. The non-motile colony, inoculated along a line, is visible near the bottom of the image. B: Positions of the swelling front as a function of time, for four plates with different thicknesses of gel. C: Left: Snapshot of the swelling front advancing on a tilted gel. Right: Positions of the swelling front as a function of time, for 4 locations depicted with arrowheads (top, bottom, left, and right). D: Two-face Petri dish confirms rhamnolipids spread through the gel. i) Schematic of a two-face Petri dish. ii) Shadowgraph of the swelling front emerging from three colonies, located on the other side of the gel. iii) Height profile obtained from optical profilometry. iv) Sessile droplet experiment confirms an significant difference in wetting property on either side of the swelling front. Error bars are standard deviation.

Figure 3 :

 3 Figure3: A: Snapshots of micrographs showing rhlA -cells before and after the swelling front passed through. A red arrowhead depicts the swelling front, visible as a white halo in phase contrast imaging, and moving from the left to the right of the field of view. B: Motility-density analysis reveals rhamnolipids greatly facilitate single-cell motility. Color code is defined in panel C (one curve every 5 minutes). Shading represents standard deviations. C: The effect of rhamnolipids on motility is abrupt, and corresponds to the passage of the swelling front (depicted by the red arrowhead). Error bars represent standard deviations.

Figure 4 :

 4 Figure 4: Swarming rescue experiments. A: On one-face Petri dish, rhlA -pBAD-RhlAB and rhlA - colonies are grown on the same side. B: On two-face Petri dish, rhlA -pBAD-RhlAB are grown on one side (within the small hole) and rhlA -are grown on the opposite side. Control experiment results (with rhlA -instead of rhlA -pBAD-RhlAB) are provided in Supplementary Figure S3. Snapshots are taken 18 hours after inoculation. Timelapse videos of the rescue experiments are available in Movie S5.

Figure 5 :

 5 Figure 5: Physical alteration of the gel occurs across a range of agar concentration. A: Snapshots of P. aeruginosa swarming colonies, 22 hours after inoculation. Each snapshot is surrounded by a square whose color consistently represents the agar concentration across panels. B: Snapshots of macroscopic rescue experiments, 22 hours after inoculation. (Note one agar patch fell from the two-face Petri dish on the 0.4% plate, just before image acquisition.) C: Footprint diameters for sessile droplet experiments. Droplets were deposited within the rhamnolipid-imbibed area (denoted "Inside") or outside the swelling front (denoted "Outside"). Data points positions were randomized on the horizontal axis for better visualization. Data points originates from 2 biological replicates. D: Jump height measured 1 mm from the swelling front. Inset: Height profiles obtained by optical interferometry. E: Position of the swelling front 15 hours after inoculation. Data points originates from 3 technical replicates. Inset: Time evolution of the front position, from one replicate. For all panels, horizontal bars are dataset averages and vertical bars are standard deviations.

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2022.06.29.498166	this version posted September 2, 2022.	The copyright holder for this preprint

Acknowledgments

I thank members of the Laboratoire Jean Perrin for fruitful discussions. I am grateful to Franc ¸oise Brochard-Wyart for pointing me to the reference [52]. This project has received financial support from the CNRS through the MITI interdisciplinary program, from the Alliance Sorbonne Université through the Emergence program, and from the ANR (ANR-21-CE30-0025).


Arthur Matthys

Sophie Demeret

Delphine Leclercq

Lucas Di Meglio

Anti-PD-L1 therapy-associated hypophysitis and limbic encephalitis

teaching and research institutions in France or abroad, or from public or private research centers.

TEXT

A 71-year-old woman with history of neuro-endocrine tumor was admitted to our intensive care unit because of a fluctuating level of consciousness preceded by the subacute onset of a working memory deficit. She had been treated five months before with two cycles of 1500 mg Durvalumab -an anti-PD-L1 (Program Death-Ligand 1) antibody. Initial MRI found bilateral FLAIR hyperintensities of the hypothalamic and limbic regions (Figure 1). Remarkably, ASL exhibited a hyperperfusion suggestive of hypermetabolism, which has been described in encephalitis. CSF analysis showed a lymphocytic pleocytosis (8 cells/µL) with high protein rate (183 mg/dL) and positive oligoclonal bands. Antineuronal antibodies including onconeuronal antibodies were absent in serum and CSF. No evidence of cancer relapse was found. Exploration of the hypothalamic-pituitary-adrenal axis showed central hypothyroidism, hypogonadism, and diabetes insipidus. She was diagnosed with autoimmune hypophysitis and limbic encephalitis associated with immune checkpoint inhibitor treatment. Rapid improvement was observed after high dose IV steroids and therapeutic plasma exchange. One-month control MRI found improvement of limbic lesions with regression of pituitary swelling (Figure 2). Encephalitis is an increasingly identified complication of immune checkpoint inhibitors. Metabolic consequences of hypophysitis as well as impaired consciousness or status epilepticus frequently lead these patients to intensive care units. Early clinical and radiological diagnostic is important to promptly start immunomodulation treatment.

  FIGURES LEGEND

Figure 2 .

 2 Figure 2. Regressive Durvalumab-induced hypophysitis after treatment

TITLE PAGE Manuscript title: Anti-PD-L1 therapy associated hypophysitis and limbic encephalitis Authorship list:

  Arthur Matthys 1 , Sophie Demeret 1 , Delphine Leclercq 2 , Lucas Di Meglio 1 Sorbonne Université, AP-HP, Hôpital de la Pitié-Salpêtrière, Département de Neurologie, Unité de Médecine intensive -Réanimation à orientation neurologique, Paris, France 2. Sorbonne Université, AP-HP, Hôpital de la Pitié-Salpêtrière, Neuroradiologie, Paris, France

	Author affiliations:
	1. Corresponding author:
	Lucas Di Meglio
	16-digit ORCID: 0000-0002-5158-8612
	Sorbonne Université
	Unité de Médecine intensive -Réanimation à orientation neurologique,
	Département de Neurologie
	AP-HP. Sorbonne Université -Hôpital Pitié-Salpêtrière
	47-83 Bd de l'Hôpital, 75013 Paris, France
	Phone: +33 (0)1 42 16 18 31
	Mail: lucas.dimeglio@aphp.fr
	Funding: none.
	Manuscript features:
	Article type: Images
	Title: 66 characters
	Text: 187 words
	Figures: 2
	Figures legend: 57 words

Conflict of interests: the authors declare no conflict of interest.


F Bachelier

J P Amiet

V Daële

D Bazin

DEVELOPMENT AND VALIDATION OF ON-LINE AND IN-FIELD AUTO-GC FOR THE ANALYSIS OF TRACE-LEVEL OVOC

Keywords: On-line monitoring, gas chromatography, OVOCs, Mass spectrometry

INTRODUCTION

Oxygenated Volatile Organic Compounds (OVOCs) are an important fraction of the VOCs and are emitted in the atmosphere from anthropological (solvent, industry, transport) and biogenic sources [START_REF] Hj ; Legreid | Specifications and Test Procedures for Ambient Air Quality Continuous Monitoring System with Gas Chromatography for Volatile Organic Compounds[END_REF][START_REF] Placet | Emissions of ozone precursors from stationary sources:: a critical review[END_REF]. Also, oxidation process in the atmosphere can lead to the formation of OVOCs [START_REF] Atkinson | Atmospheric chemistry of VOCs and NOx[END_REF]. Aldehydes and Ketones are generally measured following the ISO 16000-3 method where DNPH cartridges are used to preconcentrate the sample. The method is very sensitive and allows identification and quantification from low ppt to high ppb depending on flow and sampling time [START_REF] Uchiyama | Simultaneous Determination of C1-C4 Carboxylic Acids and Aldehydes Using 2,4-Dinitrophenylhydrazine-Impregnated Silica Gel and High-Performance Liquid Chromatography[END_REF]. Nevertheless, this technique is time consuming as it is needed to manually elute the DNPH cartridge before HPLC-UV analysis. Other techniques allow specific quantification of OVOCs such as formaldehyde but cannot quantify all OVOCs [START_REF] Becker | Development and Optimization of an Airborne Formaldehyde Microfluidic Analytical Device Based on Passive Uptake through a Microporous Tube[END_REF]. Therefore, there is a need for automated and continuous measurement of Aldehydes, Ketones and also Alcohols (which are not measured with ISO 16000-3 method). Automated gas chromatograph are widely used for the characterization of VOCs in ambient air [START_REF] Martin | airmoVOC C6-C12 -MCERTS Performance Standards for Continuous Ambient Air Quality Monitoring Systems[END_REF]. Nevertheless, OVOCs and particularly light OVOCs are often difficult to measure without interference. In this article, we present a study on the performance and limitation of a dual thermo-desorption gas chromatograph equipped with flame ionisation detector and mass spectrometer for the characterization of OVOCs in ambient air. Also, we will present results on a specific module dedicated to the measurement of light OVOCs.

METHODS

auto-TDGC-FID/MS for C2 to C6 Monitoring

An automatic gas chromatograph (airmoVOC C2-C6, Chromatotec, France) equipped with a flame ionization detector (FID) and mass spectrometer has been used for the monitoring of non-polar molecules containing from 2 to 6 carbon atoms. For each analysis, 107 mL of ambient air was drawn into the system with a flow rate of 12 mL.min -1 (air sample is integrated over 10 min). The air sample passed first through a dryer (airmoDry, Chromatotec, France) to remove the humidity and then hydrocarbons were preconcentrated at -10°C on a trap filled with a mixture of Carboxen and Carbopack. The pre-concentrated air sample was thermally desorbed at 220°C for 4 minutes and directly injected in a 25 m Al2O3/Na2SO4 column (PLOT column, 0.53 mm ID, 10.0 µm dF) located inside the heated oven of the GC. Within the first minute of the analytical procedure, the oven temperature rose from 36°C to 38°C. Afterwards a constant heating rate of 15°C.min -1 was applied and the temperature reached 202°C. Then the temperature is kept at 202°C for 630s before cooling. C2-C6 hydrocarbons were detected by FID and the detection limit has been determined to be 0.065 µg.m -3 for N-butane.

auto-TDGC-FID/MS for C6 to C12 Monitoring

For the monitoring of molecules containing from 6 to 12 carbon atoms an automatic gas chromatograph (airmoVOC C6-C12, Chromatotec, France) equipped with FID and mass spectrometer has been used. For each analysis, 440 mL of ambient air was drawn into the system with a flow rate of 20 mL.min-1 (air sample is integrated over 20 min). The compounds were pre-concentrated at room temperature on a trap filled with Carbopack mixture. The pre-concentrated air sample was thermally desorbed at 380°C for 4 minutes and directly injected in a 60 m MXT 1 column (0.28 mm ID, 1.0 µm dF) located inside the heated oven of the GC. During the first 7 minutes of the analytical procedure, the oven temperature rose from 36°C to 50°C. Afterwards a constant heating rate of 10 °C.min -1 was applied for 3 minutes followed by a heating rate of 15 °C.min -1 for 8 minutes. The temperature reached 200°C by the heating time. Then the temperature is kept at 200°C for 240 s before cooling. C6-C12 compounds were detected by a FID and the detection limit has been determined to be 0.045 µg.m -3 for Benzene.

Mass spectrometer for C2-C12 monitoring

Low-resolution mass spectra (MS) were recorded in the electron impact mode (EI, 70 eV) with a single quadrupole mass spectrometer (DET QMS, Chromatotec, France). The system is equipped with a heated multiplexing system for two streams (200°C) and two heated transfer lines (200°C). The measured intensities were computed using VistaMS software and concentration calculations were performed using hight intensity on selected ion for each species. The determination of validated results was performed with VistaMS. The tuning of the mass spectrometer was performed using Perfluorotributylamine (PFTBA) from Sigma-Aldrich. All electronic parameters were saved and used for the whole time of the experiment.

auto-TDGC-FID with methanizer for light OVOCs

The monitoring of formaldehyde, acetaldehyde and methanol was performed using automatic gas chromatograph equipped with a hydrogenating reactor to convert all OVOCs to methane before quantification using FID. The OVOCs were pre-concentrated at room temperature on a trap filled with Carbopack. The pre-concentrated air sample was thermally desorbed at 380°C for 4 minutes and directly injected in a two-dimensional columns system (polar and apolar capillary columns) located inside the heated oven of the GC. The first column is used for separation of light compounds according to their molecular mass. The polar column separates polar from non-polar compounds before identification and quantification.

Gas supply

Air generators (airmopure, Chromatotec, France) and Hydrogen generators 99.9999% with dew point below -15°C (Hydroxychrom, Chromatotec, France) were used for the flames of FIDs, valve actuations of the auto-TDGC-FIDs and drying of samples for C2 to C6 analysis. The VOCs content of gas generated by both generators was verified experimentally using auto-TDGC-FID and Non-Methanic Hydrocarbon Concentration (NMTHC) for both analysers was below 0.1 µg.m-3.

Standards used for calibration

Quality controls of the instruments were performed using NIST certified cylinders. TO15 (64 compounds) and PAMS 58 (Takachiho, Japan) were used for the calibration of instruments and response factor calculations.

Calibration setup

For the calibration of OVOCs, a certified gas mixture of TO15 compounds was used at different concentrations in the range 0.5 -10 ppbv. The concentration of each compound in the cylinder is 100 ppbv ± 10%. To perform the dilution of the cylinder with zero air, 2 mass flow controllers (MFC) from Bronkhorst were used. The flow range of the Cylinder MFC was 0-10 mL.min -1 and the flow range of Dilution MFC was 0-1 L.min -1 . The sample flow rate was 30 mL.min -1 . Calibration curves were performed with 6 different concentrations with 10 replicates for the first calibration point and 6 replicates for the others. Precision and accuracy were evaluated at 4 ppbv.

CONCLUSIONS

The aim of this study was to analyse specifically oxygenated VOCs in ambient air, using an on-line system including 2 automatic TDGC-FID coupled to a mass spectrometer, and an auto-TDGC-FID with a methanizer.

C2 to C6 measurement

Light non-polar VOCs containing 2 to 6 carbons were analysed using the first on-line auto-TDGC-FID/MS equipped with an Al2O3/Na2SO4 PLOT column. The system can analyse all light compounds from PAMS 58 cylinder and some of the molecules included in the TO15 cylinder such as freons, chlorinated compounds and carbon disulphide. Nevertheless, light OVOCs such as Methanol, Ethanol, Formaldehyde and Acetaldehyde cannot be analysed with the analyser because they cannot be eluted from Al2O3/Na2SO4 PLOT column.

C6 to C12 measurement and performance studies

The second on-line auto-TDGC-FID-MS equipped with an MXT-1 column is designed to analyse molecules containing 6 to 12 carbons and was used for the measurement of OVOCs heavier than Acetaldehyde. 1. Among these 12 compounds only ethanol is not retained enough by the trap to be detected. The FID chromatogram of TO15 standard mix at 4ppb is showed in Fig. 1. The 11 OVOCs detected were identified with their retention times. To confirm identifications, mass spectrometry detection and NIST database were used.

It is noticeable that coelutions occur between some OVOCs and the other compounds of the cylinder. As shown in Fig. 1, MTBE and Vinyl acetate are co-eluted, Ethyl acetate is co-eluted with Chloroform and Methyl methacrylate with N-heptane. Fortunately, we can avoid these coelutions with MS detection by selected the specific ion for each compound.

Calibration curves for each OVOCs were performed with six different concentrations of standard mixture. The range of concentrations is 0.5 to 10 ppb. The linearity of calibration curves was determined with determination coefficient (R²) for FID and MS detection. As reported in Table 2, the determination coefficients varied between 0.9829 and 0.9992 for FID and 0.9871 and 0.9995 for MS showing linear calibration curves for every compounds.

The limits of detection (LOD) of the systems were calculated according to the Chinese standard norm for ambient air measurement (HJ 1010(HJ -2018[START_REF] Hj ; Legreid | Specifications and Test Procedures for Ambient Air Quality Continuous Monitoring System with Gas Chromatography for Volatile Organic Compounds[END_REF]. Thus, the LODs were determined with the standard deviation of 10 replicates of the standard gas at 0.5 ppb (Table 2). The detection limits ranged from 0.065 to 0.325 ppb and 0.068 to 2.110 ppb for FID and MS respectively. Except for MEK and MBK, all detection limits are at least 50% higher with MS detection.

The precision and accuracy were calculated at 4 ppb according to the Chinese standard method. The majority of the compounds presented relative standard deviations below 10%, but the non-gaussian peak shape of oxygenated compounds could be responsible of accuracies over 10% (Table 2). In comparison, Benzene accuracies are 0.68% and 3.00 % for FID and MS respectively. As shown in Table 2, although the LOD and repeatability are overall better for FID than MS, FID detection could not provide analysis of MTBE, Vinyl acetate, Ethyl acetate and 1.4-Dioxane due to the coelutions. However, by selecting a specific ion for each species, MS detection allows accurate quantification of these compounds.

Specific gas chromatograph for light OVOC

As shown previously, methanol, ethanol, formaldehyde and acetaldehyde cannot be quantified using the C2-C12 FID/MS system. Therefore, a specific analyser has been designed for the separation and quantification of these molecules. The three main challenges for this application are: preconcentration, separation and capability to measure these molecules with very low response on FID. Analytical solutions have been found for all three challenges and we have shown that the system is perfectly suited for the measurement methanol, ethanol, formaldehyde and acetaldehyde [START_REF] Mascles | Development and comparison of three analytical systems for detection and quantification of formaldehyde and other aldehydes for process, indoor and ambient air emissions[END_REF].

In this work, the performances and limitations of detection of OVOCs in ambient air by dual thermodesorption gas chromatograph equipped with flame ionisation detector and mass spectrometer were described. Using a TO15 standard gas mixture, 11 OVOCs from acrolein to methyl butyl ketone were identified and accurately quantified on the C6C12 system with MS detection. However, for the detection of light OVOCs such as Methanol, Ethanol, Formaldehyde and Acetaldehyde a specific module dedicated to the measurement of these compounds is required.

Figure 1 :

 1 Figure 1: FID chromatogram of 4 ppb of 11 OVOCs contained in TO15 standard mix

Table 1 :

 1 List of OVOCs in TO15 standard gas mixture The TO15 gas mixture (64 compounds) contains 12 OVOCs that are listed in Table

	VOCs
	1 Ethanol
	2 Acrolein
	3 Acetone
	4 Isopropyl alcohol
	5 Methyl tert-butyl ether
	6 Vinyl acetate
	7 Methyl ethyl ketone
	8 Ethyl acetate
	9 1,4-Dioxane
	10 Methyl methacrylate
	11 Methyl isobutyl ketone
	12 Methyl butyl ketone

Table 2 :

 2 Determination coefficients, detection limits, precision and accuracy of OVOCs using FID and MS detection

	OVOCs	FID Detection			MS detection
		Determination		Precision at		Determination		Precision at
		coefficient	LOD	4 ppbv	Accuracy at	coefficient	LOD	4 ppbv	Accuracy at
		(R²)	(ppbv)	(%RSD)	4 ppbv (%)	(R²)	(ppbv)	(%RSD)	4 ppbv (%)
	Methanol	ND	-	-	-	-	-	-	-
	Ethanol	ND	-	-	-	-	-	-	-
	Formaldehyde	ND	-	-	-	-	-	-	-
	Acetaldehyde	ND	-	-	-	-	-	-	-
	Acrolein	0.9992	0.093 2.04	2.62	0.9990	0.277 10.6	1.67
	Acetone	0.9960	0.065 5.79	8.27	0.9995	0.253 5.45	2.22
	Isopropyl alcohol (IPA)	0.9829	0.130 1.12	13.6	0.9871	0.404 7.76	7.87
	Methyl tert-butyl ether (MTBE) Coeluted	-	-	-	0.9995	0.216 2.78	2.09
	Vinyl acetate	Coeluted	-	-	-	0.9992	0.431 7.49	0.73
	Methyl ethyl ketone (MEK)	0.9960	0.124 2.52	14.2	0.9899	0.068 4.37	19.8
	Ethyl acetate	Coeluted	-	-	-	0.9928	0.179 6.79	19.28
	1,4-Dioxane	0.9894	0.150 2.44	29.2	0.9978	0.285 7.29	14.9
	Methyl methacrylate	Coeluted	-	-	-	0.9984	0.273 3.43	5.92
	Methyl isobutyl ketone (MIBK) 0.9986	0.325 1.46	7.36	0.9979	0.760 8.12	2.34
	Methyl butyl ketone (MBK)	0.9970	0.270 2.77	7.68	0.9988	0.159 7.79	2.47

ACKNOWLEDGEMENTS

This work was carried out as part of a thesis with a CIFRE agreement from the National Association for Technical Research (ANRT) REFERENCES