NEW MILLI-TO-MICRO-FLUIDICS-BASED PROCESS TO SCREEN MICROBIAL GROWTH IN DROPLETS

The present invention concerns a method for screening the growth of microorganisms in a high throughput droplet milli-to-micro-fluidic system based on the detection of fluorescence of compounds released by microorganisms during growth and kept into droplets.

Microbes represent a gold mine for prospecting and engineering new functions of biotechnolological interest. However, the majority of the species that make up microbial ecosystems are not cultivated yet, and constitute a darkmatter whose functioning is difficult or impossible to decipher. Besides, culturomics, as well as strain and enzyme engineering, necessitate fastidious and costly steps of screening to explore a sufficiently large diversity to identify hits.

Here, the inventors of the present invention have discovered a new method based on droplet milli-to-micro-fluidics to speed up and miniaturize by several orders of magnitude the process of screening microorganisms growth. This new milli-to-micro-fluidic workflow is extremely economical, and it is not based on the use of fluorogenic substrates or fluorescent libraries, or on the addition of any exogeneous compound in the growth medium, contrary to all previously described workflows allowing to screen microbial growth using microfluidics. In addition, the sorting is made at the droplet level and not on the microorganism level.

The principle is indeed based on the detection of fluorescence produced by molecules released by microorganisms during growth into droplets that are generated in milli-to-micro- fluidic systems and sorted at kHz rates. As soon as the screened function is correlated to microbial growth, the process developed by the inventors could thus be exploited to discover or optimize novel enzymes, metabolic pathways, species and consortia using functional metagenomics, culturomics, or enzyme and strain engineering. It could also be used to screen antimicrobial compounds.

The principle of this invention is based on the fact that the droplet medium becomes fluorescent during/after microorganism growth.

The present invention relates to a method for screening the growth of microorganisms in a high throughput droplet milli-to-micro-fluidic system, said method comprising:

(a) generating a droplet batch in a carrier fluid to form a plurality of individual bioreactors, at least one droplet of the droplet batch encapsulating one or several cell(s) of microorganisms in growth media, (b) incubating said droplets over time,

(c) detecting the droplets in which the microorganisms have grown by detection of the fluorescence of the growth media, and

(d) selectively recovering said droplets using the fluorescence of the growth media.

In particular, the present invention relates to a method for screening the growth of microorganisms in a high throughput droplet milli-to-micro-fluidic system, said method comprising:

(a) generating a droplet batch in a carrier fluid to form a plurality of individual bioreactors, at least one droplet of the droplet batch encapsulating one or several microorganisms in growth media, wherein the microorganisms secrete fluorescent compounds during their growth,

(b) incubating said droplets over time,

(c) detecting the droplets in which the microorganisms have grown by detection of the fluorescence of the fluorescent compounds released by the microorganisms in the growth media during their growth, and

(d) selectively recovering said droplets using the fluorescence of the growth media.

In particular, the method according to the invention further comprises:

(e) recovering the microorganisms; and optionally

(f) submitting said microorganisms to biological analysis.

Even more particularly, the method according to the invention comprises repeating steps (a) to (d). More particularly steps (a) to (d) are repeated between 1 and 100 times, for example from 1 to 75, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10 or 1 to 5 times or once.

By “high throughput droplet milli-to-micro-fluidic system” is meant, in the context of the present invention, one system/one machine that enables formation of droplets (more than 100), at least one measurement on each droplet and the manipulation of the droplets. The manipulation comprises: preparing the droplets, with possibly one or several microorganisms inside, sorting the droplets.

In particular such a system comprises:

• at least one reservoir of growth media fluidically connected to a capillary injection tube; • at least one reservoir of a carrier fluid that is immiscible with the growth media, fluidically connected to a capillary reaction tube;

• an emulsifier module: the capillary injection tubes are connected to microfluidic device for droplet formation containing growth media with possibly one or several microorganisms in carrier fluid. Droplets form individual bioreactors;

• at least one detector of fluorescence.

The droplet batch is made up of a plurality of droplets of an internal fluid dispersed in a carrier fluid. Each droplet makes up a closed compartment filled with internal fluid, in which chemical or biological reactions may for example occur that may result in revealing the growth of microorganisms. For example, droplets are spherical.

According to other embodiments:

• the droplets are bioreactors for culture of microorganisms;

• each droplet is in essence a bioreactor within the droplet batch and each can be individually analyzed during its passage through lasers. After a certain stage of the organism’s lifecycle is reached, a ranking based of fluorescence and sorting can be performed using a cytometric cell-sorter, such as FACS or on on-chip-sorter. The carrier fluid comprises oil and in particular fluorinated oil containing surfactant for example RAN fluorosurfactant for droplet production.

The internal fluid of each droplet is made up of a base and optionally of one or several microorganisms. The proportions of the microorganisms and/or the natures of the microorganisms may vary from one droplet to the next.

The base comprises growth media.

The growth media used in the method according to the invention will vary depending on the nature of the microorganisms studied in the droplet bioreactor. The man skilled in the art is able to adapt the media to the type of microorganism. For example, growth media could be rich media or defined media such as LB broth, MH or M9 medium supplemented with carbon sources for bacteria, TAP medium for algae, YPD or YNB supplemented with carbon sources for yeast or any cell culture medium.

The microorganisms that are encapsulated into the droplets can be of the same nature or be different.

For example, depending on the droplet that is considered, the microorganisms can be different or different microorganisms can be encapsulated into the same droplet.

Alternatively, only the same microorganism can be encapsulated.

It means that the following exemplary cases are covered: - One or several microorganisms are encapsulated in at least one droplet, the microorganisms all being the same one (for example the same bacteria or the same yeast);

- One or several microorganisms are encapsulated in at least one droplet, the microorganisms being encapsulated in the same droplet being different (for example: two different bacteria species or the same bacteria specie but with different properties);

- Several droplets contain different microorganisms (for example one droplet comprises one or more of the bacteria 1 and the next droplet comprises one or more of the bacteria 2, bacteria 1 and 2 being different);

- Several droplets contained different microorganisms and the microorganisms encapsulated into one droplet are different (for example, the first droplet comprises one or more of bacteria 1 and one or more or more of bacteria 2 and the next droplet comprises one or more of bacteria 1 and one or more of bacteria 3).

In particular, the microorganisms can be non-fluorescent microorganisms, or fluorescent ones if the detection of their intrinsic fluorescence does not require the same excitation and emission wavelengths as those used for the detection of the compound secreted during microorganism growth, for example flavins. Such microorganisms are well- knowns by the skilled person. In addition, said property can be easily checked by the skilled person, for example by detecting fluorescence of the strains using fluorimeter. In particular, by “non-fluorescent microorganisms” is meant microorganisms with an internal fluorescence which is not sufficient to be detectable in the droplet at the encapsulation step.

The microorganisms used in the context of the process according to the invention secrete a fluorescent compound during their growth. Said fluorescent compound can be flavin, FMN or FAD for example. In one embodiment, said fluorescent compound is not NADH. In the context of the process according to the invention, flavin is particularly contemplated.

Such kind of microorganisms are also well known by the skilled person. Some of them are for example described in (Canstein et aL, 2008; Kotloski & Gralnick, 2013; Mihalcescu et aL, 2015; Wilson & Pardee, 1962; Yurgel et aL, 2014). Said microorganisms can be unicellular or multicellular microorganisms. They can be genetically modified or non- genetically modified.

In the context of the present invention, the compounds secreted by the microorganisms are naturally fluorescent and the microorganism naturally secretes those compounds, i.e the microorganisms of the present invention have not been genetically modified to secrete a fluorescent compound.

Still more particularly, the microorganisms used in the context of the process according to the present invention are selected from bacteria and yeasts.

In particular, the bacteria are chosen from Escherichia genus and the yeasts are chosen from Saccharomyces or Yarrowia genus.

In the context of the present invention, the microorganisms having grown in the droplets that are detected in step (c) can be dead or living microorganisms.

In one embodiment, the Poisson distribution can be used to determine the concentration of microorganisms to use to generate drops in order to obtain to set the average number of microorganisms per droplet: in which :

A is the average number of microorganisms in one droplet volume, and

- p(k) is the probability to have k microorganisms in a droplet.

More particularly, in the case where the number of microorganisms in the droplets is distributed following a Poisson distribution to obtain only one microorganism per droplet, there is a probability to obtain some droplets that only encapsulate growth media.

In one embodiment of the method according to the invention, each droplet of the batch encapsulates one microorganism.

In another embodiment of the method according to the invention, each microorganism is founded by from 1 to 20 cells, in particular from 1 to 4 cells.

In still another embodiment of the method according to the invention, said microorganisms grow in each droplet for between 1 and 20 generations or for between 1 and 25 generations, or more, like 30 or 35 generations for example. In particular, said microorganisms grow in each droplet for between 1 and 5 generations, for example for 2, 3, 4 or 5 generations.

For example, up to 5 generations can be carried out for 6 days.

In still another embodiment of the method according to the invention, the microorganisms in each droplet are incubated for at least one hour until few days, for example for 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 hours or for 2, 3, 4, 5 or 6 days. A droplet can thus encapsulate one or more microorganisms, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, etc. The maximum number is dictated by the maximum carrying capacity of the media for the chosen microorganism.

In particular, part of the generated droplets can be incubated at controlled temperature.

Still particularly, droplets can be kept static in one single tube, called an incubation tube.

Said droplet can contain a various number of droplets.

In particular, it contains between 100 and 10 ⁸ droplets, more particularly between 100 and 10 ⁷ or between 10 ⁵ and 10 ⁷ .

Still particularly, the volume of each droplet is from pL to pL. By “from pL to pL” is meant in particular a few picoliters, such as from 1 to 10 pL, in particular from 3 to 8 pL andmore particularly around 3pL.

Still particularly, the droplets are chosen from water/oil/water (w/o/w) double-emulsion droplets and water/oil (w/o) single-emulsion droplets.

The method of screening according to the invention, as soon as the screened function is correlated to microbial growth, the process could be used to discover or optimize novel enzymes, metabolic pathways, species and consortia using functional metagenomics, culturomics, or enzyme and strain engineering. It could also be used to screen antimicrobial compounds. The screening is carried out without the need to add a new label as the microorganisms secrete naturally fluorescent compounds, these compounds being naturally fluorescent. In addition, sorting is made at the droplet level and not at the microorganism level.

In particular, in the context of the process according to the invention, step (c) consists of the detection of a difference in the fluorescence of the growth media between the empty droplets or the droplets containing non growing microorganisms, and droplets with growing or grown microorganisms.

As previously mentioned, the fluorescence that is detected in step (c) is the fluorescence of the fluorescent compounds released by the microorganisms in the growth media during their growth.

Advantageously, during the detection step, the droplets are advantageously arranged in a single train in a sorter, so that droplets are sorted individually.

Said detection is carried out by any device known by the skilled person and coupled with the sorting of the selected droplets. The recovery device is indeed able to allow a droplet to be recovered based on the detection of fluorescence. For example, the recovery device comprises a cells/particules sorter by flow cytometry or another sorting device. The sorting device is not an imaging device. A cytometric cell-sorter, notably FACS (Fluorescence Activated Cell Sorting) can in particular be cited.

To “sort a droplet” means to address it into a specified tube or a specified microwell of a microtiter plate. Selected droplets can be sorted by diverting said fraction of interest into a sorting tube or microwells of a microtiter plate.

Sorting may lead to single droplet isolation in a specified tube or a specified microwell of a microtiter plate, for example using a cytometric cell-sorter, notably a FACS. Sorting of all the positive droplets mixed together in a tube can also be performed whatever is the sorting device used.

In one embodiment, the volume of each droplet is from 3 to 8 pL and the sorting is conducted with a FACS.

Recovering of the microorganisms as mentioned in step (e) can be conducted by any method know by the man skilled in the art. For example, by incubation of sorted droplet and microorganism it contains in rich media to allow microorganism growth.

In particular, the biological analysis of step (f) comprises but are not limited to the DNA sequencing, taxonomical and functional annotation of the genes, phenotypic characterization or enzymatic activity characterization of the recovered microorganisms.

The man skilled in the art is able to determine how to conduct these analyzes based on its routine work.

The present invention is illustrated in more detail in the following figures and examples.

Figures

Figure 1 : Fluorescence of droplets after cell growth, w/o droplets containing E. coli strains on minimum or rich media were observed in the same conditions at initial time of culture and day 4 after encapsulation. Negative control consists in a control strain with no ability to grow on encapsulated media.

Figure 2: Fluorescence of droplets after cell growth, w/o droplets containing a metagenomic clone on minimum medium + xylooligosaccharide mixture (XOS) observed at day 4 after encapsulation with different fluorescent filters. LP = long pass. Figure 3: FACS results obtained with a negative control (upper panels) and a positive control strain (lower panels). Strains were encapsulated on minimum media + xylooligosaccharide mixture as carbon sources. On the left, schematic representation of droplets at initial time and after incubation. At day 6, samples were analysed using the same parameters and same gates.

Figure 4: FACS results obtained with a mixture of 99% of negative control and 1 % of positive control strains. Same as Figure 3.

Figure 5: Density blot results obtained with the same sample of droplet containing metagenomic positive clone grew with 4g/L xylose after 6 days of growth with different sets of excitation and emission wavelengths.

Figure 6: Results obtained after cell growth with different xylose concentrations. The same strain was encapsulated on minimum medium + different xylose concentrations. Density blots were obtained with FACS. Arrow indicated level of fluorescence for condition at 5g/L xylose. Droplets were imaged at the same day.

Figure 7: Medians of autofluorescence of the “high autofluorescence” gate on FACS density blots and number of cells per droplet with cells grew at different xylose concentrations.

Figure 8: Fluorescence of droplets after cell growth, w/o droplets containing S. cerevisiae (upper panels) and Y. lipolytica (lower panels) strains on minimum or rich media were observed in the same conditions at initial time of culture and day 4 after encapsulation.

Figure 9: Density blots obtained with S. cerevisiae strain. S. cerevisiae strain was encapsulated on minimum media with and without carbon source (glucose) and incubated for 6 days to let growth occurred. Samples were analysed with two different sets of excitation and emission wavelength ( _ex 405nrn/ _em 513±13nm, upper panels and _ex 488nrn/ _em 576±10nm, lower panels).

Figure 10: Density blots obtained with Y. lipolytica strain. Y. lipolytica strain was encapsulated on minimum media with and without carbon source (glucose) and incubated for 6 days to let growth occurred. Samples were analysed with two different sets of excitation and emission wavelength ( _ex 405nrn/ _em 513±13nm, upper panels and _ex 488nrn/ _em 576±10nm, lower panels)

Figure 11 : Density blots obtained with a mixture of S. cerevisiae strains. Two S. cerevisiae strains were mixed (ratio 1 :1 ) and encapsulated on rich media supplemented with 200pg/mL G418 and incubated for 9 days to let growth occurred. Sample was then analysed with FACS and population with higher autofluorescence (gate “high AF, high SSC”) was sorted on non-selective rich solid medium.

Figure 12: Excitation and emission spectra of flavins. Figure 13: Mass spectrum results of riboflavin, lumichrome, FMN and FAD using LC/HRMS. Standard molecules were analysed individually and in mixture.

Figure 14: Autofluorescence density blot and image obtained for bovine rumen bank grew on xylooligosaccharide mixture.

Figure 15: Secondary screen of hit clones of bovine rumen bank grew in liquid on minimum medium with xylooligosaccharide mixture as carbon sources. Clones were grown in microplate over a week and optical density was regularly measured. Black line represents the threshold we fixed for a clone positivity (final OD of the negative control + 0.2), black bars represent final OD of hit clones with negative growth and empty bars final OD of hit clones with positive growth. Controls: bar with dots for negative control with empty fosmid; dashed bars for positive control clones expressing either transporter and CAZyme activities or expressing CAZymes activities but no transporter, respectively.

Figure 16: Secondary screen of hit clones of bovine rumen bank grew on solid minimum medium with XOS as carbon sources.

Clones were plated on agar medium with xylooligosaccharide mixture as carbon sources. On the left, image of the clones on plate. On the right, quantification of growth by measuring the area of each clone using Imaged. Black line represents the threshold we fixed for a clone positivity (1 .5 x area occupied by the negative control). Bars represented area of each clones, empty bars represented clones defined as positive after growth on liquid medium. The clones with black square have area ratio lower than 1 .5 compared with negative control area. Controls: bar with dots for negative control with empty fosmid; dashed bars for positive control clones expressing either transporter and CAZyme activities or expressing CAZymes activities but no transporter, respectively.

Figure 17: Repetition of secondary screen on solid medium with XOS as carbon sources. 15 negative clones were re-plated and incubated. Black line represents the threshold we fixed for a clone positivity (1 .5 x area occupied by negative control). Bars represented area of each clones, dashed bars represent result of first screen, black bars represent clones still negative after the second assay on solid plates and empty bars, positive ones.

Figure 18: Autofluorescence density blot and image obtained for bovine rumen bank grew on xylopentaose, cellopentaose and xyloglucooligosaccharide mixture (DP7 to DP9).

Figure 19: Autofluorescence density blot and image obtained for bovine rumen bank grew on xylo-oligo-saccharides DP2 to DP4.

Figure 20: Autofluorescence density blot and image obtained for bovine rumen bank grew on cello-oligo-saccharides DP3 and DP4.

Figure 21 : Results of sorting of bovine rumen metagenomic bank grew on arabinofuranosyl-xylo-oligosaccharides. Examples

Growth of E. coli in droplets induces fluorescence of the medium

In all experiments water-in-oil droplets (w/o, also called single emulsion droplets) and water- in-oil-in-water droplets (w/o/w, also called double emulsion droplets) were used. Those monodisperse droplets were generated using microfluidic PDMS chips. For single emulsion droplets, the carrier phase was HFE-7500 oil supplemented with 1 % RAN surfactant (RAN biotechnologies).

For double emulsion droplets production, the carrier phase was 150mM NaCI + 1 % Tween 80 (Tauzin et aL, 2020). Such droplets contain around 3pL of medium.

As microorganisms were encapsulated in droplets, the droplet-cell occupancy was controlled.

Around 74% of droplets contained no cell, meaning that maximum 26% contained cells (values estimated using https://www.desmos.com/calculator/j8eiciw7ds?lang=fr).

To image the phenomenon of fluorescence, an E. coli metagenomic clone, named F5min_MFS, already characterized in the lab (Cecchini et aL, 2013; Tauzin et aL, 2016) was encapsulated in picoliter droplets with maximum 1 cell per droplet according to the Poisson distribution ( 0.3). This clone has the ability to grow on minimum medium with xylo-oligo-saccharides (XOS) as carbon sources. Indeed, this fosmidic clone expresses transporter-specific for XOS and cytoplasmic CAZymes to degrade them into xylose, a carbon source usable by E. coli bacteria and was chloramphenicol resistant (due to pCCI fos fosmid). We transformed this clone with an empty plasmid to confer it kanamycin resistance, in order to identify the strain in some experiments. Rich and minimum media were tested (Table 1 ) and droplets were incubated at optimal temperature to allow cell proliferation for 4 days.

Table 1 : List of conditions tested

Cell growth was then imaged using a wide-field fluorescent microscope. Images showed that the medium of some droplets became fluorescent with Aex 435±40nm/Aem 470nm, longpass filter (Figure 1 ). This phenomenon can clearly be observed on minimum media and the fluorescence was exclusively observed in droplets containing several cells. On rich media, this phenomenon was also observed even if the medium was already highly fluorescent from the beginning of the culture. To test if this fluorescence of the droplet can be associated with cell growth in droplet or with the presence of cells in the droplet for 4 days (even without growth), a negative control E. coli strain containing the empty fosmid (chloramphenicol resistance) was used. This clone was transformed with an empty plasmid to confer it ampicillin resistance, in order to identify it in some experiments. Two sets of droplets were prepared, one containing the positive control, the other containing the negative control on selective medium with XOS as carbon sources. After a 4-day incubation, the droplets were observed (Figure 1 ). As expected, only the positive control grew whereas negative control strain did not. Furthermore, fluorescence of the medium inside the droplets was only observed when cell growth occurred and no fluorescence has been detected in the condition without growth, showing that secreted fluorescence could only be associated to cell growing.

At day 4, the same droplets were observed with different fluorescence filters to check if fluorescence detection could be better (Figure 2). Fluorescence was only observed with another set of filters ( _ex 360±40nm; _em 470±40nm) normally used for DAPI staining. Nevertheless, this excitation wavelength was not used as detection with set ( _ex 435±2.5nm; e _m 470nm long pass) was better.

As this result was systematically observed, on rich and minimum media, it was further analysed to know if it could be used for droplet sorting. This phenomenon was called “autofluorescence” as the whole droplet, meaning all the medium which composes the droplet, becomes fluorescent when growth occurred.

The medium fluorescence signal can be detected at kHz rates by FACS and used to sort bacteria growing in droplets

To assess if autofluorescence can be useful to sort droplet based on cell growth and fluorescence of the droplet, it was kept using the same clones as previously. First, these two clones were individually encapsulated in droplets on minimum medium with XOS as sole carbon sources. After 6 days, as expected, the metagenomic clone grew while the negative control one did not. We analysed the suspensions of droplets in the FACS system, Moflo Astrios sorter from Beckman Coulter (Figure 3). Two sets of filters were first applied to screen the single droplet (first set: complexity vs. size; second set: complexity vs. time of flight). The autofluorescence level of the droplet was then measured ( _ex 405nm; _em 450±30nm) vs. complexity. The threshold on the complexity parameter was 0.1. For the negative control clone, one main population pretty compact (Figure 3, upper right panel) was observed. A second population, with lower complexity, lower level of autofluorescence and more diffuse, corresponded to oil-in-water droplet that may be formed during the step of water-in- oil-in-water droplet production.

For the positive clone, the same populations were observed but a third population, with higher fluorescence level and also quite compact, was measured (Figure 3, lower right panel). This second population represented 20 to 25% of the total droplet population, which corresponds to the expected percentage of filled droplet (according to Poisson distribution, lambda around 0.3).

As both strains showed different fluorescence patterns, it was decided to test sorting based on this autofluorescence. T o identify the strains, after sorting, their ability to resist to different antibiotics was used (as described previously): Ampicillin resistance for the negative clone and kanamycin resistance for the positive clone. The two bacterial strains were mixed (with a ratio of 99% of negative clone and 1 % of positive clone) just before encapsulation on minimum medium with XOS as sole carbon sources. Again, after 6 days, sorting was performed based on parameters as previously described (Figure 4). The same pattern of fluorescence was observed than the one observed for the positive control in Figure 4, meaning two compact populations with different levels of autofluorescence.

The two populations were then sorted based on autofluorescence intensity (Table 2). To identify the strains composing both populations, the sorted droplets were first plated on non- selective rich medium and then, pick clones out on rich medium plates with selective antibiotic (ampicillin or kanamycin) and quantified the growing clones. It was found out that 95 % of the population with the lowest fluorescence intensity level contained the negative control clone (Table 2) whereas the population with the highest fluorescence level was almost exclusively (95 %) composed of the positive clone, demonstrating than sorting on autofluorescence intensity of droplets after cell growth was a highly efficient process.

Table 2: Quantification of strains present in each gate. Populations sorted from FACS were firstly plated on rich medium with only chloramphenicol as antibiotic. Each individual clone was then plated on 3 different plates: rich medium with only chloramphenicol as positive control of growth; rich medium with chloramphenicol and kanamycin and rich medium with chloramphenicol and ampicillin as selective media. Autofluorescence level is correlated to microbial growth

At last, it was wanted to know if autofluorescence intensity inside the droplet could be correlated to cell growth level and to test if a better separation of negative and positive populations could be obtained by changing couples of excitation and emission wavelengths. So, the E. coli metagenomic clone that was already used on minimum medium with different xylose concentrations, from 1 g/L to 5g/L, as carbon source (non-selective carbon source) was encapsulated. At day 6, the level of autofluorescence of all the samples with five couples of excitation and emission wavelengths was analysed and then calculated the stain index obtained for each condition (Table 3). This index is the ratio of the separation of the positive population and the negative one, divided by two times the standard deviation of the negative population. The higher is this index, the better the separation of populations is. The settings of the sorter have to be modified to have equivalent level of fluorescence for the negative gate.

For all samples, no signal was obtained with set of _ex 355nm; _em 450±30nmn (data not shown) whereas, worst separations and low stain indexes were measured with filters _ex 488nm; _em 526±26nmn and _ex 488nm; _em 576±10nmn. At last, good separations were found with _ex 405nm; _em 450±30nmn, wavelengths initially used and with _ex 405nm; _em 513±13nmn (Table 3 and Figure 5 for 4g/L).

Table 3: Stain index obtained with different wavelengths of excitation and emission after growth at several xylose concentrations.

It was decided to work with set of _ex 405nm; _em 513±13nmn as stain indexes were high at low and at high concentrations of xylose.

At day 6, the droplets were also imaged and analysed them with FACS to measure the level of fluorescence signal of the positive gate (Figure 6). To do so, we first defined the positive gate with the analysis of the sample of droplets prepared with minimum medium (M9) + 5g/L xylose, a carbon source concentration usually used for E. coli, and measured the median of fluorescence of this population. We then analysed the sample prepared with the same strain on minimum media without xylose (negative control). In this condition, as expected, no growth was observed and almost no population was in the positive gate (less 1%). At last, samples prepared with 1 g/L to 4g/L xylose were analysed. On the microscope an increase of the droplet fluorescence and the number of cells per droplet could be seen. Nevertheless, no quantification could be done this way. On the other hand, with FACS analysis, the increase of droplet autofluorescence by following the medians of fluorescence in positive gate was clearly observed and measured (Figure 7, left panel) while no change of the fluorescence median occurred in the negative gate (Figure 6).

To go further, we analysed the evolution of the number of cells inside the droplet as a matter of the initial xylose concentration. To do so, the samples of droplets prepared with minimum medium (M9) and all the xylose concentrations using FACS were re-analysed. Droplets belonging to the defined positive gate were then sorted, one-by-one directly on Petri dishes. This way, the content of one droplet was plated on one Petri dish. The content of the droplet was then spread out using spreader. This sorting was repeated 20 times for each sample (Figure 7, right panel). The plates were then incubated at 37°C overnight and the number of clones, which corresponds to the number of cells per droplet, was counted. It was found out that with an initial concentration of xylose at 1 g/L, the fluorescence level of the droplet was sufficient for sorting and that the number of cells per droplet was around 8±4. The value increased as the concentration of xylose increased to reach 26±8 cells per droplet at 5g/L.

In conclusion, it was demonstrated that autofluorescence of the medium is directly linked to cell growth inside the droplet and can be easily used for FACS sorting, meaning that:

- When cells do not have the ability to grow, no fluorescence is detected on minimum medium and no increase of fluorescence is observed on rich medium, already auto- fluorescent.

- Fluorescence intensity is directly correlated to the efficiency of growth and to the final number of cells in the droplet,

- Sorting based on secreted fluorescence can be used at least to screen for growing E. coli strains,

- The secreted fluorescent molecule is trapped inside the droplet in which it is produced and does not escape, at least for six days, showing that it is a good marker for FACS sorting of droplets.

Yeast sorting based on autofluorescence

The two species mentioned in Table 4 were encapsulated on minimum and rich medium. Table 4: List of strains and conditions tested

As for E. coli test, the droplets were observed on wide-field microscope, just after encapsulation and at day 4. For both species and both medium, firstly yeast growth wasobserved. This growth was lower for S. cerevisiae compared to Y. lipolytica and was lower on minimum medium compared to rich medium (Figure 8). Furthermore, rich medium was fluorescent. Nevertheless, secreted fluorescence was observed in all conditions even if secreted fluorescence was lower with S. cerevisiae strain.

To do first test on FACS, both species were encapsulated on minimum medium with and without carbon source (20g/L glucose) and incubated droplets at 28°C for 6 days before analysis (Figure 9 for S. cerevisiae and Figure 10 for Y. lipolytica). Sample autofluorescence was first analysed using the best set of wavelengths defined for droplet containing E. coli strain ( _ex 405nm; _em 513±13nmn). In parallel the samples were also analysed with the other set of wavelengths than we used for E. coli.

As encapsulation level was low, the population of droplets with slightly higher fluorescence level was quite low (around 3.8% for S. cerevisiae and 1.2% for Y. lipolytica strains). Nevertheless, it seemed that, with carbon source, part of the droplet has higher fluorescence intensity than without carbon source. This phenomenon was more detectable with the set of wavelengths _ex 488nm; _em 576±10nmn and could be measured for both yeasts.

To test if the population with higher fluorescence really corresponds to droplets where growth occurred, the experiment was repeated with a mixture of two S. cerevisiae strains: wild type BY4741 strain, used as negative control, whereas the second has been transformed to be resistant to G418 (BY4741 from YKO collection with G418 cassette integrated at Erc1 genome location).

As the encapsulation level was quite low, the mixture was done with 50% of each strain and was encapsulated on rich medium supplemented with 200pg/mL G418. In these conditions, only the second strain, resistant to G418 could grow. To improve growth, droplets were incubated for 9 days at optimal temperature and then analysed by FACS (Figure 1 1 ). The same pattern was observed and the population of droplets was sorted with the highest fluorescence level, first on non-selective solid rich medium following a 96-well plate pattern. Individual clones, corresponding to individual droplets, were then picked out on rich medium plates with and without selective antibiotic (G418), to quantify the number of growing clones. One hundred and fifty clones were picked out and all of them (100%) grew on medium with G418, demonstrating that all the sorted droplets contained the G418 resistant strain.

Secreted fluorescence, associated with growth of microorganisms in droplets, is thus also efficient to sort yeasts such as S. cerevisiae.

Identification of the origin of fluorescence

It was first tried to detect secreted fluorescence from batch cultures in Erlenmeyer using a fluorometer and spectra obtained with commercially available flavin standards (All products from Sigma-Aldrich, reference 47861 , F2253, F6625 and 103217 for riboflavin, FMN, FAD and lumichrome respectively, Figure 12). Solutions of flavin were freshly prepared at 1 to 10pM and excitation spectrum with emission at 530nm was done. As already described, we observed that riboflavin, FMN and FAD have excitation maxima around 450nm while lumichrome’s was around 410nm. Emission spectrum with fixed _ex at 450±4.5nm and 410±4.5nm confirmed that maximum of excitation was obtained around 530nm and 510nm respectively.

The secretion of fluorescence during the culture in batch, at _ex 450±4.5nrn/ _em 530±1 Onm, and _ex 410±4.5nm/ _em 510±10nm (Table 5). The fold of fluorescence of medium was calculated with cell growth on fluorescence of medium only kept in the same condition. In most of conditions, a secretion of fluorescent molecules during cell growth was measured, particularly on minimum media. After 72 hours of culture, the maximum of fluorescence was measured (Table 5) except for S. cerevisiae and Y. lipolytica cultures on rich media where no secretion was measured.

Nevertheless, with this technic, the nature of molecules was still unknown.

Table 5: Fold of fluorescence from E. coli, S. cerevisiae and Y. lipolytica cultures after 72

Hours In parallel, a method to detect and quantify flavins was used. Flavins could be analysed by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC/HRMS) using HS F5 Discovery column (150 x 2.1 mm, particle size 5pm, Supelco).

Samples were kept at 4°C in autosampler. Separation was done at 30°C with a gradient of 0.1 % formic acid in water (solvent A) and of 0.1% formic acid in acetonitrile (solvent B). The flow rate was set at 0.25mL/min and the multi-step linear gradient was: 2% B at 0 min, 2% B at 2 min, 5% B at 10 min, 35% B at 16 min and 100% B at 20 min. The injection volume was between 1 pL to 5pL. Mass spectra were acquired in positive mode with electrospray ionization (resolution 60,000, m/z 400) and recorded for a range of m/z 150-1 ,000. First tests showed that, under these conditions, we could detect and separate riboflavin, FMN, FAD and 7,8-dimethylalloxarine (lumichrome, Figure 13).

A range of concentration for each standard was then done to obtain the limit of detection for the compounds. Riboflavin, FMN and lumichrome were properly detected from 10 nM until 250 nM but for FAD, correct detection started from 62.5 nM up to 250 nM.

A first test was then done to quantify flavins after cell growth in droplets. To do so, the E. coli metagenomic clone was encapsulated (F5min_MFS) on minimal medium with or without carbon source, or on rich medium, with maximum 1 cell per droplet (lambda 0.3). Droplets were incubated to allow cell proliferation for 10 days. Emulsions were then broken with an antistatic gun (Karbaschi et aL, 2017). This gun has the huge advantage to break emulsion with no chemical product or solvant that may affect cell viability. After centrifugation, aqueous phases were then collected and analysed by LC/HRMS to quantify secreted flavins. As the droplets with cell growth (meaning autofluorescent) from empty droplets (or droplets with a non-growing cell) were not sorted before breaking the emulsion for this experiment, the concentration of the flavins was diluted with the medium contained in the empty droplets. For this reason, for E. coli results, a correction factor of 3.85 was applied (corresponding to the ratio of total droplets divided by filled droplets, 100/26, Table 6). In minimum medium, FAD and lumichrome were not detected whereas riboflavin and FMN were secreted in medium by E. coli. On rich medium, no secreted flavin was detected.

Table 6: Extracellular flavin secreted from E. coli strain encapsulated. Cell were encapsulated on indicated medium and incubated for 10 days for cell growth. Emulsions were then broken using an antistatic gun and aqueous phase collected for flavin quantification using LC/HRMS. Volume of injection 3pL. For yeast, results were given without correction to compensate empty droplets, meaning that the flavin concentration values were highly underestimated (Table 7).

Table ?: Extracellular flavin secreted from S. cerevisiae and Y. lipolytica encapsulated. Cell were encapsulated on indicated medium and incubated for 10 days for cell growth. Emulsions were then broken using an antistatic gun and aqueous phase collected for flavin quantification using LC/HRMS. Volume of injection 3pL.

For S. cerevisiae, no flavin was detected on rich medium. Only lumichrome, resulting of riboflavin degradation, was quantified. In Y. lipolytica cultures, on all media, only riboflavin secretion and lumichrome was detected.

Application to functional metagenomics

The process according to the invention was used to screen a bovine rumen metagenomic bank (Ufarte et aL, 2017).

The bank, composed of 21 ,200 clones, was mined for identifying new CAZymes, oligosaccharide transporters and polysaccharide utilization pathways.

To screen this bank on different substrates, it was decided to analyse around ten times the bank, to avoid loss of potential clones; to define the positive gate at the 0.1% highest autofluorescent droplet. Moreover, highly restrictive conditions were applied for the sorting and selected only droplets with the highest autofluorescence level to reduce the false positive clones. For each bank sorting, positive droplets were sorted in two 96-well plates with one well empty to control that sterility conditions were correct.

Sorting on XOS

We first choose to use XOS (xylo-oligo-saccharide mixture) as carbon sources and encapsulated the bank with maximum one cell per droplet (lambda 0.3). Before sorting the droplets, were first imaged and it was observed that growth occurred with different efficiencies in droplet, as expected for a bank (Figure 14). The bank was then analysed with the FACS and the same was observed as with microscope: an important population in gate “low autofluorescence” which corresponded to empty droplets and droplets with one cell without the ability to grow on this substrate. Above this negative population, the positive one was quite diffuse with different level of autofluorescence, which reflected, as we previously demonstrated, the different level of growth inside the droplets. For this substrate, the bank was analysed around 3.4 times and 85 hit clones were obtained. Next, we identified clones using their position in the storage plate meaning A1 to F8 and A10 to G12 (no clone in column 9).

These hit clones were used to do secondary screens:

- First, the 85 hit clones were grown in 48-well microplates on the same medium than the one used in droplet, meaning minimum medium with XOS as carbon sources. The growth was monitored by following optical density at 600nm (OD600nm). As controls, we used the negative clone only containing the empty fosmid and the positive one named F5min_MFS (dotted bar and left dashed bar respectively on Figure 15). A third control was added which was another already characterised metagenomic clone. This one, named P4, contained a fosmid with all the CAZymes needed to degrade XOS but no transporter meaning it did not have the ability to properly grow on this substrate except if some cells lysed and released CAZymes in medium to directly degrade the sugars (right dashed bar on Figure 15) (Cecchini et aL, 2013). It was estimated that hit clones have effective growth when the final OD600nm value of the cultured clone was higher by 0.2 than the negative control one. The growth over a week was measured, and in these conditions, after 7 days of culture, 48 clones of 85 grew (56.5%), meaning they expressed a functional transporter for these oligosaccharides and specific CAZymes to degrade them (Figure 15).

In parallel all the clones, were also plated on solid medium (same medium as the liquid one with 15g/L agar) as it was already observed that some clones which only expressed CAZymes but no transporter may not properly grow in liquid medium but would on solid one (Cecchini et aL, 2013). Growth on solid medium was slower than on liquid medium, so the growth was assessed after 11 days by measuring the area occupied by each clone compared to the one occupied by the negative control. It was estimated that hit clones have effective growth when the clone area was higher by 1 .5-fold than the negative control area (Figure 16,).

Most of the clones that were positive after validation screening in liquid medium grew on solid plates, but also almost all the ones that were not positive after the validation screening in liquid medium properly grew on solid medium (Figure 16).

At this step, 69 clones out of 85 sorted by droplet microfluidcs (81 .2%) have positive growth efficiency. It was decided to re-plate the 15 negative clones. These clones were plated with further space and not closed to the outlines of the plate to reduce putative biais. After 12- days incubation, growth was quantified with the same methodology as previously described (Figure 17).

This test showed that 8 out of the 15 clones tested have proper growth.

To conclude, after growth assays on solid plates, 78 hit clones selected after FACS sorting were positive (91 .8%).

Sorting on other oligosaccharides

The same process was then applied exactly to mine the bovine rumen metagenomic bank on plant cell wall oligosaccharides (xylopentaose, cellopentaose and xyloglucooligosaccharide mixture, DP7 to DP9).

Only the 0.1 % highly autofluorescent droplets was then sorted (Figure 18 and Table 8).

Table 8: Results of sorting of bovine rumen metagenomic bank grew on xylopentose, cellopentose and xyloglucan mixture (DP7 to DP9).

Eight or nine of the hit clones were then sequenced for each oligosaccharide with Sanger method. This method was used to determine short DNA sequence (around 1 ,000 nucleotides) at the extremities of the insert, in order to identify the redundant clones. For cellopentase, good sequencing results were obtained for 7 clones with 2 redundant clones. For xylopentaose and xyloglucooligosaccharides, good sequencing results were obtained for 4 and 5 clones respectively and no redundancy was observed.

In parallel, it was continued to mine the bovine rumen library for ability to grow on other plant-derived oligosaccharides. In complement to the previous analyses, cellooligosaccharides, arabinofuranosyl-xylo-oligosaccharides and xylo-oligosacharides with different polymerisation degrees were used to potentially identify specific transporters for short or long oligosaccharides (Figures 19 and 20 and Table 9 and 10, Figure 21 and Table 1 1 ).

Table 9: Results of sorting of bovine rumen metagenomic bank grew on xylo- oligosacharides.

*data not collected.

Table 10: Results of sorting of bovine rumen metagenomic bank grew on cello- oligosaccharides Table 11 : Results of sorting of bovine rumen metagenomic bank grew on arabinofuranosyl- xylo-oligosaccharides (DP2 and DP3/DP4).