Nathalie Mouheb

Wéry

Kévin Lequette
email: kevin.lequette@gmail.com

Nassim Ait-Mouheb

Nathalie Wéry

Hydrodynamic effect on biofouling of milli-labyrinth channel and bacterial communities in drip irrigation systems fed with reclaimed wastewater

Keywords: Drip irrigation, biofilm, flow behaviours, Optical Coherence Tomography, High-throughput DNA sequencing

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The scarcity of water resources is driving the development of water-saving irrigation techniques that enable integrated management of water resources. The reuse of reclaimed wastewater (RWW) is an appropriate way to alleviate the problem of scarce water resources especially in arid and semi-arid regions [START_REF] Worako | Evaluation of the water quality status of Lake Hawassa by using water quality index , Southern Ethiopia[END_REF]. Using RWW has several advantages including reducing pressure on freshwater resources and on the need for nutrients (e.g. nitrogen, phosphorus) for plant growth [START_REF] Lazarova | Water Reuse for Irrigation[END_REF] and can improve crop yields [START_REF] Wang | Impact of drip and level-basin irrigation on growth and yield of winter wheat in the North China Plain[END_REF]. Drip irrigation is the most water efficient (up to >90%) and safest technique for irrigation using RWW [START_REF] Lamm | Maintenance[END_REF]. Drippers are usually composed of a labyrinth-channel and an outlet basin compartment. The geometry of the channel promotes the dissipation of the turbulent kinetic energy due to the zigzag flow path and allows water to be delivered in drip form in an optimum quantity for crop growth. However, the labyrinth channel is narrow (cross-section of around 1 mm) [START_REF] Zhang | Anti-clogging performance evaluation and parameterized design of emitters with labyrinth channels[END_REF] and thus sensitive to clogging [START_REF] Liu | Laboratory experiment on drip emitter clogging with fresh water and treated sewage effluent[END_REF][START_REF] Niu | Influence of fine particle size and concentration on the clogging of labyrinth emitters[END_REF] which reduces drip irrigation advantages by reducing irrigation uniformity [START_REF] Dosoretz | Fouling in Microirrigation Systems Applying Treated Wastewater Effluents[END_REF] and by increasing maintenance (e.g. cleaning, replacing irrigation lines).

The process leading to clogging of the drippers is complex and the phenomenon is influenced by several parameters including water quality (concentration of suspended solids and chemical composition) [START_REF] Lamm | Maintenance[END_REF][START_REF] Oliveira | Influence of Concentration and Type of Clay Particles on Dripper Clogging[END_REF], the geometry of the dripper [START_REF] Li | Formation mechanism for emitter composite-clogging in drip irrigation system[END_REF][START_REF] Wei | Application of RP and Manufacturing to Water-Saving Emitters[END_REF] as well as biological processes [START_REF] Katz | Fouling formation and chemical control in drip irrigation systems using treated wastewater[END_REF], the latter are the most difficult to control when RWW is used [START_REF] Lamm | Maintenance[END_REF]. Moreover, biofilms in distribution networks can harbour contaminants and may impact the quality of the water released to agroecosystems [START_REF] Jjemba | Regrowth of potential opportunistic pathogens and algae in reclaimed-water distribution systems[END_REF]. Therefore, identifying and understanding the mechanisms leading to biofilm development is necessary to optimize control strategies. Previous studies have shown that flow velocity has an impact on the growth of biofilm in both drip-irrigation pipes [START_REF] Li | Effects of average velocity on the growth and surface topography of biofilms attached to the reclaimed wastewater drip irrigation system laterals[END_REF][START_REF] Mahfoud | Disruption of biofilms from sewage pipes under physical and chemical conditioning[END_REF] and in drippers [START_REF] Gamri | Biofilm development in microirrigation emitters for wastewater reuse[END_REF].

Previous studies have been carried out on the effect of hydrodynamics on the development of biofilm in straight pipe systems. Thus, an increase in flow rate or shear stress tends to promote the development of a thin and dense biofilm in the pipe wall [START_REF] Lehtola | The effects of changing water flow velocity on the formation of biofilms and water quality in pilot distribution system consisting of copper or polyethylene pipes[END_REF][START_REF] Percival | The effect of turbulent flow and surface roughness on biofilm formation in drinking water[END_REF]. However, the flow behaviour in a dripper is more complex and heterogeneous than in pipes, where the flow is simplified, due to the narrow labyrinth flow path. This milli-labyrinth channel induces non-isotropic conditions by the development of turbulent regime with a main high velocity flow and vortex zones in the channel corners [START_REF] Al-Muhammad | Experimental and numerical characterization of the vortex zones along a labyrinth millichannel used in drip irrigation[END_REF], 2016;[START_REF] Wei | Flow behaviour analysis and experimental investigation for emitter micro-channels[END_REF][START_REF] Zhang | Numerical and experimental study on hydraulic performance of emitters with arc labyrinth channels[END_REF]. [START_REF] Qian | Water quality and daily temperature cycle affect biofilm formation in drip irrigation devices revealed by optical coherence tomography[END_REF] studied the development of fouling in a milli-fluidic system fed with reclaimed wastewater using the Optical coherence tomography (OCT).OCT method allows to monitor biofilm formation non-invasively at a mesoscale (µm to mm range) and without staining [START_REF] Derlon | Predation influences the structure of biofilm developed on ultrafiltration membranes[END_REF][START_REF] Dreszer | In-situ biofilm characterization in membrane systems using Optical Coherence Tomography: Formation, structure, detachment and impact of flux change[END_REF][START_REF] Qian | Water quality and daily temperature cycle affect biofilm formation in drip irrigation devices revealed by optical coherence tomography[END_REF][START_REF] Wagner | Investigation of the mesoscale structure and volumetric features of biofilms using optical coherence tomography[END_REF][START_REF] West | Optical coherence tomography for the in situ three-dimensional visualization and quantification of feed spacer channel fouling in reverse osmosis membrane modules[END_REF]. They reported that fouling occurs mainly in inlet channel regions and in low velocity vortices areas as found by [START_REF] Ait-Mouheb | Impact of hydrodynamics on clay particle deposition and biofilm development in a labyrinth-channel dripper[END_REF].

Flow behaviour not only influences the development of biofilms but also the microbial composition of the biofilms. Previous research on biofilms has shown that hydrodynamic conditions can drive the microbial community of the biofilms [START_REF] Besemer | Biodiversity, community structure and function of biofilms in stream ecosystems[END_REF][START_REF] Hou | Variations in the microbial community of biofilms under different near-wall hydraulic shear stresses in agricultural irrigation systems[END_REF][START_REF] Rickard | Shear Rate Moderates Community Diversity in Freshwater Biofilms Shear Rate Moderates Community Diversity in Freshwater Biofilms[END_REF]. [START_REF] Rochex | Role of shear stress on composition, diversity and dynamics of biofilm bacterial communities[END_REF] shown that increased shear stress reduced bacterial diversity and slow down biofilm maturation and tend to maintain a young biofilm in a Taylor-Couette reactor. However, in the context of the use of RWW, few authors have examined the influence of dripper parameters (i.e. flow rate, cross section, and geometry of the milli-channel) on microbial composition. Previous studies have described the microbial communities present in biofilms of different drippers by analysing phospholipid-derived fatty acids (PLFAs) [START_REF] Yan | Biofilm Accumulation and Structure in the Flow Path of Drip Emitters Using Reclaimed Wastewater[END_REF][START_REF] Yan | Biofilm structure and its influence on clogging in drip irrigation emitters distributing reclaimed wastewater[END_REF][START_REF] Zhou | Effects of microbial community variation on bioclogging in drip irrigation emitters using reclaimed water[END_REF] and indicated that dripper flow rate influenced the PLFAs distribution. These authors showed that three to seven types of PLFAs associated with Gram-negative bacteria were common in biofilms inside drippers, suggesting a significant role for these bacteria in biofilm development. However, in order to develop effective biofilm control strategies, deeper community characterization is required. Recent study used high-throughput sequencing to describe the bacterial communities present in dripper biofilms to improve knowledge on fouling and to identify the microorganisms responsible for fouling [START_REF] Xiao | Electromagnetic fields for biofouling mitigation in reclaimed water distribution systems[END_REF][START_REF] Zhou | Critical controlling threshold of internal water shear force of anti-clogging drip irrigation emitters using reclaimed water[END_REF].

The objectives of the present study were to determine the effect of 3 commercial drippers with different discharges (1) on biofouling in the dripper channel and (2) on the bacterial communities in biofilms formed in drip irrigation systems fed by RWW. A nondestructive microscopic time-monitoring observation system was developed to study biofilms in commercial drippers with different flow rates (1, 2 and 4 l.h -1 ) and known cross-sections.

The combined use of the Optical Coherence Tomography method and high-throughput sequencing made it possible to monitor the development of the biofilm under different hydraulic conditions while assessing the impact of these conditions on microbial structure.

Materials and Methods

Experimental setup

Experimental setup and irrigation procedure

The development of biofilm in drippers cannot be observed over time because drippers are walled inside opaque irrigation pipes (black polyethylene tubes). Thus, commercial nonpressure compensating drippers (NPC) (model D2000, Rivulis Irrigation SAS, Lespinasse, France) with different flow path geometry and different flow rates (1, 2 and 4 l.h -1 ) (Table 1) were placed in a transparent tube (internal diameter 15mm, TubClair, Vitry-le-François, France), allowing to see biofilm development along the channel over time. A solid tube with an internal diameter of 19mm (ABS+, Stratasys U-print SE plus, Stratasys Ltd, Eden Prairie, Minnesota, US) pressed the dripper against the walls of the transparent tube (Figure 1.A,C).

Finally, an outlet hole (internal diameter 2mm) was pierced in the transparent tube above the outlet basin. Nine drippers were connected to each other according with the type of dripper using polyethylene tubing (length 10cm, internal diameter 20mm) and valves (internal diameter 20mm) (total length 3m). Then, 3 lines were built (one per dripper type). The valves were used to keep the drippers under water when they were disconnected from the irrigation line for analysis. Each of the three lines was connected to a separate tank (total volume 60l) and a pump (JetInox 82M, DAB, Saint-Quentin-Fallavier, France) (Figure 1 Table 1 lists the dripper characteristics (D1 to 3) commonly used in drip irrigation system.

The geometric parameters, including flow path length (L), width (W), depth (D) and crosssectional area (Dh) are specific to the dripper type, which influence the type of flow in the dripper path. These parameters were further integrated as two dimensionless parameters, including the width-depth ratio (W/D) and the relative radius (A 1/2 /L), as proposed by [START_REF] Li | Formation mechanism for emitter composite-clogging in drip irrigation system[END_REF]. Then, the hydraulic parameters, including the outflow (q), the Reynold number (Re) and the inlet average cross-sectional velocity (v), are related to these geometric parameters.

In order to know the hydraulic conditions at the inlet channel, the flow regimes were characterized by a Reynolds number (Re).

The Reynolds number (Re) was computed using the following formula:

= (Equation 1)
where is water density (kg.m -3 ), v the water velocity across the pipe (m.s -1 ), Dh the hydraulic diameter (m), and µ the water viscosity (Pa s).

The Reynolds number of the studied drippers is between 305 and 926 at the inlet of labyrinth.

It should be noted that these Re correspond to laminar flow regime in the case of straight pipes for example. However, due to the labyrinth geometry and small flow cross-sections, it is assumed that flow behaviours in studied drippers correspond to turbulent regime [START_REF] Li | Hydraulic Characterizations of Tortuous Flow in Path Drip Irrigation Emitter[END_REF][START_REF] Zhang | Flow regime and head loss in a drip emitter equipped with a labyrinth channel[END_REF].

The hydrodynamic radius (or hydraulic diameter), Dh, was calculated for a rectangular crosssection as:

ℎ = (Equation 2)
where A: area (mm) and P for the perimeter (mm). L: length of the flow path, W: width of the flow path, D: depth of the flow path, q: flow rate, v: average cross-section velocity Dh: hydraulic diameter, .x: pressure's exponent, CV: Coefficient of variation

Physical-chemical and microbiological quality of the RWW

The irrigation lines were supplied with reclaimed wastewater from the Murviel-Les-Montpellier in the South of France, (43.605034° N, 3.757292° E). The wastewater treatment plant is designed around stabilisation ponds with three successive lagoons (13 680 m 3 , 4784 m 3 and 2700 m 3 ) and a nominal capacity of 1,500 Inhabitant Equivalent. The RWW was placed in a 60l tank and changed twice a week to maintain the quality close to that of the wastewater from the treatment plant.

Image acquisition and processing

Image acquisition

Optical coherence tomography (OCT) was used to study the biofilm kinetics in drippers and along the milli-channel throughout the experimental period. The measurements were performed in situ and non-invasively through the transparent tube. For the measurements, the valves of the dripper systems were closed to keep the drippers in water, and then the dripper in its tubing was disconnected from the irrigation line. Measurements were taken at least once every two weeks and each time all drippers were analysed. After the OCT measurement, each dripper was replaced on the test bench at its initial location. Threedimensional OCT measurements were acquired using a Thorlabs GANYMEDE II OCT (LSM03 lens, lateral resolution: 8µm; Thorlabs GmbH, Lübeck, Germany). The axial voxel size in water (n = 1.333) of GANYMEDE II is 2.1 μm. OCTs have a centre wavelength of 930 nm. Due to the length of the drippers, the labyrinth channel was divided into eight parts (inlet, part 2, part 3, return, part 6, part 7, and end; Table S1 in Supplementary material) to study biofouling in different parts of the channels (Figure 2). S1 in Supplementary material.

Image processing

First, 3-D OCT datasets were processed in Fiji (running on ImageJ version 1.50b, [START_REF] Schindelin | Fiji -an Open platform for biological image analysis[END_REF]) and converted into 8-bit grayscales. The datasets were resliced from top to bottom into image stacks and regions of interest (inlet and return areas) were selected.

The remaining parts were allocated to the background (black). Secondly, an in-house code was used to detect the pixels associated with the plastic tube and removed using MATLAB R2018r (MathWorks ®, version 2018b). A threshold (adapted to each dataset) was applied to binarize the dataset and the region above the interface was quantified as biofilm. For each position (x, y), the pixels associated with the biofilm (up to the threshold) were summed (on z) to obtain the thickness of the biofilm. The volume of the baffles differed with the dripper (e.g. 4.7, 5.8 and 10.7 mm 3 for B1 and 3, 5 and 8.5 mm 3 for B2 for 1, 2 and 4 l.h -1 respectively, Figure 2). Therefore, to compare the level of fouling between drippers, the biofouling volume was normalized by the volume of the corresponding baffle. The volumetric coverage of the biofilm (%) was calculated for each baffle according to Equation 3:

!" % = $ %&'(&)* $ %+((),
× 100 (Equation 3)

where Vbiofilm is the biofilm volume and Vbaffle is the volume of the baffle.

Analysis of the microbial communities

Sampling the biofilm and reclaimed wastewater

Three drippers in each line were chosen at 32, 72 and 115 days and replaced by new ones to ensure the functioning of the system. However, these new drippers have not been

DNA extraction

DNA was extracted using the PowerWater® DNA Isolation Kit (Qiagen, Hilden, Germany).

Samples (drippers or filters) were placed in 5 mL tubes containing beads. The manufacturer's instructions were then followed. The DNA concentration was measured, and purity checked by spectrophotometry (Infinite NanoQuant M200, Tecan, Austria). Extracted DNA was stored at -20°C.

Bacterial quantification by qPCR

Total bacterial quantification was performed by qPCR on biofilms from drippers targeting the V9 region from 16S rDNA. The amplification reactions were performed in triplicate, and at two dilutions to check for the absence of inhibition of the PCR reaction. Reaction mixes (12µl) contained 2.5µl of water, 6.5µl of Super Mix qPCR (Invitrogen), 100nM forward primer BAC338 (5'-ACTCCTACGGGAGGCAG-3'), 250nM of reverse primer BAC805 (5'-GACTACCAGGGTATCTAAT CC-3') and 50nM of probe BAC516 (Yakima Yellow-TGCCA GCAGC CGCGG TAATA C -TAMRA) [START_REF] Yu | Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction[END_REF]. The cycling parameters were 2 min at 95°C for pre-incubation of the DNA template, followed by 40 cycles at 95°C

for 15 sec for denaturation and 60°C for 60 sec for annealing and amplification.

Illumina sequencing

PCR amplified the V4-V5 region of 16S rRNA genes with 30 cycles (annealing temperature 65°C) using the primers 515U (5′-GTGYCAGCMGCCGCGGTA-3′) and 928U (5′-CCCCGYCAATTCMTTTRAGT-3′) [START_REF] Wang | Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies[END_REF]. Adapters were added for multiplexing samples during the second amplification step of the sequencing. The resulting products were purified and loaded onto the Illumina MiSeq cartridge for sequencing of paired 300 bp reads according to the manufacturer's instructions (v3 chemistry). Sequencing and library preparation was performed at the Genotoul Lifescience Network Genome and Transcriptome Core Facility in Toulouse, France (get.genotoul.fr). Mothur (version 1.39.5) [START_REF] Schloss | Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities[END_REF] was used to associate forward and reverse sequences and clustering at four different nucleotides over the length of the amplicon. Uchime [START_REF] Edgar | UCHIME improves sensitivity and speed of chimera detection[END_REF] was used to identify and remove chimera. Sequences that appeared less than three times in the entire data set were removed. In all, 16S rRNA sequences were aligned using SILVA SSURef NR99 version 128 [START_REF] Schloss | Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities[END_REF]. Finally, sequences with 97% similarity were sorted into operational taxonomic units (OTUs) [START_REF] Nguyen | A perspective on 16S rRNA operational taxonomic unit clustering using sequence similarity[END_REF]. The chloroplast sequences were removed from the raw data. Finally, BLAST (http://www.ncbi.nlm.nih.gov/BLAST/)

was used to locate publicly available sequences closely related to the sequences obtained from the samples. A total of 11,357,162 reads were grouped in 7730 OTUs at the 97% similarity level. The rarefaction curves indicated that the sequencing depths of all samples were adequate (Figure S4 in Supplementary material).

Statistical analyses

The effect of the different commercial types of drippers (with specific geometry and hydraulic parameters) on the kinetics and bacterial composition of the biofouling was evaluated. The kinetics of biofilm development were evaluated by modelling the biofilm formation rate using linear and spline models. The sequencing data were processed under R v3.4 (www.rproject.org) using the R-Studio (http://www.rstudio.com/) phyloseq package [START_REF] Mcmurdie | PHYLOSEQ: a biocondutor package for handling and analysis of high-throughput phylogenetic sequence data[END_REF]. Kruskal-Wallis tests were performed to compare diversity and richness indices over time and between the dripper types. For the comparison of bacterial community structure, a dissimilarity matrix (Bray-Curtis) was performed and visualised using principal coordinate analysis (PCoA). A one-way analysis of similarities (ANOSIM) was used to identify significant differences in community assemblage structure between samples based on the origin of the sample [START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF]. The OTUs that contributed most to the divergence between two types of dripper were identified using Similarity Percentage (SIMPER) analysis [START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF].

Results

Areas favourable for the development of biofilm

The optical methods and discharge measurements are performed in order to analyse the effect of flow topologies along the three dripper channels. During the four month experiment, dripper outflows was higher than 75% of expected flow rate and the drippers never became clogged (Figure 3). In the first two weeks, the thickness of the biofilm was too low to be measured and the first measurements were made after one month. Based on OCT analysis (Figure S1, S2 and S3 in Supplemental material), the biofouling volume after 4 months tended to be higher at the inlet areas (2.45, 3.32 and 2.19 mm 3 for 1, 2 and 4 l.h -1 drippers respectively) and decreased after until the return area (with 1.62 and 2 mm 3 of biofilm respectively, there is no return area in 4 l.h -1 ). This means that inlet and return were the most sensible areas for clogging. Figure 4 shows the increase in biofouling thickness in the inlet and return dripper areas at one and four months. Over time, biological fouling at the inlet area increased, mainly in the first baffle and in corners, and gradually spread to the following corner baffles. After 4 months, the biofilm thickness was higher in the baffle corners for all the drippers, up to 0.5 mm while the thickness at the center of the channel was < 0.3 mm. The biofilm thickness in the return area increased mainly after the large bend (B7) in both types of drippers (Figure 4C). The volume of the baffles differed with the dripper. Therefore, to compare the level of fouling between drippers, the biofouling volume was normalized by the volume of the corresponding baffle (Equation 3). Mean biofouling of the inlet baffles increased over time in all three types of drippers (Figure S5 in Supplemental material). The level of biofouling were significantly influenced by the type of dripper (Chi²=115, p-value < 0.05) and the time (Chi²=54, p-value < 0.05) and increased more rapidly in 1 and 2 l.h -1 drippers than in 4 l.h -1 dripper types (Wald test, p-value < 0.05). After 4 months, the mean biofouling level at the inlet were higher in the 1 l.h -1 (18%) and in 2 l.h -1 (15.9%) than in the 4 l.h -1 (11.8%) drippers (conover test, p-value < 0.05). Therefore, the 1 l.h -1 dripper with lowest average cross-section velocity (v), Reynolds number and the ratio W/D and Dh 1/2 /L were the most sensitive to clogging.

Although the biofouling level of the first baffle tended to be higher, there was no statistical effect of baffles on volumetric coverage over time. However, the volume of biofouling in the first baffle was significantly higher in 1 l.h -1 drippers than in 2 l.h -1 and 4 l.h - 1 dripper types after four months with a mean volumetric coverage of 22.6%, 15.8% and 14.1

% for 1 l.h -1 , 2 l.h -1 and 4 l.h -1 drippers, respectively (Kruskal test, p-value < 0.05).

The mean increase in biofouling in the 1 l.h -1 drippers presented a sinusoidal dynamic with up and down phases at the inlet baffle (Figure S5 in Supplemental material). This could mean several detachment events occurred. The same cycle was observed in the 2 and 4 l.h -1 dripper types but only in the first baffle. The detachment event occurred at ~ 3 months in the 4 l.h -1 drippers whereas the first event appeared at 1 month in the 2 l.h -1 dripper types. After this baffle, the increase in biofouling was linear. At the return zone in 1 and 2 l.h -1 drippers, the volumetric coverage increased over time but was heterogeneous. Thus it was not possible to define kinetic models.

The type of dripper (flow rate and flow cross-section) influenced the level of fouling of the labyrinth with a higher fouling rate in 1 l.h -1 drippers (narrower flow cross-section and lower average cross-section velocity).

Microbial diversity differed between dripper biofilms and reclaimed wastewater

The structure of the bacterial communities in biofilms collected in the three types of drippers were compared using 16S rDNA Illumina sequencing. The bacterial communities in the biofilms were also compared with the communities present in reclaimed wastewater. The chloroplast sequences were removed from the raw data and represented 1.6% and 5.4% of the total sequences in biofilms and in RWW samples, respectively. Microscopic observations of the biofilms in the pipes (data not shown) also revealed the presence of eukaryotic microorganisms (bacterial predators) but the biofilms were still mainly composed of bacteria.

Mean DNA concentration increased significantly between the sampling events for each dripper types drippers (Table 3, p<0.05) but there were no differences between the dripper types (p>0.05). On the other hand, the number of bacteria remained unchanged over time and were similar between the drippers (Table 3, p>0.05), meaning that the increasing of the DNA concentration was probably due to other types of organisms that we observed in pipe biofilms in previous experiments (i.e. eukaryote, algae). 346.1±34.5 a 5.4±1.4 x 10 9a Kruskal-test and Wilcoxon ad hoc tests were performed on each sampling event to compare the values between the drippers (n= 3 for each type of dripper on each sampling event); letters show Wilcoxon test results.

Although fouling levels were higher in the 1 l.h -1 dripper type, the richness and diversity indices of the biofilms did not differ significantly and increased similarly between the dripper over time (p-value > 0.05) (Table 4) meaning that the arrival of new species in biofilms could settled regardless of hydrodynamic conditions. The richness and diversity indices of RWW were lower than those in the dripper biofilms. The majority of operational taxonomic units (OTUs) were shared by all the biofilms (Figure S6 in Supplemental material). Some OTUs and bacterial genera were specific to the dripper flow, but their relative abundance was less than 0.1% (Table S2 and Figure S6 in Supplementary material). Kruskal test and the Wilcoxon ad hoc test were performed for each dripper (n= 3 at each sampling); the letters show the results of the Wilcoxon test.

Proteobacteria, Bacteroidetes, Firmicutes and Chloroflexi were the main phyla in the dripper biofilms (Figure 4A). The main phyla found in RWW were Proteobacteria (25-55%) mainly composed of β-Proteobacteria (10-34%) and γ-Proteobacteria (2-34%), followed by Bacteroidetes (22-40%) and Actinobacteria (7-14%) (Figure 5B). These differences indicate that among bacteria in the reclaimed wastewater, bacteria able to attach to the surface of the dripper under the corresponding flow regime were selected, especially among Firmicutes and Chloroflexi (Figure 5). From 49 days onwards, cyanobacteria phylum appeared in reclaimed wastewater, explaining the increase in cyanobacteria in the drippers on the second sampling events (72 days). The same observation was made concerning Spirochaetae and Chlorobi phyla. Thus, RWW influenced the bacterial structure and composition of the dripper biofilms.

The structure of the biofilms in the drippers evolved over time according to changes in the microbial diversity in the reclaimed wastewater, and with the selection of some adapted taxa able to adhere to the surfaces, such as filamentous Chloroflexi bacteria.

Figure 5. Relative abundance of bacterial phyla (>1%) in dripper biofilms (A) and in reclaimed wastewater (B).

For each sampling event, 3 drippers per dripper type were sampled.

The dripper flow parameters influenced the composition of the bacterial communities

The impact of the type of dripper, defined both by geometric and hydraulic parameters, on the structure of bacterial communities was first investigated at the phyla and familly taxonomic levels. Proteobacteria phylum was the most abundant phylum in the biofilms (Figure 5) and was mainly composed of α-Proteobacteria, β-Proteobacteria (mainly composed of members of the Comamonadaceae family), γ-Proteobacteria and δ-Proteobacteria. At 32 days, the mean abundance of Proteobacteria was similar in all dripper types, with 49, 47 and 45% in the 1, 2 and 4 l.h -1 dripper biofilms, respectively (Kruskal-test, p-value > 0.05). At 115 days, the mean relative abundance of Proteobacteria (and more specifically β-Proteobacteria) was significantly lower in the 1 l.h -1 dripper biofilms than in the 2 and 4 l.h -1 dripper biofilms (Kruskal-test, p-value < 0.05) and decreased significantly until 28% versus 33 and 37% for the 2 and 4 l.h -1 dripper biofilms, respectively.

The phylum of Bacteroidetes was mainly composed of Sphingobacteriia (Chitinophagaceae, Lentimicrobiaceae, Saprospiraceae and Sphingobacteriaceae class members), Bacteroidia (mainly Draconibacteriaceae and Rikenellaceae member families) and Bacteroidetes_vadinHA17. There was no effect of the type of dripper on the mean relative abundance of Bacteroidetes (~ 30% over time in each dripper type, Kruskal test, p-value > 0.05). Over time, the relative abundance of Sphingobacteriia and Bacteroidia decreased while the relative abundance of Bacteroidetes_vadinHA17 increased in the three types of drippers.

The mean relative abundance of Chloroflexi (mainly composed of Anaerolineaceae and Caldilineaceae member family) increased significantly over time and was significantly higher in 1 l.h -1 dripper biofilms after 115 days (16% versus 14% and 10% in 1, 2 and 4 l.h -1 dripper biofilms, respectively, Kruskal test, p-value < 0.05). At 115 days, the mean relative abundance of the Anaerolineaceae family was significantly higher in 1 l.h -1 dripper biofilms than in the 4 l.h -1 dripper biofilms (Kruskal test, p-value < 0.05). There were no significant differences in the Firmicutes phylum (mainly represented by Christensenellaceae, Clostridiaceae and Ruminococcaceae), according to the dripper types or over time (Kruskal test, p-value > 0.05).

The impact of flow rates on bacterial communities was then investigated at the genus level.

Table S3 lists the 10 top genera found in all the dripper biofilms over time. At 32 days, the 1 l.h -1 dripper biofilms were dominated by the Comamonadaceae family (6%) including Hydrogenophaga genus (9%) and Pseudoxanthomonas (3%). The mean relative abundance of the Hydrogenophaga genus was significantly influenced by the flow rate (Kruskal test, pvalue < 0.05) and was higher in the 1 l.h -1 dripper biofilms (3% and 4% for 2 and 4 l.h -1 dripper biofilm, respectively). Over time, the mean relative abundance of members of the Comamonadaceae family and of the Hydrogenophaga genus decreased significantly in the 1 l.h -1 (Kruskal test, p-value < 0.05), close to the relative abundance in 2 and 4 l.h -1 dripper biofilms at the end (Kruskal test, p-value > 0.05). Although Proteobacteria phylum dominated at 115 days, some members belonging to Bacteroidetes phylum increased over time as

Lentimicrobiaceae family and Bacteroidetes_vadinHA17 and dominated in all three drippers at 115 days (Kruskal test, p-value > 0.05).

Even if the majority of the genera were the same in the different drippers and on the sampling occasions, some genera with low abundance (<1%) depended specifically on the dripper flow rate and the sampling time (Table S2, Figure S6 in Supplemental material). For instance, only Vulcaniibacterium and Microvirga genera were found in the 1 l.h -1 dripper at 32 and 72 days. This means that hydrodynamic conditions influenced both the dominant and less abundant bacterial taxa. 21

Dripper hydraulic properties impact bacterial community structure

PCoA was performed to compare the structure of bacterial communities between the three types of dripper over time and confirmed that the bacterial community in the dripper biofilms changed depending on hydraulic dripper properties (Figure 6). At 32 days, the bacterial populations in 2 and 4 l.h -1 were clustered (one-way ANOSIM, R=0.71, p < 0.05).

The bacterial communities in the 1 l.h -1 dripper biofilms formed a second cluster (Figure 6, A). This means that the hydraulic conditions had little influence on the structure of the microbial community in 2 and 4 l.h -1 compared to that in the 1 l.h -1 dripper biofilms at the first stage of development. At the end of the experiment (Figure 6, C) the bacterial community in 2 and 4 l.h -1 diverged and formed specific groups (one-way ANOSIM, R=0.65, p<0.05).

The main contributors of the bacterial community were identified using SIMPER and Fusibacter genera (OTU 60) were the main contributors and were associated with the 1l.h -1 drippers. These genera were also the main genera found in 1 l.h -1 dripper biofilms at 32 days (Table S3 in Supplemental material). At 115 days, 15 OTUs significantly contributed to divergence. Hydrogenophaga (OTU 5) and Pseudoxanthomonas (OTU 25) were still strong contributors associated with 1 l.h -1 dripper biofilms. Aquabacterium (OTU 10), Ideonella (OTU 15) and Bacteroidetes members (OTU 19, OTU 85) were associated with the 4l.h -1 dripper biofilms, whereas Sphingobacteriales family members (OTU 3, OTU 136) and

Terrimonas genus (OTU 24) were associated with 2l.h -1 dripper biofilms.

Discussion

The main problem with the use of the drip irrigation supplied with reclaimed wastewater is biofouling. Understanding the development of biofouling is the only way to limit the clogging of drip irrigation systems. The labyrinth milli-channel drippers favour the formation of heterogeneous flow with vortex zones. In this study, a laboratory experiment was performed to investigate the kinetics of biofouling and changes in the bacterial communities over time as a function of the hydrodynamic parameters (flow rate, cross-section) of the drippers. Three types of commercial flat drippers used in agriculture with specific channel geometry and different flow rates (1, 2 and 4 l.h -1 ) were installed on a test bench and supplied with treated urban wastewater.

Based on OCT results, the thickness and the volume of the biofouling were higher in the inlet of the channel (mostly in the first baffle) in all the drippers and close to the return areas of the labyrinths in 1 and 2 l.h -1 drippers. The biofouling at the inlet of the dripper increased over time in all three dripper types (1, 2 and 4 l.h -1 ) tested here but the 1 l.h -1 dripper type was more sensitive. Previous studies had found that clogging capacity is more influenced by the geometrie of the dripper channel than flow rate (G. Y. [START_REF] Li | Hydraulic Characterizations of Tortuous Flow in Path Drip Irrigation Emitter[END_REF]Li et al., 2019). In this study, as [START_REF] Lavanholi | Methodology to Evaluate Dripper Sensitivity to Clogging due to Solid Particles: An Assessment[END_REF] and [START_REF] Li | Formation mechanism for emitter composite-clogging in drip irrigation system[END_REF], the dripper flow path with smaller cross-section and average cross-section velocity (v), W/D or Dh 1/2 /L parameters were relatively weaker in the anti-clogging risk. Dripper flow path with larger v would locally induce a greater turbulence. Thus, the greater near-wall shear stress would accelerate the detachment of biofouling, which would facilitate the removal of the biofouling [START_REF] Ait-Mouheb | Impact of hydrodynamics on clay particle deposition and biofilm development in a labyrinth-channel dripper[END_REF][START_REF] Al-Muhammad | Experimental and numerical characterization of the vortex zones along a labyrinth millichannel used in drip irrigation[END_REF]. Previous numerical studies have shown that the greatest head loss occurs in the first inlet baffles [START_REF] Al-Muhammad | Experimental and numerical characterization of the vortex zones along a labyrinth millichannel used in drip irrigation[END_REF], 2016) and that the water velocities and turbulent kinetic energy is characterized by low values in corners of dripper channel [START_REF] Al-Muhammad | Modeling a weak turbulent flow in a narrow and wavy channel: case of micro-irrigation[END_REF]. Thus, the reduction of velocity in inlet channel can enhance the adhesion and deposition on the wall surface [START_REF] Ait-Mouheb | Impact of hydrodynamics on clay particle deposition and biofilm development in a labyrinth-channel dripper[END_REF], explaining the sensitivity to fouling of these areas. In addition, using OCT we observed the biofouling level from the 1 and 2 l.h -1 drippers tended to decreased after the inlet areas as observed by [START_REF] Ait-Mouheb | Impact of hydrodynamics on clay particle deposition and biofilm development in a labyrinth-channel dripper[END_REF] but re-increased close to the return areas. The return areas was present in the 1 and 2 l.h -1 drippers only and seemed to be sensitive to fouling. One explanation to explain the biofouling close to the return area is that the large bend modifies abruptly the flow behaviour which becomes similar to the inlet area. Studies on biofilm from straight pipe shown that these differences can influenced the mass transfert (nutrients) and so the biofilm development [START_REF] Araújo | Influence of Flow Velocity on the Characteristics of Pseudomonas fluorescens Biofilms[END_REF]. Then, CFD studies would be performed to link the biofouling accumulation and local hydrodynamic conditions.

In the present study, biofouling was monitored by OCT in each baffle of the inlet channel. The dripper biofouling alternated between up and down phases locally, mainly for the 1 l.h -1 dripper type with smaller cross-section and low flow rate. This suggests that monitoring the development of local biofouling should be considered to optimize control methods. The down phases may be linked with detachment events [START_REF] Flemming | The biofilm matrix[END_REF]. Detachment events appeared to occur more frequently in the 1 l.h -1 drippers. Several studies have shown that there is a link between biofilm development, production of extracellular polymeric substances (EPS) and hydrodynamic conditions. EPS are essential to ensure the maintenance and stability of the biofilm on a surface. Biofilms grown under low velocities are subject to lower shear stress forces which favour faster growth [START_REF] Melo | Physical stability and biological activity of biofilms under turbulent flow and low substrate concentration[END_REF]. However, biofilms subjected to low flow constraints have limited mechanical strength and are more prone to sloughing than those formed at higher flows [START_REF] Teodósio | Flow cell hydrodynamics and their effects on E. coli biofilm formation under different nutrient conditions and turbulent flow[END_REF]. In this study, EPS characterisation and quantification were not performed due to low biofilm quantity inside a dripper. However, even if the hydrodynamic forces associated to the dripper types drive the bacterial composition of the biofilm, the response could also be in the modification of the structure and composition of the biofilm matrix. Further research using OCT combined with EPS characterisation methods and with microbial composition analysis will advance our understanding of biofouling in drippers.

The structure of bacterial community of the RWW has a direct influence on the structure and composition of the dripper biofilms. Members of the Comamonadaceae family (β-Proteobacteria including Hydrogenophaga genus) dominated in the biofilm samples taken at 32 and 72 days, but were replaced by the Bacteroidetes_VadinHA17 wastewater sludge group belonging to Bacteroidetes at the end (Table S3). This gram-negative, non-mobile anaerobic group is often found in sewage treatment plant sludge and indicates that over time, bacteria commonly found in wastewater have become established and have become the majority in communities. The Comamonadaceae family, which includes many denitrifier members, has been found in wastewater treatment plants and was involved in the first phases of fouling of membrane bioreactors fed with wastewater [START_REF] Ziegler | Dynamics of the fouling layer microbial community in a membrane bioreactor[END_REF]. Other phyla such as Spirochaetae and Cholorobi appeared in the RWW over time and eventually in the biofilms (Figure 4). The RWW used in this study was obtained after treatment by lagooning and the treatment process can affect the structure and bacterial composition of biofilms.

Further research are needed to study the effect of the wastewater treatment processes on biofilm composition in irrigation lines in order to improve management of biofilm development in the drippers.

DNA concentration increased over time in the dripper biofilms whereas the bacterial concentration remained stable. This could be explained by the presence of other types of organisms as algae and eukaryotes. Moreover, bacterial predators can influence the structure and composition of the biofilm [START_REF] Böhme | Protists with different feeding modes change biofilm morphology[END_REF][START_REF] Parry | The use of ultrasonic imaging to evaluate the effect of protozoan grazing and movement on the topography of bacterial biofilms[END_REF]. As perspective, a metagenomic approach of the function of microorganisms involved in the biofilm formation and the effect of other microorganisms (algae, predators) can complete the understanding of dripper clogging.

Bacterial diversity and richness were not influenced by hydraulic parameters of the drippers. This is not consistent with [START_REF] Rochex | Role of shear stress on composition, diversity and dynamics of biofilm bacterial communities[END_REF] who shown that increased shear stress (from 0.055 to 0.27 Pa), corresponding to an increase of the Reynolds number, reduced the bacterial diversity in a Conical Couette-Taylor Reactor. Then differences between the drippers in terms of flow behaviour may not be sufficient to observe an effect on these indices. Although bacterial diversity was not influenced by the hydraulic parameters, the structure of the bacterial community was. These changes are in agreement with the results of previous studies performed on pipe and reactors [START_REF] Ai | Mechanism and kinetics of biofilm growth process influenced by shear stress in sewers[END_REF][START_REF] He | Hydrodynamic shear force shaped the microbial community and function in the aerobic granular sequencing batch reactors for low carbon to nitrogen (C/N) municipal wastewater treatment[END_REF][START_REF] Saur | Impact of wall shear stress on initial bacterial adhesion in rotating annular reactor[END_REF]. The level of biofouling decreased with an increase of the cross-section velocity (0.34, 0.61 and 0.78 m.s -1 for 1, 2 and 4 l.h -1 drippers respectively) in the channel. However the structure of bacterial communities appeared to be less affected by the cross-section velocity above 0.61 m.s -1 (2 l.h -1 dripper). Even though previous studies have shown that the increase in velocity and shear forces influence the spatial organization and structure of the bacterial community [START_REF] Saur | Impact of wall shear stress on initial bacterial adhesion in rotating annular reactor[END_REF], the differences between the 2 and 4 l.h -1 drippers may not be sufficient to influence this community in the first phases of biofilm development. This could explain similarities in the structure of the bacterial communities sampled in 2 and 4 l.h -1 drippers compared to the 1 l.h -1 dripper biofilms until 115 days, and this in spite of the flow differences due to the geometry of the labyrinths (e.g. return zone).

Proteobacteria, Bacteroidetes, Firmicutes and Chloroflexi were the most abundant phyla in all three types of dripper (Figure 4). These phyla have been already been described in drippers supplied by treated wastewater [START_REF] Lequette | Drip irrigation biofouling with treated wastewater: bacterial selection revealed by high-throughput sequencing[END_REF][START_REF] Song | The influence of chlorination timing and concentration on microbial communities in labyrinth channels: implications for biofilm removal[END_REF][START_REF] Zhou | Quantitative relationship between biofilms components and emitter clogging under reclaimed water drip irrigation[END_REF]. Over time, the relative abundance of Proteobacteria and more specifically β-Proteobacteria decreased significantly in 1 l.h -1 dripper biofilms compared to 2 and 4 l.h -1 dripper biofilms. β-Proteobacteria have been reported to be a dominant group in both drinking water biofilms [START_REF] Douterelo | Influence of hydraulic regimes on bacterial community structure and composition in an experimental drinking water distribution system[END_REF] and wastewater biofilms [START_REF] Biswas | Microbial community composition and dynamics of moving bed biofilm reactor systems treating municipal sewage[END_REF][START_REF] Ma | Microbial communities in an anaerobic dynamic membrane bioreactor (AnDMBR) for municipal wastewater treatment: Comparison of bulk sludge and cake layer[END_REF] and could play an important role in the formation of biofilm in drippers. This group can easily attach to surfaces and can withstand high water velocity and high shear force [START_REF] Douterelo | Influence of hydraulic regimes on bacterial community structure and composition in an experimental drinking water distribution system[END_REF], which may explain their dominance in drippers 2 and 4 l.h -1 .

Chloroflexi and Bacteroidetes phyla were also abundant in dripper biofilms.

Anaerolineaceae family members (Chloroflexi) were more abundant in 1 l.h -1 dripper biofilms at the end than in 2 and 4 l.h -1 dripper, meaning that Chloroflexi are sensible to the high Reynolds number. These phyla are commonly found in activated sludge of wastewater treatment plants and include filamentous bacteria as Anaerolineaceae family members (Chloroflexi). Research has shown that filamentous bacteria are essential to the formation of activated sludge but when they are over-concentrated, they are responsible for foaming and bulking events [START_REF] Kragelund | Identity, abundance and ecophysiology of filamentous bacteria belonging to the Bacteroidetes present in activated sludge plants[END_REF][START_REF] Kragelund | Identity, abundance and ecophysiology of filamentous Chloroflexi species present in activated sludge treatment plants[END_REF][START_REF] Nielsen | Identity and ecophysiology of filamentous bacteria in activated sludge[END_REF] and for fouling membrane bioreactors in wastewater treatment plants [START_REF] Li | Impact of filamentous bacteria on properties of activated sludge and membrane-fouling rate in a submerged MBR[END_REF][START_REF] Rehman | Metagenomic analysis of sludge and early-stage biofilm communities of a submerged membrane bioreactor[END_REF]. But the increase of Anaerolineaceae over time can also be explained by the interaction with other bacteria. Chloroflexi and Hydrogenophaga spp (Proteobacteria phylum), mainly found in 1 l.h -1 drippers, were also observed by [START_REF] Ziegler | Dynamics of the fouling layer microbial community in a membrane bioreactor[END_REF] who studied the biofouling of a pilot-scale membrane bioreactor fed with wastewater. From a physiological point of view, Hydrogenophaga spp. are able to synthesise polymeric substances [START_REF] Calderer | Effects of enhanced denitrification on hydrodynamics and microbial community structure in a soil column system[END_REF].

Filamentous Chloroflexi produce a complex of enzymes able to degrade EPS, in turn, enabling the cells to use EPS as substrate to maintain their activities [START_REF] Kragelund | Identity, abundance and ecophysiology of filamentous Chloroflexi species present in activated sludge treatment plants[END_REF].

EPS produced by Hydrogenophaga spp could facilitate fouling due to filamentous bacteria and flow parameters. Thus, the increase in Chloroflexi abundance over time could be explained by a sufficient EPS synthesis rate and by the presence of Hydrogenophaga spp at the early stage of biofilm formation. Thus, further research on the role of filamentous bacteria in biofouling development is required, especially by focusing on the effect of the hydraulic and geometric parameters of the flow path on their abundancy.

The divergence between the 1 l.h -1 drippers and the 2 and 4 l.h -1 drippers prevailed over time and was mainly driven by Hydrogenophaga and Pseudoxanthomonas genera (Figure 5). Although no clear information is available on the effect of hydraulic conditions on the installation of these two bacterial genera in drippers, they are often found in biofilms associated with membrane reactor fouling supplied by wastewater [START_REF] Zheng | Characteristics and formation mechanism of membrane fouling in a full-scale RO wastewater reclamation process: Membrane autopsy and fouling characterization[END_REF][START_REF] Ziegler | Dynamics of the fouling layer microbial community in a membrane bioreactor[END_REF]. Contrary to the 1 l.h -1 dripper biofilms, biofilms from the 4 l.h -1 dripper were mainly driven by Aquabacterium (Proteobacteria) and Ideonella (Proteobacteria, Comamonadaceae family) genera after 4 months. These genera were already found associated to wastewater biofilms [START_REF] Lequette | Drip irrigation biofouling with treated wastewater: bacterial selection revealed by high-throughput sequencing[END_REF][START_REF] Luo | Succession of biofilm communities responsible for biofouling of membrane bioreactors (MBRs)[END_REF][START_REF] Mccormick | Microplastic in surface waters of urban rivers: Concentration, sources, and associated bacterial assemblages[END_REF] and included species able to metabolize plasticizers used in plastics [START_REF] Kalmbach | Aquabacterium gen. nov., with description of Aquabacterium citratiphilum sp. nov., Aquabacterium parvum sp. nov. and Aquabacterium commune sp. nov., three in situ dominant bacterial species from the Berlin drinking water system[END_REF][START_REF] Tanasupawat | Ideonella sakaiensis sp. nov., isolated from a microbial consortium that degrades poly(ethylene terephthalate)[END_REF]. Thus the type of material and the hydraulic parameters of the drippers could influence the presence of bacteria with specific metabolic activities over time.

Conclusion

The combined use of OCT and high throughput sequencing highlighted the influence of hydrodynamic parameters (flow rate, cross-section) of the drippers supplied with RWW on biofilm development and bacterial communities:

1) Biofouling was facilitated close to the inlet and in the vortex zones of the dripper channels, more specifically in 1 l.h -1 drippers where the Reynolds number, crosssection and average cross-section velocity (v), W/D or Dh 1/2 /L were smaller.

2) The dripper geometry influence the hydraulic flow behaviour (inlet Reynold number and average cross-section velocity) and the structure of bacterial communities in biofilms. However, the structure of the bacterial community from the 2 and 4 l.h -1 drippers were more similar than the 1 l.h -1 dripper biofilms.

3) The relative abundance of filamentous bacteria belonging to Chloroflexi phylum was lower in 1 l.h -1 compared to 2 and 4 l.h -1 .

  .B). A screen filter (mesh size 0.13mm) was installed to reduce the physical clogging of emitters following the technical recommendations of this type of dripper. The inlet pressure was set at 0.08 MPa with a pressure gauge. A gutter was installed below each lateral line to collect the water discharged from the drippers during the experiments. The gutters returned the discharged RWW to the respective tanks, enabling the recycling of water. The lines were supplied twice a day five days out of seven for 1h, with an interval of 6h off. The outlet flow rate from the drippers was measured weekly to evaluate emitter performance. Drippers are considered clogged when there is a decrease of at least 25% in the expected flow rate. The system was not flushed during the entire four-month experiment (from April to August 2018).

Figure 1 .

 1 Figure 1. Dripper system (A, C) and test bench (B). The drippers were placed in a transparent tube to enable optical measurements. The test bench was composed of 1. a tank (60l); 2. a water pump; 3. a 0.13mm mesh screen filter; 4. a pressure reducer; 5. a

Figure 2 .

 2 Figure 2. Schematic diagram of the geometric channel from the drippers. The dotted boxes correspond to the study areas. The length and width (in mm) of each baffle are listed in the TableS1in Supplementary material.

  analyzed. Each dripper in the transparent tube was extracted with sterile clamps and placed directly in a sterile tube and stored at -20°C until DNA extraction. RWW was filtered (15 to 30 mL) each week through 0.2 µm (Supor® 200 PES Membrane Disc Filter, Pall Corporation) to analyse the bacterial community in the reclaimed wastewater. Filters were stored at -20°C until DNA extraction.

Figure 3

 3 Figure 3 Mean flow rate (and standard error) according to the dripper type. The dotted line of discharge of less than 75% corresponds to the limit usually set up to consider that a dripper is clogged. n refers to the number of dripper of the lines.

Figure 4 .

 4 Figure 4. Biofilm thickness measured using OCT method at the inlet of 1, 2 l.h -1 drippers (A) and 4 l.h -1 dripper (B) and in the return areas of drippers (C) measured after 1 and 4 months.

  analyses and were shown to change over time. Those explaining 50% of the global bacterial divergence are indicated in Figure 5. At 32 days, 11 OTUs played a significant role in bacterial community divergence. Pseudoxanthomonas (OTU 25), Hydrogenophaga (OTU 5)

Figure

  Figure 6. PCoA ordination plot of bacterial communities found in 1 l.h -1 (•), 2 l.h -1 (▲) and 4 l.h -1 (■) dripper biofilms at 32 days (A), 72 days (B) and 115 days (C). The analysis was based on Bray-Curtis similarity coefficients. Arrows indicate the orientation and contribution of the OTUs (most significant contributors explaining 50% of the global bacterial divergence).

Table 1 . Dripper parameters

 1

				Geometric parameters of the path		Hydraulic parameters
		Structure of the dripper
	Dripper	flow path	L	W	D	Dh	Volume of the path	/	q (l.h -1 )	Re	v (m.s -1 )	.x	CV (%)
				(mm)			(mm 3 )
	D1		103.4 1.01 0.8 1.02	127	1.26 0.009	1	305	0.34 0.4	1.4
	D2		94.1 1.03 0.9 1.12	142	1.14 0.011	2	579	0.60 0.4	1.9
	D3		47.4 1.35 1.1 1.17	129	1.23 0.023	4	926	0.78 0.4	2.5

Table 2 . Physico-chemical characteristics of the RWW

 2

	Each week (n=16), several physical-chemical and
	microbiological analyses were performed to evaluate the RWW quality. Chemical oxygen
	demand (COD), ammonia, nitrate, and phosphorus concentrations (mg l -1 ) were measured
	with a spectrophotometer (DR1900, Hach Company, Loveland, CO, USA) using Hach
	reagents ® . Conductivity and pH were measured with probes (TetraCon ® 925 and pH-

Table 3 . DNA concentration and quantification of bacteria by qPCR in dripper biofilms

 3

	Sampling	Dripper	Mean DNA	Mean of bacteria
	time	flow rate	concentration	/dripper
	(days)	(l.h -1 )	(µg/µl)	(16S copies/dripper)
		1	137.4±67.9 a	6.2±0.9 x 10 9a
	32	2	113.9±42.7 a	5.7±0.6 x 10 9a
		4	118.1±26.8 a	9.9±1.2 x 10 9a
		1	220.4±34.6 a	9.2±0.5 x 10 9a
	72	2	287.4±113.8 a	1.2±0.05 x 10 10a
		4	194.4±20.2 a	4.3±2.3 x 10 9a

Table 4 . Estimated species richness and diversity indices according to the dripper flow rate and the RWW Richness indices Diversity indices Estimated OTUs Samples Flow rate (l.h -1 ) Age (Days) Observed Chao1 Shannon 1/Simpson

 4

			32	786 ± 36 a	786 ± 36 a	4.7 ± 0.1 a	40 ± 7 a
		1 (n=3)	72	1215 ± 18 b	1215 ± 18 b	5.5 ± 0.1 b 88.4 ± 15.8 b
			115 1640 ± 135 c	1640 ± 135 c	5.9 ± 0.1 c 149.5 ± 17.4 c
			32	752 ± 61 a	752 ± 61 a	4.7 ± 0.1 a 42.3 ± 2.7 a
	Dripper	2 (n=3)	72	1286 ± 188 b	1286 ± 188 b	5.5 ± 0 b 90.3 ± 6.6 b
			115	1758 ± 92 c	1758 ± 92 c	5.8 ± 0.1 c 114.2 ± 9.4 c
			32	783 ± 54 a	783 ± 54 a	4.6 ± 0.1 a 40.4 ± 7.7 a
		4 (n=3)	72	1360 ± 23 b	1360 ± 23 b	5.6 ± 0 b 106.7 ± 3.9 b
			115	1726 ± 30 c	1726 ± 30 c	5.8 ± 0 c 116.6 ± 3.3 c
	RWW (n= 11)		-	836 ± 310	836 ±310	4.2 ± 0.7 32.6 ± 22.9

Acknowledgements

The authors gratefully acknowledge the financial support of the French Water Agency, project "Experimental platform for the reuse of reclaimed wastewater in irrigation, Murviel-lès-Montpellier". We thank Guillaume Guizard (LBE-INRAE, Narbonne, France) and Jean-François Bonicel (UMR G-Eau-INRAE, Montpellier, France) for their contribution to the development of the dripper system, Geoffrey Froment (UMR G-Eau-INRAE, Montpellier, France), François Liron (UMR G-Eau-INRAE, Montpellier, France) and Marine Muffat-Jeandet (UMR G-Eau-INRAE, Montpellier, France) for their contribution to the development of the test bench, Annabelle Mange (UMR G-Eau-INRAE, Montpellier, France) and Valérie Bru-Adam (LBE-INRAE, Narbonne, France) for their assistance in the field and at laboratory.

Water velocity evolves along the channel with low velocities at the inlet and in the vortex zones. These changes can influence the transport of nutrients to the biofilm along the channel.

Further studies on nutrient transport and on the structure of the bacterial community of the biofilms along the drippers should increase our understanding of fouling phenomena in sensitive areas. Another point is that biofouling can include physical and chemical processes.

In this study, the physico-chemical composition of the biofouling was not analysed. Exploring the link between the different types of clogging (physical, chemical and biological) in different dripper types is an important future research path

Disclosure statement

No potential conflict of interest was reported by the authors. Authors have approved the final article.


MD, MPH Ardalan Sharifzadehgan

MD Marc Laurans

MD Marine Thuillot

MD Andres Huertas

Pierre Baudinaud

Kumar Narayanan

MD Mariana Mirabel

MD Jean-Emmanuel Bibault

Pierre Frey

Victor Waldmann

MD, MPH Emilie Varlet

Denis Amet

MD Christophe Juin

MD Thomas Lavergne

MD Xavier Jouven

MD Philippe Giraud

MD Catherine Durdux

MD Eloi Marijon
email: eloi_marijon@yahoo.fr

Radiotherapy in Patients With a Cardiac Implantable Electronic Device

Keywords: Radiotherapy, Cardiac Implantable Electronic Device, Remote monitoring, Risk stratification

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Because of substantial progress in the fields of both cardiology and oncology, the prevalence of patients with cardiac electronic implantable device (CIED), such as pacemaker (PM) or implantable cardioverter-defibrillator (ICD), who also require radiotherapy (RT) for cancer is now increasing dramatically. 1 2 3 Globally speaking, out of the 3.4 million new cancer cases diagnosed in European countries in 2012, half would have received at least 1 course of RT. [START_REF] Borras | The optimal utilization proportion of external beam radiotherapy in European countries: An ESTRO-HERO analysis[END_REF] Although not addressed in the most recent cardio-oncology expert consensus and guidelines, RT can affect significantly the electronic components of CIED resulting in malfunction and/or damage, especially among those who are totally pacing dependent or at high-risk of ventricular arrhythmias. This involves a substantial concern in daily practice requiring a multidisciplinary discussion to balance benefits and risks of RT. 5 6 Since the first guidelines published by the American Association of Physicists in Medicine in 1994, several have been published and more recently the Heart Rhythm Society (HRS) (2017). 7 8 9 10 In the present paper, we aimed to report the experience of a tertiary multidisciplinary teaching hospital, and discuss our practice in the context of recently published guidelines.

Methods

This was a retrospective observational monocentric study, carried out at the European Georges Pompidou Hospital (Paris, France), between March 2006 and June 2017. We identified all consecutive patients who underwent RT, with a PM or an ICD. Detailed information on RT plan, as well as CIED characteristics at baseline and during regular followup were collected.

Regarding RT plan, information on the tumor location, the number of fractions, the type of external beam radiation (photons, electrons or rarely protons), and energy range (units of megavolt, MV) were collected. A radiation course can range from a single fraction to 8-9 weeks of daily treatment, depending on the condition being treated. Currently, RT is mainly delivered by a medical linear accelerator (LINACS), using photons and/or electrons beams.

Secondary neutron producing beam includes 15-or 18 MV photons, whereas non-neutron producing include 6-or 10 MV photons, electrons only, and Cyberknife TM . Cumulative dose to the tumor and at the CIED were both estimated prior to RT. The unit of measurement for absorbed radiation dose (i.e., energy deposited) is the Gray (1 Gy = 1 J of absorbed energy from ionizing radiation per 1 kg of matter). When multiple regions were irradiated, the maximal prescription dose and associated fractionation were recorded. For clinical purposes, we divided the radiation exposure into 3 zones: zone 1 includes the head and the neck, zone 2 includes chest and pectoral region, zone 3 includes abdomen and pelvic regions.

Regarding CIED information, data collected at baseline (pre-RT) included the type of device (PM, ICD), device location, device manufacturer, the number of leads, the primary indication for implantation, PM dependency (defined as intrinsic rhythm ≤ 30 bpm), and history of ventricular arrhythmias (for ICD recipients). 11 12 All CIED follow-up data were systematically collected from medical reports, including overlying skin's condition, frequency of device interrogation, battery characteristics (residual voltage and impedance), lead parameters (impedance and pacing/sensing thresholds), and arrhythmia events using electrogram recording when available. Malfunction of the device included lead dysfunction, premature battery depletion, CIED reset in back-up mode or complete device failure, and inappropriate ICD therapy (as shocks or anti-tachycardia pacing).

We established a local protocol in 2006 in order to improve patient safety during RT. 13 14 All patients were systematically evaluated prior to RT initiation including CIED interrogation. CIED patients were classified as low-, intermediate-or high-risk.

Low-risk patients were those that fulfilled all of the 3 following criteria: (i) estimated cumulative dose to CIED <2 Gy; (ii) intrinsic rhythm > 30 bpm (i.e., not pacing dependent) and (iii) without an ICD. Patients deemed as being intermediate-risk did not fulfil one of the former criteria but had no high-risk criteria. We classified as high-risk, patients whose cancer was in close proximity to the CIED device and/or receiving a cumulative CIED dose ≥ 5 Gy.

In high-risk patients, repositioning of the device prior to RT was considered systematically.

After CIED repositioning, patients could be subsequently reclassified, as low-or intermediate-risk. Monitoring protocols differed according to risk stratification.

During RT sessions, it was recommended that all patients have audiovisual and ECG monitoring as well as physicians' presence. Furthermore, a cardiologist or CIED clinician was available within a 10 minutes reach and an automatic external defibrillator was available on site. In addition, for intermediate-and high-risk patients, it was advised to systematically apply a magnet over the CIED to avoid any external interference leading to pacing inhibition or inappropriate ICD therapy, as well as to perform an ECG before and after each session.

Regarding follow-up, CIED interrogation was planned to be performed at the end of the RT treatment course, and at 1, 3, and 6 months post-RT follow-up visits. In addition, for intermediate-and high-risk patients, a systematic device interrogation was planned on a weekly basis regardless of the RT scheme.

Categorical data were reported as numbers and proportions. Continuous data were reported as mean ± standard deviation (SD) or median [IQR], when appropriate. All data were analyzed using R Project for Statistical Computing software (v 3.3.2). The authors had full access to and take full responsibility for the integrity of the data.

Results

Over the 11-year period, a total of 12,736 patients underwent RT in our center. Ninety (1%) patients had a CIED, including 82 PMs and 8 ICD. Principal indications for pacing were high-level atrioventricular block in 23 (31%), and sinus node dysfunction in the remaining patients. (Table 1)

Prior to RT and according to our local protocol, 50 (56%) patients were assessed as being at low-risk of CEID dysfunction, 35 (39%) were classified as intermediate-risk and 4 (5%) patients were considered as high-risk. Two patients were originally classified in highrisk group, requiring PM extraction prior to RT for ipsilateral breast cancer. In 1 of those 2 patients, device replacement was not deemed necessary and was then excluded of the evaluation; the PM had been initially inserted for sinus node dysfunction with subsequently permanent atrial fibrillation with preserved atrioventricular conduction. The second patient required contralateral CIED reimplantation prior to RT, and was then reclassified in the intermediate-risk group. All patients in high-risk group received a cumulative dose to the CIED device of more than 5 Gy.

Overall, the median total dose delivered to the tumor was 49.5 [31.5;66.0] Gy with a median cumulative dose to the CIED device was 0.0 [0.0;0.7]. Neutron producing beams were delivered in 49 (55%) patients and non-neutron producing beams in 40 (45%). In the latter group, 1 received electron-only treatment, 2 received a combination of photons and electrons, and 2 received Cyberknife TM . Systematic cardiac monitoring during RT delivery was provided for all patients, and electrocardiograms (before and after radiation exposure, as recommended in the local policy) during RT were performed in 12 of the intermediate-and high-risk patients (31%). Only 3 patients in the intermediate-risk group and none in the highrisk group received magnet over the device during the RT sessions (3 out of 39, 8%). No significant clinical event was detected during and immediately after RT delivery.

CIED evaluation was available for 87 patients (97%). Overall, 56% had appropriate surveillance during follow-up. In high-and intermediate-risk groups, weekly CIED interrogations during RT were carried out as recommended by our local policy in 22 out of 39 (56%) patients. In addition, 20 of the 50 low-risk patients (40%) were followed on a weekly basis, though not required by our local protocol.

The follow-up at 1 month was available in 73 patients (91%), at 3 months in 66 (87%), at 6 months in 65 (81%) and at 12 months in 55 (79%). Overall, CIED malfunction occurred in 5 patients (6%), after a mean period of 21.5 ± 16.7 days from the start of RT. Of the 5 patients with CIED malfunction, 1 had been classified as low-risk and 4 as intermediate-risk.

None of them had permanent device damage (Table 2). CIED reset to back-up mode was observed in 4 cases, symptomatic in only 1 case (exercise-induced dyspnea). Where weekly CIED interrogation was carried out (cases 1, 3 and 4), the evaluations preceding the malfunction diagnosis were normal. During the median follow-up of 24 [3;102] months, no case of sudden cardiac death was reported in the entire population.

Discussion

In our series, we observed that severe CEID malfunction during and after RT were infrequent. The most frequent event was a resetting in back-up mode which was most often asymptomatic. Our findings also illustrate the inadequate adherence of physicians to local policies to detect potential adverse events in a timely fashion, and support the need for a more practical protocol as suggested by the recent 2017 HRS recommendations.

Several factors influence CIED malfunction associated with RT delivery. First, maximum cumulative dose to the CIED device is important and cut-offs have been proposed since the 1980s, with a 2 Gy threshold mentioned in the first guidelines supported by the American Association of Physicists in Medicine in 1994. 9 10 13 15 In line with our findings, where majority of CIED malfunctions occurred with very low cumulative dose delivered to the device, the 2 Gy cut-off remains debated and is no longer part of the recent HRS recommendations. [START_REF] Indik | HRS expert consensus statement on magnetic resonance imaging and radiation exposure in patients with cardiovascular implantable electronic devices[END_REF] Second, a correlation between dose rate (defined as the absorbed dose per unit of time, Gy/min) and CIED malfunction has been proposed by Mouton et al. [START_REF] Mouton | Influence of high-energy photon beam irradiation on pacemaker operation[END_REF] Nevertheless, Rodriguez et al. reported dysfunctions due to dose rate to be mostly transient and reversible. [START_REF] Rodriguez | Radiation-induced effects in multiprogrammable pacemakers and implantable defibrillators[END_REF] Third, more recent studies have focused on the importance of stochastic effects related to interactions with ionized particles, with especially neutrons produced from high energy beams (energy > 10 MV) recently being identified as the strongest predictor of dysfunction. 18 19 20 21 Neutrons are generated mainly in LINACS head through interactions of photons with nuclei of high atomic number materials. [START_REF] Naseri | A review on photoneutrons characteristics in radiation therapy with high-energy photon beams[END_REF] Grant et al. reported in a retrospective clinical study that all CIED malfunctions occurred with high-energy particles producing neutrons. [START_REF] Grant | Radiotherapy-Induced Malfunction in Contemporary Cardiovascular Implantable Electronic Devices: Clinical Incidence and Predictors[END_REF] Although counter intuitive, the occurrence of CIED malfunction in patients undergoing pelvic RT, by definition far from the device, is explained by the secondary production of neutrons with high-energy particles. [START_REF] Grant | Radiotherapy-Induced Malfunction in Contemporary Cardiovascular Implantable Electronic Devices: Clinical Incidence and Predictors[END_REF] In our study, 3 out of the 5 patients with CIED malfunctions had pelvic RT with 18 MV photon beam. Finally, in addition to the direct effect of radiation, the level of electromagnetic interference may play a role in the occurrence of transient oversensing, which could cause pacing inhibition or inappropriate ICD therapies (not observed in our study); however, this mechanism appears to be rare.

The infrequency of CIED malfunction (6%) reported in our study is in line with the largest published series of CIED patients undergoing RT. [START_REF] Zaremba | Radiotherapy in patients with pacemakers and implantable cardioverter defibrillators: a literature review[END_REF] CIED malfunctions with a median absorbed dose of 1.0 Gy [2.90;29.5]. 19 21 Several national guidelines have been proposed in different countries. 3 8 9 24 25 26 Although existing algorithms differ to variable extents, the main criteria usually considered for risk stratification are (i) location of the CIED, (ii) maximal cumulative dose to the CIED (usually > 2 Gy), (iii) pacing dependency or presence of an ICD. In these patients, continuous monitoring during RT delivery is usually recommended with an CIED clinicians available within a 10 minutes reach, as well as weekly CIED interrogation during follow-up. [START_REF] Hurkmans | Management of radiation oncology patients with a pacemaker or ICD: a new comprehensive practical guideline in The Netherlands. Dutch Society of Radiotherapy and Oncology (NVRO)[END_REF] Recently, Indik et al. under the auspices of the HRS have published a North-American consensus statement for better standardization. [START_REF] Indik | HRS expert consensus statement on magnetic resonance imaging and radiation exposure in patients with cardiovascular implantable electronic devices[END_REF] Similar to previous guidelines, CIED relocation is not recommended for devices expected to receive a maximum cumulative incident dose < 5 Gy. By contrast, the energy beam is considered very early in the risk stratification algorithm (Figure 1). A non-neutron producing treatment is preferred, and if not possible, RT should be performed with asynchronous pacing (in pacing dependent patients) and/or ICD therapies deactivated and a weekly CIED interrogation during the course of RT is recommended. 8 24 Authors have published a checklist for allowing both electrophysiologists and RT physicians to better evaluate patient risk prior to RT (Figure 3). Considering our data, 62 (69%) patients would have required weekly follow-up according to 2017 HRS recommendation versus the 39 (43%) recommended using our local policy.

With regard to these current guidelines, availability of CIED clinicians and the public health resources needed for implementation in routine clinical practice need to be considered.

In fact, a more sensitive risk stratification with frequent device monitoring could increase the workload for CIED clinicians and pose difficult organizational challenges for both cardiology and RT departments. This would explain why half of all patients in the intermediate-risk group in our study were not followed weekly as recommended while, a high proportion of patients in the low-risk group were followed closer than recommended. Bravo-Jaimes et al.

suggested recently that all CIED malfunctions could have been identified with remote monitoring. [START_REF] Bravo-Jaimes | CIED malfunction in patients receiving radiation is a rare event that could be detected by remote monitoring[END_REF] In the future, remote CIED monitoring appears to be a safe solution for patients requiring weekly CIED interrogation, simplifying and optimizing the follow-up for patients and clinicians without increasing significantly their workload.

Although observational data on CIED and RT are scarce, we need to acknowledge several limitations of our study. First, it is an observational retrospective study. Second, it is a single center experience providing data on a relatively small number of patients with an even smaller event rate, not allowing further analysis of factors associated with CIED malfunction.

Finally, some proportion of patients were lost to follow-up. However, no sudden cardiac death occurred, making undetected late severe CIED malfunction unlikely.

In conclusion, our findings underline the lack of rigorous monitoring and follow-up of CIED patients undergoing RT, although CIED malfunction remains a rare event, relatively benign in nature. Optimization of local policies, especially the consideration of the energy beam type, could potentially simplify monitoring without compromising patient safety. In this setting, a close collaboration between CIED clinicians and radiation oncologists, and finally, more systematic use of remote CIED monitoring may be of substantial value.

Figure 1 .

 1 Figure 1. Comparative Risk Stratification Algorithm for patients with CIED undergoing RT. Left Panel: Algorithm at our Hospital. Right Panel: HRS 2017 Guidelines 10

Figure 2 .

 2 Figure 2. Checklist prior to radiation treatment (from HRS 2017 Guidelines) 10

Figure 1 . 1 Figure 2 .

 112 Figure 1. Comparative Risk Stratification Algorithm for Patients with CIED Undergoing RT. Left Panel: Algorithm at our Hospital. Right Panel: HRS 2017 Guidelines 10

  19 21 Zaremba et al. reported 3.1% CIED malfunctions, most frequently transient resetting, and Brambatti et al. reported 1.5%

Table 1 . Patients CIED and RT characteristicsTable 2 . CIED and RT characteristics in patients with CIED malfunction

 12

Table 1 . Patients CIED and RT characteristics

 1

	Variable