M Petris

F Naldini

P Pellegrini
email: paola.pellegrini@univ-eiel.fr

R Pesenti
email: pesenti@unive.it

A dynamic decomposition approach for the real-time Railway Trac Management Problem

Keywords: railway trac management, real-time scheduling, problem decomposition

INTRODUCTION

Railway services are operated following a predened timetable. However, their execution is often perturbed by unexpected events that make this timetable infeasible. Delay caused by these events is named primary delay, and it implies that trains occupy tracks at times that are dierent from the planned one. Depending on trac and track layout, these late occupations may bring to conicts, in which at least one train must slow down or even stop to preserve safe separation. This slowing down generates secondary delay, which may quickly propagate in the network. Dispatchers can take actions to limit delay propagation, as train rerouting and rescheduling. They mostly do so manually. Several optimization approaches have been proposed in the literature to tackle this problem and support dispatchers [START_REF] Cacchiani | An overview of recovery models and algorithms for real-time railway rescheduling[END_REF]. This problem is named real-time Railway Trac Management Problem (rtRTMP). The great majority of the existing approaches either focus on geographically limited infrastructures represented microscopically [START_REF] D'ariano | An Advanced Real-Time Train Dispatching System for Minimizing the Propagation of Delays in a Dispatching Area Under Severe Disturbances[END_REF][START_REF] Krasemann | Design of an eective algorithm for fast response to the rescheduling of railway trac during disturbances[END_REF][START_REF] Pellegrini | RECIFE-MILP: An Eective MILP-Based Heuristic for the Real-Time Railway Trac Management Problem[END_REF], or merge some microscopic aspect to a macroscopic representation to deal with large infrastructures [START_REF] Lamorgese | An Exact Decomposition Approach for the Real-Time Train Dispatching Problem[END_REF]. One of the main challenges of the microscopic based approaches is their computational performance on large-scale networks. To deal with this challenge, few papers try to coordinate trac management decisions made on several microscopic parts of infrastructures [START_REF] Corman | Optimal inter-area coordination of train rescheduling decisions[END_REF], or on a somehow obtained decomposition of the overall problem. For example, [START_REF] Luan | Decomposition and distributed optimization of real-time trac management for large-scale railway networks[END_REF] presents various approaches to dene decompositions a priori and to force the various trac management decisions to be coherent. A rather dierent problem conception is proposed by [START_REF] Van Thielen | Considering a dynamic impact zone for real-time railway trac management[END_REF]: the decomposition of the problem changes with trac evolution. Specically, the paper denes an algorithm based on the so-called dynamic impact zone. As soon as a conict is detected, rst rerouting possibilities for the two involved trains are assessed. If no rerouting possibility exists to eliminate conicts, the further conicts that will be generated by the possible resolutions of the considered one are identied and included in the dynamic impact zone. The conict resolution is then decided by assessing its consequence in terms of delay propagation in the dynamic impact zone if rst-come-rst-served is applied.

In this work, we propose a neighborhood-based trac management algorithm, following to some extent the problem conception of [START_REF] Van Thielen | Considering a dynamic impact zone for real-time railway trac management[END_REF]. We model the infrastructure microscopically. Our algorithm consists in making asynchronous trac management decisions. Asynchronous decisions are made also in [START_REF] Sasso | The Tick Formulation for deadlock detection and avoidance in railways trac control[END_REF], where train movements are split in so called temporal ticks to assess the presence of deadlocks in a single track network with passing loops. In our case, these decisions allow trains to reach their destination aiming at the minimization of delay propagation. Specically, we identify the neighborhood of a reference train whenever a decision is to be made on its route or on the precedence with respect to another train. This neighborhood includes only the trains that may use track sections in common with the reference one, in the vicinity of the latter's current location and in the very near future. Trac is then managed by applying an optimization approach as the one by [START_REF] Pellegrini | RECIFE-MILP: An Eective MILP-Based Heuristic for the Real-Time Railway Trac Management Problem[END_REF], only considering trains in the neighborhood and the identied possibly common track sections. By doing so, alternative routes are taken into account, but they are as short as possible. This allows limiting the size of the instance and hence the optimization time. Moreover, whenever possible, it avoids making decisions that may have to be modied in the future, when the neighborhood is recomputed. However, routes must be long enough to guarantee that no deadlocks occur right out of the neighborhood due to the decisions made here. The algorithm starts identifying the concerned trains and their shortest but long enough routes. Then, it follows the principle that has proved to be successful in [START_REF] Pellegrini | RECIFE-MILP: An Eective MILP-Based Heuristic for the Real-Time Railway Trac Management Problem[END_REF], simultaneously optimizing routes and schedules. We theoretically show that, in networks with some specic characteristics, this algorithm guarantees the achievement of a deadlock-free network-level solution, if it exists.

MODELING PRINCIPLES

We denote by BS and T C the set of block sections and track-circuits composing the infrastructure, respectively. We consider a set T of n trains traveling in the network. They may use dierent routes to reach their nal destination: the set of the available routes for train t is denoted by R t . A train makes a decision upon alternative routes once it has reserved the block section where the switch that gives rise to the alternatives is located. We call route decision block section (RDBS) a block section where such decision is to be made: in a three-aspect system, it is the block section such that a further one exists between it and the block section including the switch. Given a route r ∈ R t , a sub-route r bs→bs ′ of r is the sequence of block sections of r from bs to bs ′ , where bs and bs ′ are block sections of r such that bs has to be traversed before bs ′ when traveling along r. To indicate that a sub-route is available for train t, we will write r bs→bs ′ ⊆ r ∈ R t . Moreover, given a subset of routes R ′ ∈ R and a subset of block sections BS ′ ∈ BS, we denote by Ext(R ′ , BS ′ ) the set of block sections composing sub-routes r bs→bs ′ ⊆ r, r ∈ R ′ , where bs ∈ BS ′ and bs ′ ∈ BS \ BS ′ is the next RDBS on r. As an example, consider Figure 1: let R ′ contain routes r ′ and r ′′ and BS ′ the block sections up to the one identied by the pair of signals s1 and s2. Set Ext(R ′ , BS ′ ) includes all the block sections along r ′ until the RDBS identied by s3 and s4 and those along r ′′ until the RDBS identied by s5 and s6.

TRISTAN XI Symposium

Original abstract submittal We describe railway trac as a discrete event system. Events occur at the latest times at which route or precedence decisions must be made. They are the situations in which a train must exit a RDBS. Hereinafter, with a little abuse of notation, when we write time k we understand the time instant of the occurrence of event k. The state of the network at time k = 0, 1, 2, . . . is identied by: (i) the vector p(k) = [p t (k) : t ∈ T ] of the positions of all trains, that is the last block section reserved by t; (ii) the set Y (k) = {y t,t ′ ,tc (k) : t, t ′ ∈ T, tc ∈ T C} of the precedences previously dened and, hence, in force between times k -1 and k, on common track-circuits. Specically, a value y t,t ′ ,tc ∈ Y (k) is set equal to 1 (respectively to 0) if train t is planned to use tc before (respectively after) train t ′ . If no precedence has been xed yet, y t,t ′ ,tc is undened. At a certain time k, the system is in its nal state if all the trains reserved or crossed their nal destination.

To conclude the section, let us introduce some sets of block sections that will be exploited in our algorithm (Section 3). Given the state of the system at time k, let S t (k) be the set of block sections that must be considered in the trac management decisions involving train t. These are the block sections claimed by t at k. They are the ones that t may use between p t (k) and further RDBSs. Given S t (k) and a route r ∈ R t such that p t (k) ∈ r, we denote bs(r, S t (k)) the last block section in S t (k) that t encounters when traveling along r. We denote by R t (k) = {r ∈ R t : p t (k) ∈ r} the set of routes available for t at k and by BS(S t (k)) the set of all bs(r, S t (k)). For example, in Figure 1, position p t (k) of t is the block section identied by signals s1 and s2. Assuming S t (k) contains all block sections shown in the gure, including those derived by the extension discussed there, then R t (k) = {r ′ , r ′′ }. Here, bs(r ′ , S t (k)) and bs(r ′′ , S t (k)) are, respectively, the ones identied by pairs of signals (s3, s4) and (s5, s6).

SOLUTION ALGORITHM

The algorithm is based on a dynamic decomposition of the problem. Specically, at time k, train t for which decisions are to be made is identied. Its neighborhood is dened and the rtRTMP is locally solved by iteratively calling the optimization algorithm RECIFE-MILP. The neighborhood constitutes a sub-instance of the overall problem. A sub-instance is dened by a pair (Q t (k), S(k)), where Q t (k) ⊆ T is a subset of trains and S(k) ⊆ BS is the subset of block sections claimed by the trains in Q t (k), i.e., those along which they may travel.

The procedure to determine a short-term strategy, i.e., a local solution to manage trac when event k is triggered for train t is outlined in Algorithm 1. To lighten the notation, we drop the dependency on k when possible. In the initialization phase the algorithm takes a snapshop of the entire network. Given the positions of all trains t ′ ∈ T , it determines sets S t ′ of block sections claimed by t ′ (lines 2-4). Subset of trains Q t is set to be equal to the singleton containing t. Each iteration starts with a call to function Sub-instance_generating_procedure, where some easy extensions of sub-instance (Q t , S) are performed in order to avoid trivially infeasible iterations (line 7). For example, trains in T \ Q t sharing claimed block sections with trains in Q t are included in Q t . Then, we call RECIFE-MILP to determine a short-term strategy ST S for sub-instance (Q t , S) (line 8). The set of previously established precedences Y (k) is also given as input to RECIFE-MILP. If a non-empty short-term strategy ST S is found, the algorithm stops and returns ST S (line 13). Otherwise, we try to further extend the sub-routes of some trains TRISTAN XI Symposium Original abstract submittal

Figure 1

 1 Figure 1 Graphical representation of relevant sets of block sections: round brackets show set BS ′ , curly brackets indicate set Ext(R ′ , BS ′ ) for train t having routes r ′ and r ′′ available.

5

set Qt := {t}, S := t ′ ∈Q t S t ′ and ST S := ∅; 6 while ST S = ∅ do 7

(Qt, S) := Sub-instance_generating_procedure(Qt, S);

(lines 10-12). If an extension is possible we start the next iteration. Otherwise, the algorithm stops and we return failure. We observe that in the worst-case scenario the sub-instance may be extended to consider the entire network and all the trains travelling on it. Finally, this algorithm enjoys an important property if the network of interest can be modeled as a series-parallel graph [START_REF] Dun | Topology of series-parallel networks[END_REF]:

Theorem 1 The algorithm always allows to reach the nal state of the railway trac system on a network that can be modeled as a series-parallel graph, if at all possible.

In the paper, we will formally prove the validity of this theorem and we will provide a proof-ofconcept of the applicability of the algorithm.


Sufang Li

Andrea Fanesi

Thierry Martin

Filipa Lopes
email: filipa.lopes@centralesupelec.fr

Physiological transition of Chlorella vulgaris from planktonic to immobilized conditions

Keywords: Acclimation, Chlorella vulgaris, Immobilized state, Physiological transition, Planktonic state, Microalgae biofilms

Biofilm-based technologies present many advantages (e.g., higher productivity, lower water demand and harvesting costs) compared with the conventional planktonic approaches for microalgae cultivation. A better understanding of photosynthetic biofilm formation is though still needed in order to develop and run biofilm-based systems at large-scale. In this study, for the first time the physiological transition of C. vulgaris cells from planktonic to immobilized state was tracked (cell number, morphology, photosynthetic performance, and cellular composition) during the first 24 hours of immobilization. The results clearly confirmed that microalgae rapidly respond to immobilization via physiological adjustments. Over very short time-scales (3 hours), cells used photosynthesis to grow (increase in size, 140%), at expense of cell division, and adjusted their carbon allocation patterns (increase in the relative carbohydrates pool, 135%). The experiments confirmed that this behavior is specific to cells in the immobilized state. Triggering factors such as water and/or nutrient availability may be responsible for this fast acclimation process. Additionally, lipid content doubled by the end of cultivation, possibly due to imbalanced carbon and nitrogen metabolisms. These results offer new insights for understanding the mechanisms involved in microalgae biofilm formation and development, further helping the operators to optimize the biofilm-based systems.

Introduction

The use of biofilm-based technology for microalgae cultivation has recently become a growing field of research due to their advantages in comparison to conventional photobioreactors or raceways. Indeed, the possibility to grow microalgae attached to a substrate reduces the overall water requirements and the energetic expenses related to biomass harvesting and drying, making the cultivation process more cost-effective. Nevertheless, their use at the commercial scale is still limited. Understanding the mechanisms of biofilm development and physiological adjustments during the cultivation phases will certainly help establishing standard operative procedures, and ultimately setting up this emergent technology at large scale.

At present, most of the knowledge we have about physiological responses to external perturbations in microalgae comes from the phytoplankton. Typical response mechanisms include, depending on the time scales, regulation (over minutes) which describes the adjustments of catalytic efficiency of enzymes that occurs without net synthesis or breakdown of macromolecules, and acclimation (over hours or days) that involves a whole macromolecular reorganization at the cell level [START_REF] Halsey | Phytoplankton strategies for photosynthetic energy allocation[END_REF][START_REF] Raven | Adaptation, acclimation and regulation in algal photosynthesis[END_REF]. Both strategies have been extensively described for microalgae cultures in suspension [START_REF] Raven | Adaptation, acclimation and regulation in algal photosynthesis[END_REF], however these mechanisms are poorly studied in biofilm-based systems [START_REF] Mantzorou | Microalgal biofilms: A further step over current microalgal cultivation techniques[END_REF][START_REF] Wang | Biofilm based attached cultivation technology for microalgal biorefineries -A review[END_REF]. Indeed, it is possible that the typical acclimation processes described for suspended cultures could not fit when microalgae live in sessile mode, as cells may present different response strategies as a function of their lifestyle [START_REF] Wang | The difference in effective light penetration may explain the superiority in photosynthetic efficiency of attached cultivation over the conventional open pond for microalgae[END_REF][START_REF] Zhuang | Differences between attached and suspended microalgal cells in ssPBR from the perspective of physiological properties[END_REF].

In biofilms, microalgae cells are enclosed in an extracellular scaffold made of several polymers and water [START_REF] Fanesi | The architecture of monospecific microalgae biofilms[END_REF][START_REF] Flemming | The eps matrix: the "house of biofilm cells[END_REF], and this complex architecture creates a completely different microenvironment from that of their planktonic analogs that are typically surrounded by a less variable environment [START_REF] Zhuang | Differences between attached and suspended microalgal cells in ssPBR from the perspective of physiological properties[END_REF][START_REF] Toninelli | Scenedesmus dimorphus biofilm: Photoefficiency and biomass production under intermittent lighting[END_REF]. Gradients of nutrients and light, bulk hydrodynamics or humidity are already known to affect biofilm development [START_REF] Huang | Comparison of Chlorella vulgaris biomass productivity cultivated in biofilm and suspension from the aspect of light transmission and microalgae affinity to carbon dioxide[END_REF][START_REF] Murphy | Flux balancing of light and nutrients in a biofilm photobioreactor for maximizing photosynthetic productivity[END_REF][START_REF] Podola | Porous substrate bioreactors: a paradigm shift in microalgal biotechnology?[END_REF][START_REF] Shiratake | Air-drying of cells, the novel conditions for stimulated synthesis of triacylglycerol in a green alga, Chlorella kessleri[END_REF]. Initial adhesion, which may last several hours or days, is a key step affecting biofilm formation [START_REF] Carbone | Evaluating microalgal attachment to surfaces: a first approach towards a laboratory integrated assessment[END_REF][START_REF] Sekar | Laboratory studies on adhesion of microalgae to hard substrates[END_REF] and dramatic changes in physiological states may occur within this period in which planktonic cells need to rapidly acclimate to the new environmental conditions [START_REF] Meng | Effect of membrane blocking on attached cultivation of microalgae[END_REF][START_REF] Jungandreas | The Acclimation of Phaeodactylum tricornutum to blue and red light does not Influence the photosynthetic light reaction but strongly disturbs the carbon allocation pattern[END_REF]. From a process point of view, the inoculum step (i.e., in biofilms the adhesion of the cells) is of paramount importance to ensure healthy and productive biofilms.

Up to date, information regarding the cellular adjustments occurring during a transition from the planktonic state to biofilm have been reported only for bacteria [START_REF] Chua | Bis-(3′-5′)-cyclic dimeric GPM regulates antimicrobial peptide resistance in Pseudomonas aeruginosa[END_REF][START_REF] Qayyum | Protein translation machinery holds a key for transition of planktonic cells to biofilm state in Enterococcus faecalis: A proteomic approach[END_REF]. Some works tried to decipher the driving force behind the lifestyle transition from the perspective of molecular pathways, like the expression of biofilm-associated genes or proteins [START_REF] Qayyum | Protein translation machinery holds a key for transition of planktonic cells to biofilm state in Enterococcus faecalis: A proteomic approach[END_REF][START_REF] Waite | Transcriptome analysis of Pseudomonas aeruginosa growth: comparison of gene expression in planktonic cultures and developing and mature biofilms[END_REF]. In a previous study [START_REF] Jefferson | What drives bacteria to produce a biofilm?[END_REF], the authors listed a battery of genes implicated in cell motility, adhesion and quorum sensing that are essential players in biofilm formation. Also, phenotypic changes in e.g. cell morphology, cell surface structures and organelles (such as flagella, pili and appendages) participating in the biofilm formation were also observed from free-living to surface-attached cells [START_REF] O'toole | Biofilm formation as microbial development[END_REF][START_REF] Kodjikian | Biofilm formation on intraocular lenses by a clinical strain encoding the ica locus: a scanning electron microscopy study[END_REF][START_REF] Zhang | Morphological observation and comparative transcriptomic analysis of Clostridium perfringens biofilm and planktonic cells[END_REF]. It should be stressed that planktonic microalgae can rapidly (i.e., from minutes to hours) acclimate to shifts in nutrients and light levels [START_REF] Jungandreas | The Acclimation of Phaeodactylum tricornutum to blue and red light does not Influence the photosynthetic light reaction but strongly disturbs the carbon allocation pattern[END_REF][START_REF] Sciandra | Responses of growth rate, pigment composition and optical properties of Cryptomonas sp. to light and nitrogen stresses[END_REF]. We therefore cannot rule out a physiological transition in microalgae from planktonic to biofilm like that occurring in bacteria.

To the best of our knowledge, only one study on microalgae has characterized the metabolic differences between planktonic cells and those in a mature biofilms [START_REF] Romeu | Characterization of planktonic and biofilm cells from two filamentous cyanobacteria using a shotgun proteomic approach[END_REF]. However, information regarding how microalgae tune their physiological state to acclimate during a shift from planktonic to immobilized conditions has never been studied before.

The present work aimed at characterizing the physiological behavior of microalgae when shifting from planktonic to immobilized states. In order to do that, we immobilized C. vulgaris on filter membranes to track the physiological adjustments including growth, morphology (cell size), photosynthetic performance and macromolecular composition in the first 24 hours after immobilization. This study may give a basis and new insight for understanding the transition of microalgae from planktonic to benthic lifestyle. From a practical point of view, it may help optimizing microalgae biofilm-based technologies for biomass and/or compounds production by identifying eventual stress factors during the early phases of biofilm formation.

Materials and Methods

Inoculum -Planktonic culture maintenance

Planktonic cultures of Chlorella vulgaris SAG 211-11b (Göttingen, Germany) were grown semi-continuously in a 1 L transparent bottle with 800 mL 3N-Bristol medium at an average irradiance of 250 μmol photons m -2 s -1 (50W LED, the irradiance was measured inside the bottles by a QSL-2100 quantum scalar irradiance sensor, Biospherical Instruments, San Diego, CA, USA) at 25°C. The cultures were bubbled with filtered air and maintained within a range of chlorophyll a (Chl a) concentration from 0.25 to 1.0 mg L -1 by daily dilution. The planktonic cultures were pre-acclimated to the growth condition for at least 8 days before starting any experiment (after acclimation, the specific growth rate, µ, of the culture was calculated based on cell number (µ = 1.4 ± 0.24 d -1 ).

Immobilized growth of C. vulgaris on filter membranes

Immobilized cells of C. vulgaris were cultured in the system reported in Fig. 1a [START_REF] Li | Biomass production and physiology of Chlorella vulgaris during the early stages of immobilized state are affected by light intensity and inoculum cell density[END_REF]. The planktonic cells from the inoculum culture (section 2.1) were gently vacuum-filtered on nitrate cellulose filters (NC) (25-mm diameter, 0.2 µm pore size, Whatman, the effective colonization area was 2.01 cm -2 ) with an initial cell density of (27.0 ± 1.1) × 10 6 cells cm -2 . Membranes were then placed on the glass fiber filters (working as an absorbing material for the medium; 0.2 µm pore size, 47-mm diameter, Whatman) and illuminated at 250 μmol photons m -2 s -1 (same light intensity used for the planktonic inoculum, LI-190R Quantum Sensor, LI-COR Biosciences GmbH, Bad Houmburg, Germany). Filters were then placed in sterile petri dishes (55-mm diameter) filled with 3 mL of 3N-Bristol medium.

After immobilization, the cells were harvested after 1h, 3h, 6h and 24h, respectively. Time 0 h correspond to data of the planktonic culture (inoculum). Physiological measurements of the filtered cells just after immobilization were also carried out in order to ensure that the filtration had no effect on cell physiology. Measurements were conducted immediately after sample collection at each time point to characterize a series of physiological parameters.

Planktonic versus immobilized cultures

In order to compare the behavior of suspended and immobilized cells, suspended cultures (Fig. 1b) with the same number of pre-acclimated cells as those filtrated on the membranes were grown in small glass beakers (25 mL) with 3 mL Bristol medium (around 3.6 mm water depth).

The cultures were mixed with a magnetic stirrer and exposed to 250 μmol photons m -2 s -1 at 25℃ as the immobilized cells.

Cell number

Cell number of planktonic and immobilized cultures were measured through flow cytometry (Guava EasyCyte HT; Millipore, USA). For the immobilized cultures, the cells were recovered in two steps. First, the membranes were rinsed with fresh medium (3N-Bristol). This allowed to harvest most of the cells from the support. Second, the membranes with the remaining cells were placed in 50 mL centrifuge tubes and vortexed with fresh medium for 10 seconds. After vortexing, suspensions from both steps were combined to determine total cell numbers by cytometry, as described in [START_REF] Fanesi | Shear stress affects the architecture and cohesion of Chlorella vulgaris biofilms[END_REF]. Algal solutions were diluted to a cell concentration range of (1.0 -6.0) ×10 5 cells mL -1 before the measurement. Cells were detected by chlorophyll a fluorescence (excitation at 488 nm and detection at 680 nm). In planktonic cultures cell numbers were expressed as cell mL -1 , whereas in immobilized state the cell areal density (cells cm -2 ) on the membrane was calculated based on the effective colonization area (2.01 cm -2 = the effective filtration area).

Cell volume

Cell morphological changes were observed with an AxioSkop 2 plus microscope (Carl Zeiss, Oberkochen, Germany) using a 63× magnification lens. Average volumetric cell size was determined considering a minimum of 300 individual cells by AxioVision SE64 Rel. 4.9.1 Software (Zeiss, White Plains, NY, USA) [START_REF] Hillebrand | Biovolume calculation for pelagic and benthic microalgae[END_REF].

Photosynthetic performance

Photosynthetic activity was assessed with a Pulse amplitude modulation (PAM) fluorometer (AquaPen, AP 110-C, Photon Systems Instruments, Drasov, Czech Republic). After 10 min of dark-incubation, the samples were exposed to a stepwise increase of seven actinic lights (from 0 to 1000 μmol photons m -2 s -1 , blue led at 455 nm) with 60s intervals to construct the electron transport rate versus photon flux density (ETR/PFD) curves. The relative electron transport rate (rETR) was calculated as described in [START_REF] Guzzon | Wastewater biofilm photosynthesis in photobioreactors[END_REF], and the rETR/PFD curves were fitted with the function rETR = rETR max (1 -e -α I/rETRmax ) [START_REF] Webb | Carbon dioxide exchange of Alnus rubra[END_REF] to estimate the maximum rETR (rETR max ), the initial slope of curves α and photo-saturation irradiance E k (E k = rETR max /α).

Cell components: Chlorophyll a, macromolecular and elemental composition

Chl a was extracted with dimethyl sulfoxide (DMSO) [START_REF] Li | Biomass production and physiology of Chlorella vulgaris during the early stages of immobilized state are affected by light intensity and inoculum cell density[END_REF][START_REF] Qiu | A new method for fast extraction and determination of chlorophylls in natural water[END_REF]. Approximately 1.5 ×10 7 cells were collected by centrifugation and then incubated with 1 mL DMSO at 65 ℃ for 60 min. The absorbance at 649 nm and 665 nm were measured using an Evolution 60S UV-visible spectrophotometer (Thermo Scientific, Madison, WI, USA). The chl a concentration (μg mL -1 ) was quantified using the equation described by [START_REF] Li | Biomass production and physiology of Chlorella vulgaris during the early stages of immobilized state are affected by light intensity and inoculum cell density[END_REF][START_REF] Qiu | A new method for fast extraction and determination of chlorophylls in natural water[END_REF] and the Chl a content on a per-cellvolume basis (fg μm -3 ) was then calculated. ATR-FTIR PerkinElmer Spectrum-two spectrometer (PerkinElmer, Waltham, MA, USA) was used to track variations in cells macromolecules as described by Fanesi et al. [START_REF] Fanesi | The architecture of monospecific microalgae biofilms[END_REF]. The spectra were baselined and the relative size of the macromolecular pools was characterized based on the maximum absorption values at the corresponding spectral ranges: carbohydrates (C-O-C; 1200-950 cm -1 ), lipids (C=O; 1750-1700 cm -1 ), proteins (Amide I; 1700-1630 cm -1 ).

The ratio of carbon (C) to nitrogen (N) at 0h and 24h was measured with an elemental analyzer (Organic Elemental Analyzer FLASH 2000 CHNS/O, Thermo Scientific). Dried biomass (1~2 mg) harvested from immobilized cultures was used for the analysis after being previously washed twice with MilliQ water and dried at 100°C until a constant weight was reached. Carbon and nitrogen quotas were expressed per cell volume.

Light measurements in the biofilm and suspended culture

Light intensity at the bottom of the biofilms was estimated according to the Lambert-Beer model [START_REF] Grenier | The impact of light supply to moving photosynthetic biofilms[END_REF]:

Where I bottom (μmol photons m -2 s -1 ) corresponds to the light intensity in the deepest layer of the biofilm (in contact with the support), I 0 stands for the incident light (= 250 μmol photons m -2 s -1 ), h is the biofilm thickness at time 0 (51.2 ± 10.4 μm, [START_REF] Li | Biomass production and physiology of Chlorella vulgaris during the early stages of immobilized state are affected by light intensity and inoculum cell density[END_REF]) and b corresponds to the biofilm extinction coefficient (5.5×10 3 m -1 , [START_REF] Grenier | The impact of light supply to moving photosynthetic biofilms[END_REF]).

In order to calculate the average irradiance received by the microalgae in the suspended culture (see section 2.3), one defined optical depth λ which reflects the actual amount of light absorbed was calculated using Eq (2) [START_REF] Bernard | Modelling of microalgae culture systems with applications to control and optimization[END_REF]:

Where ξ is the light absorption coefficient and I z is the light intensity at the depth of z (distance from the illuminating surface, here z = 3.6 mm). According to the incident and transmitted light (at bottom of the beaker) intensities measured using the quantum sensor, the value of λ is 0.2033. Thus, assuming that the algal cultures were homogeneous, the average irradiance (I av ) received by cells across the cultures could be calculated by Eq (3) [START_REF] Bernard | Modelling of microalgae culture systems with applications to control and optimization[END_REF]:

Relative air humidity for immobilized culture

To monitor the changes in relative air humidity to which the immobilized cells were exposed, a humidity sensor (P750, Dostmann) was placed inside the Petri dishes. The growth chamber was immediately covered with the lid following the algal immobilization, and the humidity was recorded during the 24 hours of incubation.

Statistics

All experiments were performed on three independent biological replicates and the data are reported as mean values with standard deviations (SD). In order to explore the physiological transition of microalgae during the transition from planktonic to immobilized state over time, a principal component analysis (PCA) and a heatmap were computed using R software [START_REF] Core | R: A language and environment for statistical computing[END_REF] (combined with packages Vegan and NMF), respectively. The cell areal density, average cell biovolume, and physiological transitions in photosynthetic parameters and biochemical composition over time were statistically analyzed by one-way ANOVA using IBM SPSS Statistics 25.0 software (SPSS Inc., Chicago, IL). Scheffe's multiple comparisons procedure was carried out after the tests of normality and variance homogeneity. Student's t-test was employed to test for differences between C (or N) quotas of pre-acclimated planktonic and immobilized cells at end of the cultivation. Significant differences at a level of P<0.05 are shown with different letters.

Results and discussion

Physiological transition from planktonic to immobilized state

Environmental conditions (light, nutrients, temperature, etc.) may change rapidly on time scales that match those of cellular processes in aquatic ecosystems [START_REF] Macintyre | The effect of water motion on short-term rates of photosynthesis by marine phytoplankton[END_REF]. Besides regulation and adaptation, acclimation is the main strategy utilized by photosynthetic organisms to cope with such changes [START_REF] Raven | Adaptation, acclimation and regulation in algal photosynthesis[END_REF]. As an example, variations in pigment synthesis or breakdown in marine phytoplankton have been described due to photo-acclimation that occurs on timescales typical of water mixing in the open ocean, and rapid changes in activity of different components of the photosynthetic apparatus were observed in estuaries [START_REF] Macintyre | The effect of water motion on short-term rates of photosynthesis by marine phytoplankton[END_REF]. These mechanisms are thought to act according to the main rule of optimizing the balance between light energy absorption and the overall photosynthetic capacity of a cell to protect from excess energy and to finally maximize growth. Extensive research on the subject has been performed on planktonic cultures [START_REF] Jungandreas | The Acclimation of Phaeodactylum tricornutum to blue and red light does not Influence the photosynthetic light reaction but strongly disturbs the carbon allocation pattern[END_REF][START_REF] Anning | Photoacclimation in the marine diatom Skeletonema costatum[END_REF][START_REF] Ritz | Kinetics of photoacclimation in response to a shift to high light of the red alga Rhodella violacea adapted to low irradiance[END_REF] and on complex microphytobenthic communities [START_REF] Mouget | Migration or photoacclimation to prevent high irradiance and UV-B damage in marine microphytobenthic communities[END_REF][START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF][START_REF] Van Leeuwe | Photoacclimation in microphytobenthos and the role of xanthophyll pigments[END_REF] or in general on mature natural biofilms [START_REF] Karsten | Green algae in alpine biological soil crust communities: acclimation strategies against ultraviolet radiation and dehydration[END_REF]. However, no information concerning how microalgae acclimate during the first stages of substrate colonization exists, even though this knowledge could help understanding how microalgae biofilms form, develop and in turn to optimize culture production.

For the first time, our results revealed that in C. vulgaris the transition from planktonic to immobilized state triggered very fast changes in the cells (Fig. 2). The PCA (Fig. 2a) clearly shows a gradual separation of the samples along the PC1 (70% of variance explained) on the base of their physiological changes over time. Interestingly, such a separation was evident only for the cells that were immobilized, whereas the cells in the planktonic state did not show remarkable physiological adjustments over the first hours (0 -6h), and the separation after 24h along the PC2 (17% of variance explained) was mainly due to an increase in cell number (Fig. 2b, Table S1).

These results confirm that the short-term physiological changes were biofilm-specific. Our results are in agreement with other studies that report differences between planktonic and immobilized cultures [START_REF] Zhuang | Differences between attached and suspended microalgal cells in ssPBR from the perspective of physiological properties[END_REF][START_REF] Huang | Comparison of Chlorella vulgaris biomass productivity cultivated in biofilm and suspension from the aspect of light transmission and microalgae affinity to carbon dioxide[END_REF]. For instance, it has been demonstrated that cells immobilized on surfaces may be less prone to self-shading [START_REF] Huang | Comparison of Chlorella vulgaris biomass productivity cultivated in biofilm and suspension from the aspect of light transmission and microalgae affinity to carbon dioxide[END_REF] and more vulnerable to dehydration [START_REF] Shiratake | Air-drying of cells, the novel conditions for stimulated synthesis of triacylglycerol in a green alga, Chlorella kessleri[END_REF][START_REF] Naumann | Growing microalgae as aquaculture feeds on twin-layers: a novel solid-state photobioreactor[END_REF]. A higher availability to CO 2 compared to suspended counterparts may be also improved by the direct exposure to gas [START_REF] Huang | Comparison of Chlorella vulgaris biomass productivity cultivated in biofilm and suspension from the aspect of light transmission and microalgae affinity to carbon dioxide[END_REF][START_REF] Shiratake | Air-drying of cells, the novel conditions for stimulated synthesis of triacylglycerol in a green alga, Chlorella kessleri[END_REF][START_REF] Zhuang | The characteristics and influencing factors of the attached microalgae cultivation: A review[END_REF]. Fig. 2. Scores plot of PCA (a) and heatmap (b) depicting the differences between planktonic and immobilized cells and the patterns of the physiological parameters during the transition (24 hours), respectively. In the heatmap, yellow color represents a low value for a specific parameter, whereas dark blue color represents a high value for that parameter.

In Fig. 2b, the heatmap summarizes the changes in the physiological profile of the cells in the planktonic and in the immobilized states, showing gradual adjustments (from pale green to yellow or to dark blue) of photosynthetic parameters and macromolecules over time. Overall, the cells reacted to the immobilization with an immediate (at 3 or 6 hours) increase in cell volume (140%, Fig. 3a) and relative pool size of carbohydrates (135%, Fig. 3c and3d), together with a decrease in Chl a content per unit volume (already after 1 hour; Fig. 3a). Interestingly, no cell division was observed before 3h from the immobilized state (Fig. 3b). The decrease in the Chl a concentration (65%, Fig. 3a) was therefore linked to the increase in cell volume. This is a typical response of cells growing in an unbalanced state when one or more resources are limited [START_REF] Jungandreas | The Acclimation of Phaeodactylum tricornutum to blue and red light does not Influence the photosynthetic light reaction but strongly disturbs the carbon allocation pattern[END_REF][START_REF] Ritz | Kinetics of photoacclimation in response to a shift to high light of the red alga Rhodella violacea adapted to low irradiance[END_REF].

While photosynthesis was not inhibited (rETR max , Fig. 3b), the cells promptly tried to re-balance the absorbed to consumed energy ratio by diluting the Chl a content (through the increase in volume) and by accumulating carbohydrates (as a C and electron sink), respectively. Such temporal scales for physiological adjustments have never been described during a transition from planktonic to immobilized states. after cells immobilization. The spectra were presented as the average of measurements and normalized by Amide I band (1649 cm -1 ) which was selected as an internal reference to assess the relative lipids and carbohydrates contents. The peaks corresponding to main organic substances were marked as: carbohydrates (C-O-C; 1174 -980 cm -1 ), lipids (C=O; 1737 cm -1 ), proteins (Amide I; 1649 cm -1 ), v = stretching, δ = bending vibrational modes in infrared spectroscopy. All the results were shown as mean value ± SD (n = 3). Bars with different letters represent the statistical differences at level of P < 0.05.

Microalgae (both planktonic and benthonic) are well known to rapidly tune physiological adjustments in response to external cues [START_REF] Sciandra | Responses of growth rate, pigment composition and optical properties of Cryptomonas sp. to light and nitrogen stresses[END_REF][START_REF] Karsten | Green algae in alpine biological soil crust communities: acclimation strategies against ultraviolet radiation and dehydration[END_REF][START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF]. For the phytoplankton, several works reported rapid physiological adjustments to cope with shifts in environmental conditions [START_REF] Sciandra | Responses of growth rate, pigment composition and optical properties of Cryptomonas sp. to light and nitrogen stresses[END_REF][START_REF] Ritz | Kinetics of photoacclimation in response to a shift to high light of the red alga Rhodella violacea adapted to low irradiance[END_REF][START_REF] Fujiki | Variability in chlorophyll a specific absorption coefficient in marine phytoplankton as a function of cell size and irradiance[END_REF]. For example, planktonic cells of Phaeodactylum tricornutum subjected to a shift in light spectral quality (from blue to red light, or vice versa) exhibited changes in Chl a quota, effective quantum yield and cell macromolecules already after 2 hours from the shifts [START_REF] Jungandreas | The Acclimation of Phaeodactylum tricornutum to blue and red light does not Influence the photosynthetic light reaction but strongly disturbs the carbon allocation pattern[END_REF]. On the other hand, studies that tracked the physiological changes and biochemical pathways of bacteria during planktonic-to-biofilm transition also confirmed the specific biofilm-related genes or proteins, which were only expressed in benthic but not planktonic bacterial cells [START_REF] O'toole | Biofilm formation as microbial development[END_REF][START_REF] Chua | Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles[END_REF][START_REF] Zhao | A starvation-induced regulator, RovM, acts as a switch for planktonic/biofilm state transition in Yersinia pseudotuberculosis[END_REF][START_REF] Zou | Cellular and molecular responses of Salmonella typhimurium to antimicrobial-induced stresses during the planktonic-to-biofilm transition: Antimicrobial-induced responses of foodborne pathogens[END_REF][START_REF] Zou | Cellular and molecular responses of Salmonella typhimurium to antimicrobial-induced stresses during the planktonic-to-biofilm transition: Antimicrobial-induced responses of foodborne pathogens[END_REF]. Similarly, natural algal biofilms can rapidly adjust their metabolisms (from minutes to hours) in response to emersion, stressful light conditions, altered humidity and temperature [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF][START_REF] Van Leeuwe | Photoacclimation in microphytobenthos and the role of xanthophyll pigments[END_REF][START_REF] Häubner | Aeroterrestrial microalgae growing in biofilms on facades-response to temperature and water stress[END_REF][START_REF] Perkins | Responses of microphytobenthos to light: primary production and carbohydrate allocation over an emersion period[END_REF]. For instance, Corcoll et al. [START_REF] Corcoll | Light history influences the response of fluvial biofilms to Zn exposure[END_REF] showed rapid changes (within 6h) in photosynthetic processes of fluvial biofilms after a sudden increase or decrease in the incoming light.

In our case, the shift in external conditions related to the immobilized state was hypothesized to have stimulated the physiological response.

Factors inducing the transition from planktonic to biofilm state

Three main factors that could have induced these fast responses after immobilization are proposed: light, humidity and nutrients. While cells in suspended cultures are subjected to dynamic light patterns due to mixing [START_REF] Bernard | Modelling of microalgae culture systems with applications to control and optimization[END_REF][START_REF] Combe | Long-term adaptive response to high-frequency light signals in the unicellular photosynthetic eukaryote Dunaliella salina[END_REF], in an immobilized system, a light gradient may establish between the top and the bottom of the biofilm and on a short-time scale each biofilm layer is constantly exposed to the same light intensity. Based on our computations, the average light intensity in the immobilized and in the planktonic state was very similar (ca. 220 µmol photons m -2 s -1 , Table 1) suggesting that the physiology of the immobilized cells was affected by other factors. After immobilization, the relative water content (in the gas space of the reactor) for cells rapidly decreased from 100% in the planktonic solution to 93.5% in the immobilized stage (Table 1). As demonstrated by Häubner et al. [START_REF] Häubner | Aeroterrestrial microalgae growing in biofilms on facades-response to temperature and water stress[END_REF] and Shiratake et al. [START_REF] Shiratake | Air-drying of cells, the novel conditions for stimulated synthesis of triacylglycerol in a green alga, Chlorella kessleri[END_REF], the relative air humidity affected the photosynthetic activity, chemical composition and growth profiles of microalgae biofilms. In a water-saturated atmosphere (100% air humidity), cells presented a high growth profile and photosynthetic efficiency, while at humidity of around 93%, they were strongly inhibited [START_REF] Häubner | Aeroterrestrial microalgae growing in biofilms on facades-response to temperature and water stress[END_REF].

Table 1. Light profiles and relative air humidity of planktonic and immobilized cultures. Accordingly, although the photosynthetic activity was not affected in the first hours of immobilization, at 24h a decrease in photosynthetic efficiency (rETR max , Fig. 3b) was observed, together with a 2.0-fold increase in the lipids content in the cells (Fig. 3c and3d). The latter was also reflected in a change in cell stoichiometry: while nitrogen quota kept stable, the carbon quota (fg µm -3 ; Fig. 4a) significantly increased after 24h resulting in a greater C/N ratio (1.8 folds; Fig. 4b), which suggests an uncoupling between carbon and nitrogen metabolisms. Typically, nutrients may modulate photosynthetic activity in several ways depending on the limiting compound [START_REF] Wykoff | The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii[END_REF]. The two main ways are the low repairing rate of proteins in photosystems and the competition for ATP and reducing equivalents between C assimilation and nutrients uptake [START_REF] Beardall | Approaches for determining phytoplankton nutrient limitation[END_REF][START_REF] Turpin | Effects of inorganic N availability on algal photosynthesis and carbon metabolism[END_REF]. On the other hand, a similar trend of rETR max was found by Forster and Martin-Jézéquel [START_REF] Forster | Photophysiological variability of microphytobenthic diatoms after growth in different types of culture conditions[END_REF] who investigated microphytobenthic diatoms and by Fanesi et al. [START_REF] Fanesi | The architecture and metabolic traits of monospecific photosynthetic biofilms studied in a custom flow-through system[END_REF] who characterized a diatom biofilm. The authors proposed a link between nutrient availability and the Calvin cycle activity.

Interestingly, in another work, a more efficient carbon dioxide mass transfer, and subsequent productivity, has been suggested in C. vulgaris biofilms compared to suspended cultures [START_REF] Huang | Comparison of Chlorella vulgaris biomass productivity cultivated in biofilm and suspension from the aspect of light transmission and microalgae affinity to carbon dioxide[END_REF].

Changes in CO 2 concentration could therefore have triggered the physiological transition observed in the current study. Further experiments are required to deeply investigate the relationship between photosynthesis and nutrients uptake in photosynthetic biofilms.

On the base of these results, it is likely that humidity and nutrients played a major role in the transition from planktonic to immobilized that the cells exhibited.

On the whole, the data show that the rapid morphological and physiological changes are specific to the immobilized state. In addition, it is likely that changes in environmental conditions triggered those physiological changes. The rapid acclimation process that the cells underwent immediately after being immobilized seemed to be aimed at optimizing fitness under the new growth conditions [START_REF] Geider | Dynamic model of phytoplankton growth and acclimation: responses of the balanced growth rate and the chlorophyll a:carbon ratio to light, nutrient-limitation and temperature[END_REF][START_REF] Geider | The role of cost-benefit analysis in models of phytoplankton growth and acclimation[END_REF][START_REF] Geider | A dynamic regulatory model of phytoplanktonic acclimation to light, nutrients, and temperature[END_REF]. Such a great physiological plasticity of cells growing in biofilms, as proposed by other authors, is a key trait to cope with the external variabilities as the biofilm develops [START_REF] Perkins | Responses of microphytobenthos to light: primary production and carbohydrate allocation over an emersion period[END_REF][START_REF] Forster | Photophysiological variability of microphytobenthic diatoms after growth in different types of culture conditions[END_REF].

Further work should be carried out to identify more in detail the underlying acclimation mechanisms and the triggering factors. For instance, a full description of the pigment contents, rates of photosynthesis/respiration, nutrients levels (nitrogen and CO 2 ) and enzyme activity would certainly allow to assess the underlying biological mechanisms. Furthermore, with the developments in genomics-and proteomics-based approaches, molecular mechanisms/pathways regulating such an acclimation process (from planktonic to immobilized state, or vice versa) could be in future elucidated [START_REF] Qayyum | Protein translation machinery holds a key for transition of planktonic cells to biofilm state in Enterococcus faecalis: A proteomic approach[END_REF][START_REF] Jefferson | What drives bacteria to produce a biofilm?[END_REF].

Conclusion

This study for the first time tracked the physiological behavior of cultured microalgae from planktonic to immobilized condition. The results pointed out that cells rapidly acclimated to the new environmental conditions (surface-associated) by increasing the cell size and adjusting the carbon allocation (carbohydrates pool) (within 6h). Moreover, this behavior seems to be specific of the immobilized state of the cells. We also hypothesized that changes in the local environmental conditions to which cells are exposed might have been responsible for the described cellular behaviors. However, further assays are required to fully comprehend the behavior of cells switching from planktonic to benthic modes. This will represent a step forward to better understand biofilm communities, for the selection of suitable strains and optimize biofilm-based systems.

CRediT authorship contribution statement S. Li: Conceptualization, Investigation, Visualization, Formal analysis, Writingoriginal draft,

Fig 1 .

 1 Fig 1. Schematic representation of the cultivation systems used for the immobilized (a) and

Fig. 3 .

 3 Fig. 3. Changes in average cell volume and Chl a quota (a), maximal relative electron transport

Fig. 4 .

 4 Fig. 4. Carbon (or nitrogen) content (a) and C/N ratio (b) in the immobilized cells at the

Acknowledgments

S. Li was supported by the China Scholarship Council (CSC) and A. Fanesi was founded from the LabeX LaSIPS project Greenbelt managed by the French National Research Agency (ANR).

Funding acquisition. T. Martin: Conceptualization, Materials fabrication. A. Fanesi: Conceptualization, Investigation, Formal analysis, Writingreview & editing, Funding acquisition. F. Lopes: Supervision, Conceptualization, Formal analysis, Validation, Writingreview & editing.

Data availability

Data will be made available on request.

References:

Declaration of competing interest

The authors declare that there is no conflict of interests regarding the publication of this paper.


Nalini Rakotonandraina
email: nalini.rakotonandraina@lasalle-beauvais.fr

Loïc Sauvée

Linking marketing innovation and territorial resources: the example of "Porcilin/Saveurs en'Or", a regional brand with a nutritional attribute

Keywords: Local agrifood product, regional development, marketing innovation

This paper discusses the development and marketing of local agrifood products in a context of sustainable regional development illustrated by the case study "Porcilin/ Saveurs en'Or". The case highlights an example of marketing innovation and territorial branding strategy applied to pork meat in the Nord Pas de Calais region in France. Indeed, Porcilin/ Saveurs en'Or is a combination of two initiatives: The Porcilin brand, a nutritional brand (with omega 3) considered as innovative brand, associated with a local product (pork meat); and Saveurs en'Or, a collective regional label developed to promote local agri food products and resources in the Nord Pas de Calais region which serves as an umbrella for regional products in this region. The study focuses on differentiation strategy of local products in order to create added value and governance mechanisms implemented in order to have a successful marketing strategy.

differentiation strategy, value, nutritional brand, pork meat, governance, Porcilin/Saveurs en'Or" 1-Introduction: potentiality of regional products, evolution of consumer's views and trends, and consumers 'behaviours toward local food Since food crisis (BSE), consumer behaviours and trends towards food conception are continuously evolving. Consumers are more selective and more demanding [START_REF] Dinis | Territorial marketing: A useful tool for competitiveness for rural and peripheral areas[END_REF] about food information especially origin as well as product and raw materials, with the necessity of traceability systems. Otherwise, there is an increasing requirement of qualities, food safety, and environmental issues for consumers. They are more sensitive about health, and production practices (like animal welfare, organic, pesticides use, GMO...). In addition, consumers' attention about ecological concern has increased their preferences towards Rakotonandraina N.& Sauvée L.-LaSalle Beauvais Polytechnic Institute-France regional and organic products. Indeed, direct market channels as farmers' markets, community-supported agriculture, vegetable box schemes, and other cooperative distribution and delivery programs have proliferated, especially in the last decade, in many advanced industrial countries (Festing, 1998;Groh and McFadden, 1997;Kneen, 1993;Powell, 1995;Hinrichs, 2000). Then, circuits of proximity and a dynamic of consumption around the local products growing. This highlights the importance of local product's valuation not only to meet the expectations of consumers but also for the regional economy.

The challenges for local products are twofolds: in the one hand economic and social aspects and in the other hand environmental issues. Development of local ressources allows conservation of regional activities (local production) and is also a way of value creation for local companies. Consequently jobs are preserved or created at the regional level. Direct agricultural marketing contributes not only to the rural farm economy, but also to local tourism and small business development (Atkinson andWilliams, 1994, Hinrichs, 2000).

Besides, local supply means less transportation and favor sustainable development because of reduction of carbon emission, According to Friedmann (1993), Lockeretz (1986) andHinrichs (2000), by direct agricultural markets locality, seasonality over distance and durability are preserved, and consequently, consumers obtain fresh, high-quality farm products at reasonable prices, as well as unusual local specialty products.

Thus the main questions are : how to promote local agri-food products / how to create value for these products? What marketing strategies and what organizational management is necessary to implement?

Research methodology

The main objective of the research focuses on territorial marketing of local resources: a conceptual framework about marketing innovation, value creation strategy and governance mechanisms within chain actors and institutional environment. The study approach is qualitative and materialized by case study. In the first part of the research, a literature review will be done about marketing strategy including quality differentiation and innovation concepts for regional products. Notion of coordination mechanisms will be approached on this study based on Williamson's conceptual framework. In the second part of the paper, the case study (Porcilin/Saveurs en'Or) will be used as an empirical inquiry (Yin, 1989;Grunert and al., 2005), which allows for an investigation of a contemporary phenomenon within its reallife context, when the boundaries between the phenomenon and context are not clearly Rakotonandraina N.& Sauvée L.-LaSalle Beauvais Polytechnic Institute-France evident, and in which multiple sources of evidence are used. The case study Porcilin/Saveurs en'Or illustrates an example of territorial marketing of local products in the Nord Pas de Calais region. The case gives better understanding of marketing innovation applied to local pork resources (pork meat), and an overview of the implemented chain organization, actors' interaction, relationships and coordination throughout these initiatives considering micro and macro level (company level and at the territorial/regional level).

2-Theoretical framework

Marketing approach of local food products

To face the continual evolution of consumers' expectations and market competition, developing differentiation strategy becomes evidence for the local players to conquer niche markets through marketing mix strategy. Indeed, according to the work of [START_REF] Dinis | Territorial marketing: A useful tool for competitiveness for rural and peripheral areas[END_REF], marketing is a process of value creation (functional or emotional benefits) and product promotion, and then an operational tool (Kotler 1996:6). By this way marketing mix is a useful tool for local resources to create a competitive advantage by using a differentiation strategy and by this way to communicate and to inform consumers about them and to access market.

Consequently, to be able to create value, local actor's marketing strategy differentiation may focus on innovation issues of product or process or by segmentation strategy using globally various quality schemes called Quality Management System (including quality assurance system through official sign, private or collective brand, certification system etc.) Interests of farmers, consumers and localities together drive the resurgence in direct agricultural marketing (Kinsey, 1994;Hinrichs, 2000). Consumer demands concerning animal welfare, food safety, environmental considerations and use of genetically modified organisms (GMOs) are examples, where evolving and heterogeneous consumer demands create needs for enduser focussed market orientation to extend across the whole value chain. However, strategic differentiation choice depends on market trends and potential valuable resources [START_REF] Dinis | Marketing and innovation: Useful tools for competitiveness in rural and peripheral areas[END_REF]. the Guarantee for the Traditional Specialities (GTS), or the organic chain. Moreover, a differentiation strategy in using quality schemes may refer also to a high level of product , 1990).

Rakotonandraina N.& Sauvée L.-LaSalle Beauvais Polytechnic Institute-France

Marketing Strategy in using innovation concept

The survival of both regions and firms depends on their ability to achieve competitive advantages in the global economy and to conquer new markets [START_REF] Porter | The Competitive Advantage of Nations[END_REF]Kalantaridis, 1997) in a context of globalization. Innovation can be considered as a factor of differentiation, which creates profit (main motivation to innovate) [START_REF] Schumpeter | The Theory of Economic Development[END_REF][START_REF] Dinis | Marketing and innovation: Useful tools for competitiveness in rural and peripheral areas[END_REF].

According to [START_REF] Schumpeter | The Theory of Economic Development[END_REF] secondly process innovation and finally organizational innovation. Innovation is a crucial element for firm competitiveness and consequently its success is related to marketing strategy orientation and its capability to anticipate market tendencies (European Commission, 2003;[START_REF] Dinis | Marketing and innovation: Useful tools for competitiveness in rural and peripheral areas[END_REF].

The concept of quality governance

According to Gonzales Diaz and al (2009) governance mechanisms are essential to promote final product quality and to high quality. Consequently, an organization that offers both coordination and motivation devices must be set up. Governance structure includes firstly the chain organization or chain coordination like membership, rules etc., and secondly the process of quality management including quality definition, specification and control mechanisms [START_REF] Ponte | Quality standards, conventions and the governance of global value chains[END_REF]Gibbon, 2005, Williamson, 1991). The concept of governance structure focuses on the form of intra and inter-firm coordination, in other words the business relationships (contracts, trust) and vertical coordination between the various players [START_REF] Raynaud | Alignment between Quality Enforcement Devices and Governance Structures in the Agro-food Vertical Chains[END_REF]Peterson et al., 2001 Notions of lead firms and actors are important in the concept of governance because lead the firm has a particular functional position about decision role and control of the whole chain.

Rakotonandraina N.& Sauvée L.-LaSalle Beauvais Polytechnic Institute-France
Governance mechanisms can be subdivided in two parts: governance of transaction, and the governance of quality which will be materialized by the quality management system and related to the quality policy including some functions like quality definition, ensuring compliance, creating rules (negotiation, incentives…) (Rakotonandraina and Sauvée 2011, Williamson, 1991[START_REF] Ponte | Quality standards, conventions and the governance of global value chains[END_REF]. Indeed, QMS includes the organizational structure, responsibilities, processes, procedures, and resources that facilitate the achievement of quality management (Luning and al, 2002). Quality Management system is used by an organization to direct and control the implementation of its quality policies and the achievement of its quality objectives (Luning andMarcelis, 2007, ISO, 2005). Calais which develops a regional product of proximity offers'strategy.

Porcilin association groups together players in the pig supply chain in North Pas de Calais region composed by: two feed industries, a producer group with thirty hog producers, a slaughterhouse, a craft meat-processor and the retailer group Auchan. Its objectives include the development of the trademark Porcilin and the promotion of the regional pig producers, and product differentiation through quality. The brand operates under a private specification related to origin of pig and raw material composition. Indeed, Porcilin brand is a regional brand which guarantees to consumers a traditional product with quality differentiation composed by product organoleptic characteristics and a nutritional attribute throught the presence of omega 3. Nutritional attribute Omega 3 is from the use of flax seeds in according to Porcilin requirements specification. By this way, Porcilin appears as an innovative brand attesting pork with omega 3, meaning of Porcilin. Porcilin brand offers a wide range of processed pig meat with regional specialties.and seasonal and sold with a premium price (about less than 10% higher compared to standard product). Porcilin products are distributed exclusively on hypermarkets of the retailer group Auchan in the North Pas de Calais region.

Otherwise, the brand image of Porcilin is strengthening by the collective regional brand

Rakotonandraina N.& Sauvée L.-LaSalle Beauvais Polytechnic Institute-France
Saveurs en' OR ("Gold Taste"), a collective label which reinforces and conveys local identity of Porcilin's product. Saveurs en'OR is a collective regional brand created on 2004 by the Quality Group of North Pas de Calais (GQNPC), and the Promotion Committee Nord Pas de Calais. The brand guarantees a product manufactured and consumed in the state or a processed product in Nord-Pas de Calais region with key regional raw materials.

Saveurs en'OR aiming firstly to answer consumers' waits about identification of regional product, secondly, to develop agri-food activities and at the last to make the regional origin of Saveurs en' OR to bring more information about the regional product.

3-2 Chain organization and relationships between actors

Porcilin Association which manages and promotes Porcilin brand brings together various players in the pork chain from farmers (thirty farmer members) to distributor. Commitments of adhesion into the chain are mainly the respect of specifications and annually contribution payment by certified players including for the functioning expenses of the chain. For internal organization, the association includes an operational office and a board of direction composed by representatives of all partner companies which are feed company, farmers, slaughterhouses and meat processors, retailer group and Saveurs en'OR. The office is constituted by a chairman, a vice president, a secretary, a team for animation and communication (from regional council chamber) and pig farmers. At the regional level, the collective label Saveurs en'OR is managed and owned by APASO Adrianor, Promotion Committee). Saveurs en' OR chain operates through three major cells including: a promotion committee which is a service of the regional chamber of agriculture in North pas de Calais, and which contributes to the promotion and development of regional food in North Pas de Calais ; a pilot committee of Saveurs en'OR in charges of all decisions regarding the chain (specification, strategies…) and a committee composed by all companies members responsible in one hand of choosing and selecting members of pilot committee and in other hand managing private funds and collecting payment contribution from members.

Certified members of

4-Analysis and concluding comments

Case study highlights differentiation strategy based on product innovation, particularly on innovation of existing product by introducing a nutritional ingredient to the pork meat (pork meat with omega 3), supported by a "branding alliance" strategy through the association of the Porcilin brand with the collective regional brand Saveur en'OR. Saveurs en'OR has a role of caution for the Porcilin product as regard to the regional origin of the pork product. The alliance of both quality schemes brings more visibility to the local pork chain at the regional level and constitute a competitive advantage for involved actors in in term of product image and notoriety and product quality in one hand and in other hand bring incentives for farmers (premium price). Indeed, Porcilin brand as well as Saveurs en'OR is a quality schemesoriented towards a differentiation strategy and then strengthen each over: Porcilin developed a differentiation strategy based to nutritional product attributes and Saveurs en 'Or by the product origin and geographical process area for product including the regional know how and tradition. Another important factor is the supporting organization (institutional organization, association of promotion) that promotes the local industries and plays a significant role in the sustainable development of regional chains by coordinating collective action, training for local operators, managing funding, and grant. In other words, of quality approach achievement depends on strategic choice of marketing orientation firstly matching to consumer demands, and capabilities of owner to coordinate chain by incentives and control mechanism and a strong partnerships/collaboration between chain actors.

In conclusion, creation of added value for local products depends on capability to build a marketing innovation, and better coordination mechanism aligning with a marketing differentiation strategy . However, innovation marketing with traditional product or local resources is limited by its acceptance of consumers, especially in the case of nutritional attributes innovations.

Figure1.

  Figure1. Conceptual model of value creation strategy for local products

3-

  Empirical study: case of Porcilin/Saveurs en'OR: marketing strategy and organization 3.1. Porcilin brand's differentiation strategy According to previous work of Rakotonandraina and Sauvée (2011), Porcilin program is a quality scheme (pig farmers 'brand) in the North Pas de Calais region in France, initiated by pig producers' association named Porcilin association which managed the chain. This approach is supported by a large retailer (Auchan company group) of the region North Pas de

  Saveurs en'OR products as support for referencing in the distribution and purchase an argument for consumers. The program groups various regional actors: food industries, craftsmen, traders, retailer, institutional bodies and farmers. The brand indicates richness, expertise and knowhow of North Pas de Calais region and required food quality of each product according to specific requirements. Saveurs en'OR Regarding communication policy, Porcilin association developed direct communication by farmers in store aiming to inform consumers about the Porcilin quality scheme completed by a collective communication by

  association which pilots the collective action of the program Saveur en OR with various purposes including the promotion of agricultural products and foodstuffs in North Pas de Calais region, and public awareness about the origin of products, the collective brand and different quality schemes in the region. In addition, APASO through its various committees is in charge of the property rights, the control mechanisms, strategy decision concerning specification, market development, communication policy and coordination and supervision Rakotonandraina N.& Sauvée L.-LaSalle Beauvais Polytechnic Institute-France of various collective implemented actions and partnering. Committees are composed by a board of directors and an office and includes various chain actors (from different existing sector and local business in the region with the distributor and consumer association), Consular chamber and Saveurs en' OR support partners (example GQNPC Qualinorpa,

  Saveurs en'OR (including Porcilin association) are commited to sign a contract which consists of the respect of specification, control monitoring, and the sensory analysis of products (before obtaining the approval certification). Collaborative relationships exist between the both approaches Porcilin and Saveurs en'OR. Relationships between actors are formalized into: Institutional relationship (memberships), control and monitoring relationships and transaction relationships. Institutional exchanges can be found at two levels: firstly, relation between Porcilin association and its members and secondly exchange relationship between Porcilin association and Saveurs en'OR to develop chain. Porcilin internal relationship can be characterized as information relationship between various certified actors through meetings, and animation training for farmers. Indeed, a general meeting is held every year and the Board meets every two months about market development (volume target and incentive payment for farmers). Furthermore, relation exchanges of support and process improvement exist between players with a strong participation of the retailer group Auchan (meat department managers). It is summarized to a meeting regarding chain evolution and a visit in farms. In addition, partners of Porcilin chain maintain a regular communication to exchange about various subjetcs concerning development and problems of the program (production, process, control, market).

Figure 2 :

 2 Figure 2: Strategy differentiation of Porcilin/Saveurs en'OR

  European Commission, 2005). Besides, private quality schemes (individual company or collective) claiming various differentiation attributes (animal welfare, nutritional attribute, locally origin, etc.) can be used for local products promotion. Indeed, rural development should be based on strategies which "promote, in all possible ways,

		Marketing differentiation strategy
		Product/Process
		innovation
	Local local capacity building for sustainable development in rural areas, and in particular, private Value Niche
	resources and community-based initiatives which are well-integrated into global markets" (The Cork market
	Declaration	Quality schemes
		(QMSs)

quality (example of Label Rouge in France) or specific and constant characteristics Rakotonandraina N.& Sauvée L.-LaSalle Beauvais Polytechnic Institute-France (certificates Conformity CCP) (

Rakotonandraina N.& Sauvée L.-LaSalle Beauvais Polytechnic Institute-France