Dr L Prat
email: laurent.prat@ensiacet.fr

Handling of Polymer Particles in Microchannels

Keywords: Design principles, Microcapsules, Microreactors, Polymerization -

This paper deals with solid-liquid operations in microchannels. Continuous operations on solids (modification of frequency, change of solvent, encapsulation) in order to handle polymer particles in microchannels are described in terms of the limits of operating conditions and their possible applications. A methodology to design and implement operations on polymer particles is presented here. It is applied for the generation of onion-like structures. A microdevice completely built with Plexiglas plates and fused silica capillaries is used. The device includes droplet generation, polymerization and microparticle flow manipulation. The particles manipulated are in the range of 70 to 200 microns with a very low index of polydispersity.

Introduction

Microreactors are receiving growing interest in polymerization processes [1,2]. The micron-sized dimension is directly used for precise size distribution of the solid obtained [3,4]. The use of microfluidics allows the attainment of polymeric particles with different shapes and compositions. Directly derived from the shape of the droplets, polymer beads [5][6][7], disks, and plugs [7,8] were thus synthesized. Moreover, microfluidics enables the formation of multiphase droplets [9] from which partial polymer spheres [10], Janus [11], and ternary polymer particles [12] were obtained. In each case, synthesized polymeric particles have an extreme regularity and a broad range of size is available.

An important issue is now to be able to implement operations in continuous on the generated microparticles. This will allow the construction of more complex structures (onionstructured microparticles, assemblies of particles, etc.). From a process engineering point of view, multiple operations and their couplings are then needed (multiple operation connection, addition of a secondary fluid, solvent change or increase, or the decrease of interval length between two particles). This study is focused on a methodology to analyze a complex multi-operation system to produce a specific micro-object. From the analysis, a technological solution is proposed and validated in terms of operations performed and coupling feasibility.

Three main operations are considered: droplet generation, reaction, and successive encapsulation.

System Analysis

In order to create onion-like structures, a clear sequence to apply to the particle flow has first to be developed. Preliminary analysis of the system is proposed in order to define which steps are necessary and what could be the technological responses to meet the objectives.

With a hypothesis on the shape factor of the droplet, a geometric pattern (Fig. 1) of a droplet chain moving in a continuous flow can be described by four measurable variables: drop length (L g 1) ), width (w g ), velocity (v), and frequency of generation (f); the characterization of flow is reduced to three variables in the case of spherical droplets. From these data, it is possible to calculate the other dimensions: length of the pattern (L pattern ), length of the interval between droplets (L i ), volumes, etc. In addition, the two immiscible phases are charac-

Alain Marcati 1 Christophe Serra 2

Michael Bouquey2 Laurent Prat 1 1 Institut National Polytechnique de Toulouse, Université de Toulouse, Laboratoire de Génie Chimique, Toulouse, France. terized with their physico-chemical properties (uv): viscosity, density, and surface tension between the two phases.

The whole microsystem can thus be characterized with this geometric pattern (Fig. 2). Each block has entry variables, outlet variables, action parameters, and discrete parameters.

The generation block forms the droplet pattern; the flow rates of the continuous phase, Q c , and dispersed phase, Q d , are directly responsible of the variables of the pattern. The geometry of the reactor, for instance, T or cross-junction and the material of the channel, especially its roughness (R c ) or the contact angle with the fluid (h), also have an impact on the pattern, but this impact is much more difficult to quantify.

The reaction block transforms the droplet pattern into a particle pattern depending on the length (l p ), width (w p ), velocity, and frequency of the particle. The length, L, and the diameter, d, of the channel influence the exposition time of droplets flowing at a velocity, v. Energy brought by temperature (T°) or UV light (ht) in our case creates the particles.

The encapsulation of the particles needs the injection of a new phase at a flow rate, Q 3 . The third phase makes the pattern more complicated to characterize. New variables appear, such as the number of particles included (N int ), the length (L ext ), the width (w ext ), the velocity (v ext ), and the frequency (f ext ) of the generation of the external droplet. In this case again, the roughness and the geometry of the encapsulation area have discrete parameters.

These three blocks can be directly connected to each other, but a transfer block can also be added between them in order to change the frequency of the droplet, or particles flow, or change the solvent around the dispersion. It can be composed of a single or some secondary channels having an angle, a, with the main channel. This block is the most complex, because it has to deal with the compatibility between the phases added or withdrawn, or the compatibility between the fluids and the material. It will then define some of the limits of the operation conditions for each intervention on the flow, such as the flow rate of injection or aspiration and the global geometry of the block.

Thanks to these transfer blocks, the manipulation of the flow and the selection of phases involved in the multiple-layer structure are possible. After the encapsulation block, a new reaction block can be connected in order to create a new solid layer and then encapsulate again. Each new layer added makes the geometrical pattern more complex to describe and an increasing number of variables will be necessary to describe it.

Experimental Material

Multi-Block Microsystem

The microparticle management unit system (Fig. 3) is divided into three parts, providing three different process operations: droplet generation, the polymerization part, and the microparticle management unit. A detailed description of device manufacturing is presented in a previous article [13] but, in this article, chloroform -which has the property to dissolve Plexiglas -is used to seal the devices.

Droplet Generation Unit

The droplet generation unit can either be a T or a cross-junction. It is composed of two Plexiglas plates and standard 360micron outer diameter (OD) capillaries; different inner diameters (ID) can be used. The first plate is worked with a micromilling machine and a 300-micron drill. The channels thus created host the capillaries that are put in contact all together at the T-junction and fixed with glue when correctly set. The second plate is then sealed thereupon to close the generator thanks to chloroform. Capillaries of 180-micron inner diameters are used in this study.

Connections and Polymerization Block

The polymerization unit is composed of a single capillary whose length can vary to ensure good exposure time to UV light. Standard 360-lm capillaries made of fused silica have 20 microns-thick polyimide coating. This coating is useful to protect against capillary breaking, but absorbs UV light which prohibits polymerization, so it has to be removed. The connection between the different units is possible due to larger capillaries (320-lm ID, 465-lm OD). Polyimide coating is to be removed from 360-lm capillaries in order to be inserted into the larger ones. Capillaries from both units are set in straight contact and glued. No leakage is observed and there is neither droplet deformation nor clogging due to polymer particles.

Microparticle Management Unit

The last part of the microfluidic system is derived from previous studies on secondary channels [14], which have shown that it is possible to act on the continuous phase without dis-turbance on the dispersed phase. The secondary channels will either extract continuous phase or inject a second fluid. Fluid extraction has to face the respective pressure drop within the main or secondary channels. A right balance is to be found for effective aspiration.

As for the droplet generator, two Plexiglas plates and standard 360-lm OD capillaries are used to produce this unit. The first plate is milled with 300-lm drills according to a predefined computer-aided design. When all the capillaries are in place, the second plate is added and both plates are sealed with chloroform injected into secondary channels. All the channels have a square section of 380 × 380 lm so that they can host 360-lm capillaries. The angles between the main and secondary channels have been fixed to 90°or 20°. This last value is the minimum possible to ensure a good design during the milling process.

Chemical System and Solutions

Tripropylen-glycol diacrylate is used as the organic phase liquid. An initiator of photo-induced polymerization (hydroxylcyclohexyl-phenyl ketone) is added to the monomer to produce a 5.0-wt % mixture [5]. This chemical system is described in previous studies on polymerization in microchannels. It has the advantage to be very reactive thanks to two acrylate groups and, thus, interesting to produce microparticles with short exposition time.

An aqueous solution of bromothymol blue (BTB) is used during the manufacturing process in order to protect the main channels during the injection of chloroform. 1,4-Butanediol is used as a test fluid for the change of continuous phase around particles and 1-octanol is used as a test fluid for the encapsulation of particles.

Surrounding Equipment

The fluids are stocked into 1 to 20-mL syringes (Terumo, BD) placed on syringe pumps (Harvard 2000, Picoplus). The connection with the capillaries is made with a 22G needle (Terumo) directly glued onto them with cyanolithe glue. The observation is made with a fast CCD camera (NV1000) coupled with a microscope (Nikon SMZ-10). The image acquisition rate is 900 frames per second. Exposition time is chosen in order to have enough light for contrast distinction and short exposition for the droplet edge sharpness. A 100-W spot lamp is used to provide the necessary light. The images are acquired with transmission light through Plexiglas.

Results and Discussion

In the first part, the generation and the polymerization of droplets inside capillaries are described. Then, a microparticle management unit is connected to the reaction part in order to perform operations on the particle flow. The complete device (connections and sealing) is resistant up to 30 mL h -1 , which can be evaluated as a 0.2 bar m -1 pressure drop. In the experimental domain studied, no leakage is observed but sensible points concern the glue which ensures the sealing between the capillaries and Plexiglas channels.

Monomer Droplet Generation and Polymerization

Before polymerizing the droplets, several experiments were performed in order to find an operating domain where droplets and, consequently, particles have a spherical shape. The generation of the monomer droplets at a cross-junction is described by Fig. 4. The dispersed phase is injected from the capillary on the left and is maintained between the two inlets of the continuous phase. The generation of the droplet is obtained in the outlet capillaries; a thin film enters the capillary and starts to develop until it forms a droplet while advancing in the tube. When the drop is fully created, it is released and the film of dispersed phase goes back to the entrance of the outlet capillary.

The size of the droplets follows the same correlation observed for silicon oil in water in a previous article [13]. The operating domain is now set: the dispersed phase varies from 20 to 500 lL h -1 and the continuous phase from 4000 to 10 000 lL h -1 . Due to the very low thickness of the fluid in the capillary submitted to the UV lamp, the residence time necessary to obtain solid particles is reduces down to about 5 s or even less. Fig. 5 shows the particle size distribution through the operating conditions.

Monodisperse particles of different size and shape are obtained in the range of 70 to 200 microns. Classically, particle size decreases with the increase in continuous phase flow rate and increases when the dispersed phase is raised. Particle shape varies with monomer flow rate; a hydrodynamic break on the dimension of the particles is observed for each continuous flow rate. As water flow rate increases, the break happens for higher monomer flow rate. It is marked in Fig. 5, and corresponds to the limit between polymer beads and bulletshaped particles. At low monomer flow rate (inferior to 200 lL h -1 ), particles are thus spherical and no satellite particles are observed; when they are recovered on a glass sheet, they automatically set themselves in regular hexagonal structures. The coefficient of variation in the particle diameter is measured on over 150 particles by image analysis. For all the spherical particles, it is located between 0.8 and 1.5 %. An example is given on the upper graph of Fig. 5. At higher monomer flow rates, particles assume this bullet shape, and satellites become as numerous and visible as the flow rate is high.

Continuous Phase Aspiration

The frequency of particles flowing inside the microchannel is originally determined by the ratio of the continuous and dispersed flow rates. It can easily be regulated by withdrawing or adding continuous phase. Particle velocity is then reduced or accelerated. In order to select the best configuration and operating conditions to change the continuous phase around the particles, a comparison between a Y junction with an angle of 20°and a T-junction is made. Even if the particles are not perfectly spherical, these experiments are lead with a dense medium of particles (water flow rate of 4 mL h -1 ; monomer flow rate of 100 lL h -1 ). The flows with particles widely spaced seem indeed simpler to manipulate than particles flowing very close to each other. As particles may clog the secondary channel if they are brought in by the aspiration, it is important to find the limit in the flow rates of aspiration applied. Consequently, a comparison between a particle flow and a droplet flow is first made.

Aspiration with Droplets and Particles

The images taken during the manipulation of the flow are presented in Fig. 6. With particle flow, the withdrawal flow rate is cautiously increased. The distance between particles decreases progressively until they touch each other at 2 mL h -1 , but this state is unstable and particles begin to put themselves in staggered rows. At 2.5 mL h -1 , particles move into the withdrawal channel. The aspiration flow should then be lower than 2 mL h -1 . With droplet flow, the aspiration flow rate is increased until all the droplets are drained into the secondary channel. The distance between droplets and walls slowly decreases until some may explode with the shock. At 3 mL h -1 , flow starts to be chaotic and droplets can switch to the secondary channel. At 4 mL h -1 , droplets move into the secondary channel after a few seconds. Then flow is regular in the withdrawal channel and droplets do not touch the walls. An aspiration flow rate of 2.5 mL h -1 is then a limit to prevent droplets from directing into the secondary channel. There is a little difference in the maximum flow rate of aspiration, but the behavior of droplets and particles during continuous phase aspiration is similar. It is simpler then to study the withdrawal of the continuous phase with droplets because, even if they explode by contact with the walls, they can be cleaned, whereas particles may totally clog the channels and consequently the microsystem.

Determination of the Angle of the Aspiration Channel

The withdrawal of continuous phase around droplets is then tested in a T-junction with the same operating conditions (Fig. 7). The aspiration flow rate is progressively increased until droplets are drained into the secondary channel. At 1.5 mL h -1 , the droplets are very close to the angle of the secondary channels. When the aspiration flow rate is at 2 mL h -1 , some droplets may explode at the angle and some droplets are attracted into the secondary channel. Fig. 7 compares the aspiration efficiency with an angle of 20°a nd an angle of 90°function of the aspiration flow rate. The aspiration efficiency is defined as the difference between distance without aspiration (initial interval between droplets) and the distance with aspiration, divided by the initial interval between droplets. At low aspiration flow rate, the response is pretty similar for each angle but, when aspiration is further increased, there is a strong disparity between the two angles. Not only the angle of 20°allows recovering more continuous phase, but the limit of aspiration is even higher than that of the Tjunction. The angle of 20°seems thus much more interesting for the withdrawal of the continuous phase. This difference can be explained by the manufacturing of the channel of 20°. As the angle is narrow, the mill creates, close to the main channel, an area where the width of the secondary channel is much larger than its height. So, there is part of the main flow that is automatically driven into this area (Fig. 8) and this makes easier the aspiration of the continuous phase into the secondary channel.

Nevertheless, it also brings the droplets closer to the walls even when there is no aspiration flow rate applied. Due to the mechanical stress applied during milling, the roughness of the channels is pretty important and difficult to control. In some cases, the explosion of droplets by contact with the secondary channel walls is observed, and this will automatically interfere with the regularity of the droplet and, consequently, the particles. To prevent this, a T-junction that keeps more droplets at the centre of the channels is used in order to withdraw continuous phase for experiments during the change of solvent around particles.

Change of Continuous Phase

In order to change the continuous phase around the particles, the injection of a new phase and the withdrawal of the old phase must be coupled. The aim is to find operating conditions where carrier fluid can be easily replaced by a new one. To obtain the change of continuous phase, it is necessary to create a laminar flow between the old and the new one; the new phase must then be injected from one part of the channel and the old one withdrawn from the other part. Considering the limitations on the flow rates of aspiration, more than one secondary channel is necessary to withdraw the old phase. Preliminary tests showed that it was necessary to inject a phase miscible with the aqueous phase carrying the particles. Even if the angle of 20°has proved to create unstable flows, a first experiment is conducted in a device with this type of secondary channels (Fig. 9).

The initial particle flow was generated with an aqueous flow rate of 4 mL h -1 . A solution of BTB is used as miscible fluid and the flow rate of injection is fixed at 4 mL h -1 . As the initial aqueous phase and the BTB solution have comparable physico-chemical properties and flow is laminar, half of the channel is occupied by BTB and half by the aqueous solution. In this configuration, any change of level in the channel is easily no-ticeable. The first aspiration site is located at 10 cm from the injection site and the second aspiration site at 20 cm.

A first aspiration is applied; aspiration flow rate is increased up to 2 mL h -1 . At this flow rate, the water level has appreciably decreased to a half. Due to wall effects, some BTB is aspired though the withdrawal channel. A second aspiration is then added with the first aspiration set at 2 mL h -1 . Aspiration flow rate is again raised up to 2 mL h -1 . The main channel is practically full of BTB after the second aspiration, but again some BTB is aspired through the secondary channel. It seems then that the two aspiration channels were necessary to first remove carrier fluid after the injection of the BTB solution. This number may eventually be reduced if carrier fluid is aspired before the injection of BTB and the injection flow rate is increased. In order to reduce diffusion phenomena, withdrawal channels should be placed closer to secondary fluid injection.

A new device is then built with the T-junction as secondary channels in order to keep a regular particle flow. In this device, a third aspiration site is added because a higher aqueous flow rate is used (8 mL h -1 ) in order to manipulate spherical particles. The aspiration sites are located 5 cm from each other and from the injection in order to reduce the diffusion between both phases. An example of solvent change around particles in this new device is presented in Fig. 10. To better illustrate the transfer of particles from one phase to another, 1,4-butanediol is used because it has a clear interface with water once put in contact in a microchannel.

Before introducing particles, the flow rate of the introduction of 1,4-butanediol is determined with a droplet flow. 1,4butanediol is indeed far more viscous than the aqueous solution and, thus, it occupies a bigger part of the channel. If the level of 1,4-butanediol is too significant, the droplets and, consequently, particles will enter in contact with the wall at the bottom of the channel. With an equivalent flow rate of aqueous solution and 1,4-butanediol of 8 mL h -1 , all the droplets touch the wall and explode. At 6 mL h -1 , half of the droplets explode by contact with the wall. Finally, the flow rate of 1,4butanediol is set at 4 mL h -1 , which is the highest flow rate possible without droplet explosion.

When a particle reaches the T-junction, it is first repulsed at the bottom of the channel and close to the wall by the introduction of the new phase. Then, it automatically tries to regain the centre of the channel, troubling the interface (image at the injection site). As long as the particle advances through the channel, it passes from the aqueous phase to the 1,4-butanediol and is located at the interface between both phases at each aspiration site because of the attraction; it creates dragging on the interface (double interface appearing on the image of the second aspiration site). The level of the aqueous phase progressively decreases at each aspiration site until there is one phase left at the last T-junction. The global aspiration flow rate applied is 9 mL h -1 , whereas the initial water flow rate was 8 mL h -1 . The difference may be the result of mixing effects because of the troubles created at the interface by the particles.

As a consequence, the particles are less distant from each other: two particles are visible at the last aspiration site. The particle pattern obtained after the change of solvent is timeproof.

Particles Encapsulation

In this part, the objective is to trap one particle into a single droplet. As the chain of particles arriving at the injection point is regular, there will only be one flow rate of injection that will correspond to the entrapment of a single particle into one droplet in a stable sequence. In order to add a degree of freedom on the size of the droplet surrounding the particle, an aspiration module is positioned just before the injection of the new phase. The coupling of an aspiration module and the injection module helps regulating the size of the new droplet by acting on the distance between the particles. The modules used in this case are cross-junction so that particles are kept in the middle of the channel during injection or aspiration. In these experiments, octanol is used directly on the aqueous solution carrying particles. Two different initial flow rates of monomer are represented on Fig. 11; aqueous flow rate is imposed at 8 mL h -1 .

For each monomer and aspiration flow rate, the octanol flow rate is progressively increased: at low flow rates, one or more particles can be trapped in a single droplet. At high flow rates, the droplets can be generated without particles inside. The graph of Fig. 11 ticeable that the distance between particles strongly interacts with the synchronization flow rate and, consequently, the frequency and the size of the droplets generated. The droplets generated are the consequence of the physico-chemical properties of the two phases involved and the size of droplets obtained will change if another phase is used but adding the aspiration module can notably reduce its size. If aspiration is too important, it may induce contact between particles or the fall into the bottom of the channel. The aspiration in this case is also subject to limitations not to modify the particle pattern.

Conclusion

A complex multioperation microdevice including droplet generation, polymerization part, and particles handling is reported here. Polymer particles generated are monodisperse and in the range of 70-200 microns. This range can easily be widened by changing the inner diameters of capillaries that constitute the device. The feasibility of coupling aspiration and injection modules in order to perform continuous operations, such as solvent change or particles encapsulation in microchannels has been demonstrated. From the results, it is possible to generate complex solid structures. For instance, by coupling solvent change and particles encapsulation, onion-like structures with a chosen nature are reachable. Of course, the specific device of this study presents constraints which limit the operating domain. These constraints are mainly due to the mechanical etching that implies roughness, but this could be solved in second generation devices. The methodology and the analysis of this paper can be applied to design other devices for the generation of other micro-objects.

Symbols used

Figure 1 .

 1 Figure 1. Droplet pattern inside the microchannels.-1) List of symbols at the end of the paper.

Figure 2 .

 2 Figure 2. Characterization of the whole microsystem using the droplet pattern.

Figure 3 .

 3 Figure 3. Schematic representation of the complete microparticle management unit system.

Figure 4 .

 4 Figure 4. Generation of monomer droplets at a cross-junction inside capillaries: water flow rate at 10 mL h -1 ; monomer flow rate at 0.2 mL h -1 .

Figure 5 .

 5 Figure 5. Cartography of particles obtained in a cross-junction and measurement of the variation coefficient for the particle diameter (upper graph).

Figure 6 .

 6 Figure 6. Progressive aspiration of the continuous phase in a particle flow (left) and a droplet flow (right).

Figure 7 .

 7 Figure 7. Progressive aspiration of the continuous phase in a T-junction (left), and comparison of the aspiration efficiency between an angle of 20°(~) and 90°(ᮀ).

Figure 8 .

 8 Figure 8. Droplet explosion by contact with a wall in a Y-junction with an angle of 20°when there is no aspiration applied.

Figure 9 .

 9 Figure 9. Replacement of the aqueous phase carrying the particles by a BTB solution with two consecutive aspiration channels.

Figure 10 .

 10 Figure 10. Replacement of the aqueous phase carrying the particles (dotted white squares) by 1,4-butanediol with three consecutive aspiration channels.

Figure 11 .

 11 Figure 11. Research of the synchronization flow rate of the third phase function of the aspiration flow rate and the initial flow rate of the monomer.

Ecole Européenne de Chimie, Polymères et Matériaux de Strasbourg, Laboratoire d'Ingénierie des Polymères pour les Hautes Technologies, Strasbourg, France.

Acknowledgements

The authors acknowledge the French Ministry of Education and Research for having funded this work through the grant ANR n NT05-1_45715.

The authors have declared no conflict of interest.


Claire Joannis-Cassan

Marie-Line Delia

Jean-Pierre Riba

Limitation phenomena induced by biofilm formation during hydrocarbon biodegradation

Keywords: biofilm, hydrocarbon, bioremediation, diffusion limitation, Pseudomonas alkanolytica

Limiting factors induced by the formation of a biofilm on hydrocarbon's surface during in-situ bioremediation were identified. A liquid-liquid system (hydrocarbon-aqueous solution) and a device made of six fixed-bed columns packed with sand were successively used on laboratoryscale. In the liquid-liquid system, it was shown that biofilm growth was limiting by the diffusion of both oxygen and hydrocarbon within the biofilm. At the end of growth, the biofilm thickness reached 80±20µm. In the fixed-bed columns, additional difficulties were observed. First, distribution of oxygen in the deepest part of the sand was difficult: from 84 to 100% of the biofilm growth occurred in the upper 2/3 part of the sand. Then, the surface of the hydrocarbon available for microorganisms was found to be a limiting factor for the biofilm's development.

INTRODUCTION

Developments in the chemical industry have resulted, over time, in increasing incidences of soil contamination. Hydrocarbon pollution, resulting from the closure of old gas supply companies and oil tank leaks, has made a major contribution to this contamination causing extensive damage to the ecosystem. In the past 20 years, methods have been developed to clean up such contaminated soils [START_REF] Autry | Bioremediation: an effective remedial alternative for petroleum hydrocarbon-contaminated soil[END_REF] . Among them, in-situ biological methods are being considered more frequently since they are able to decontaminate soil in depth. So, these methods are relatively cheap with less disruption of the ecosystem than chemical or physical processes [START_REF] Atlas | Microbial degradation of petroleum hydrocarbons: an environmental perspective[END_REF] . In-situ bioestimulation consists of enhancing the growth of indigenous microorganisms, that are able to use hydrocarbons as source of carbon and energy by injecting into the soil an aqueous solution containing nitrogen, phosphate, mineral salts and oxygen.

Under favourable conditions, decontamination of several soils has been successfully carried out [START_REF] Dupont | Fundamentals of bioventing applied to fuel contaminated sites[END_REF][START_REF] Heyse | In-situ aerobic biodegradation of aquifer contaminants at Kelly Air Force Base[END_REF][START_REF] Kosson | In-situ and on-site biodegradation of industrial landfill leachate[END_REF][START_REF] Michelsen | In-situ biological oxidation of hazardous organics[END_REF] . The main difficulty of this type of process resides in the presence of two nonmiscible phases, hydrocarbon and the aqueous solution, which must be in contact with the microorganisms. Moreover, some microorganisms may excrete polymers and form a biofilm on the hydrocarbon surface [START_REF] Marin | Emulsifier production and microscopical study of emulsions and biofilms formed by the hydrocarbon-utilizing bacteria Acinetobacter calcaoaceticus MM5[END_REF] . Biofilms are usually composed of two major components: microbial cells and extracellular polymers (essentially polysaccharides and glycoproteins) [START_REF] Christensen | Physicals and chemicals properties of biofilms[END_REF] .

Biofilm formation includes 5 steps: (i) conditioning of a surface or interface, (ii) adhesion of cells, (iii) formation of micro-colonies, (iv) biofilm formation and growth and (v) possible sloughing of pieces of the biofilm due to flow rate dynamics or shearing effects of flowing fluids. If strong shear forces are applied to biofilms, thickness does not exceed more than a few micrometers whereas for free development, thickness reaches several millimetres. Thus, biofilms should strongly influence mass transfer by limiting the diffusion of nutrient or oxygen within the biofilm [START_REF] Characklis | Laboratory biofilm reactors[END_REF] . If the biofilm thickness exceeds the depth of substrate penetration, an "inactive" area could appear in the inner biofilm. For instance, when the oxygen transfer is limited by diffusion, an aerobic area could develop near the support surface. Thus, biofilm could considerably modify the bioremediation process by forming an additional barrier between the two phases and the micro-organisms. However, the presence of biofilm is rarely taken into account in bioremediation studies [START_REF] Paulsen | Visualisation of bacterial degradation and mobilisation of oil in a porous medium[END_REF] . This paper describes the effect of the development of a biofilm on a hydrocarbon surface during bioremediation process. First, growth kinetics were established in a liquid-liquid system, composed of an emulsion between a hydrocarbon and an aqueous solution, in order to identify factors limiting growth. Then, biofilm growth was studied in a six-column laboratoryscale system to reproduce the complexity of the in-situ bioremediation process.

MATERIALS AND METHODS

Micro-organisms and culture conditions

Bacteria

Pseudomonas alkanolytica ATCC 21034 was used for all experiments. The strain was maintained on an agar Petri dish at 4°C. The storage medium was composed of digested soy peptone: 5 g, meat extract: 1 g, yeast extract: 2 g, NaCl: 5 g, agar: 15g in 1 dm 3 of reverse osmosis purified water. The pH was adjusted to 7.2 with 1 mol.dm -3 NaOH.

Culture medium

The culture medium consisted of a mineral salt solution and a hydrocarbon added at 2 % v/v. O: 10 mg in 1 dm 3 of deionised water. The pH was adjusted to 7 using 1 mol.dm -3 NaOH and the medium was sterilized for 20 minutes at 121°C. The hydrocarbon was n-dodecane (Fluka). Before being added to the mineral salt solution, the dodecane was checked for sterility under a microscope (Olympus, 400×magnification). For each test, possible contamination was regularly checked by observing the morphology of the cells under a microscope (Olympus, 400×magnification).

Preparation of the starter cultures

Two different starter cultures were used for the inoculations: one for the liquid-liquid reactor and another for the soil six columns system. To obtain reproducible initial conditions, the starter cultures were prepared in two steps. First, a preculture, inoculated from the storage medium, was grown for 5 days in a flask containing 100 cm [START_REF] Dupont | Fundamentals of bioventing applied to fuel contaminated sites[END_REF] of the culture medium. The flask was shaken at 250 rpm and incubated at 30°C.

Then, for the two-phase reactor, 1 cm 3 of the aqueous phase of the preculture was introduced in 100 cm 3 of fresh culture medium. After a 3-day growth at 30°C, an appropriate volume of the aqueous phase was introduced into the liquid-liquid system to have 0.01g.dm -3 (dry weight) of biofilm at the beginning of the experiments.

For soil experiments, 3 cm 3 of the preculture were introduced in 300 cm 3 of medium culture.

After a 3-day growth at 30°C, the culture was centrifuged 5 minutes at 20,000 rpm. The pellet was washed and suspended in an appropriate volume of the aqueous solution to obtain a concentration of 0.5 g.dm -3 (dry weight). This suspension was used to inoculate the sand.

Experimental devices

Liquid-liquid system

The liquid-liquid system was a 4 dm 3 working volume cylindrical reactor (200 mm in diameter, 300 mm in high). The emulsion between the aqueous solution and dodecane (2% v/v) was obtained using a Rushton turbine (75 mm in diameter, 6 flat blades) located halfway up the culture medium (Figure 1-a). To study oxygen limitation, tests were carried out by pumping air through the culture medium. The air was sterilized by passing through a 0.22 µm filter before being introduced into the reactor at a flow rate of 8.6 cm 3 .s -1 . Unless otherwise stated, the reactor was stirred at 250 rpm and the temperature was set at 30°C. The growth kinetics were determined by withdrawing samples from the reactor every 12 hours.

Bioremediation system

Bioremediation experiments were carried out in 6 glass columns (Amersham Pharmacia Biotech, Saclay, France) (40 cm in length, 2.4 cm in diameter) simultaneously. A sand (Prolabo), with standardised size (400 µm in diameter) was used as a soil model. The porosity was 0.34 and the hydraulic conductivity was estimated at 8.10 -5 m.s -1 . The permeability was 8.2 10 -12 m 2 . Each column was packed with 150 g of sterile sand (Figure 1-b). Dodecane was first introduced into the sand from the bottom of the column. The sand was then washed with sterile water for 12 hours. The quantity of capillary-trapped dodecane in the columns was estimated by weighing the quantities introduced and recovered. The hydrocarbon was homogeneously distributed throughout the sand, as a Sudan red coloration proved. Biological inoculation was carried out by loop circulation from the top of the column of 300 cm 3 of the starter for 6 hours. The sand was then washed with the aqueous solution to recover the free bacteria.

The bioremediation process was run by trickling down 1 dm 3 of the aqueous solution into each column; the sand was kept saturated. The aqueous phase was recycled through each column with a peristaltic pump at a flow rate of 0.36 cm 3 .min -1 , so the interstitial rate was 2 mm.min -1 .

Experiments were carried out at room temperature, which was 20°C ± 2°C. The growth kinetics were determined by sampling the sand of an entire column at different times since a preliminary study proved that both initial inoculation and biofilm growth were statistically similar in the 6 columns [START_REF] Joannis | Biofilms bactériens se développant sur un hydrocarbure en milieux liquide et poreux[END_REF] . The last column was divided into three parts equal in volume (top, middle, and bottom) which were separately analysed to determine the biofilm distribution in the sand. At the end of the experiment, the biofilm sloughing from the porous medium was estimated by measuring the dry weight of biofilm in the aqueous phase.

Analytical methods

Biofilm quantification

Dry weight measurement by filtration. This technique is based on filtration through cellulose acetate filters (Sartorius) (0.2 µm pore size). For the two-phase culture, two samples were directly removed from the culture medium with a piston sampler (12.5 cm 3 ) and centrifuged for 10 min at 20,000 rpm. Three parts were observed: a pellet (biofilm), an aqueous phase and a layer floating on the top (an amalgam of dodecane, biofilm and some traces of the aqueous phase). After removing the aqueous phase, a solvent mixture (acetone-petroleum ether, 3:1 v/v) was added. This solvent made it possible to separate dodecane from the biofilm by making the hydrocarbon soluble with the aqueous phase [START_REF] Goswami | Different modes of hydrocarbon uptake by two Pseudomonas species[END_REF] and by precipitating biofilm [START_REF] Joannis | Comparison of four methods for quantification of biofilms in biphasic culture[END_REF] . The total amount of biofilm was recovered after centrifugation at 34,000g and removal of the supernatant. The resulting pellet was suspended in sterile water, filtered and dried at 70°C until a constant dry weight was achieved. For the porous system, biofilm should be extracted from the porous medium. Therefore, the amalgam between dodecane and the biofilm was recovered by mixing the sand with sterile water using the following protocol. The content of the column was pushed out into a 1 dm 3 flask. Then, five successive extractions were made by adding sterile water to the sand, mixing vigorously for 30 minutes and recovering the liquid phase (biofilm, hydrocarbon and water) after sand sedimentation. We checked that the total amount of biofilm was recovered by weighing the sand of each column before and after the experiments. The total water volume used was 750 cm 3 per column. The five extracts were combined. Five samples of this liquid phase were taken out with a 24.7 cm 3 piston sampler to be analysed as it is described above for the liquid-liquid samples.

Dry weight estimation from optical density measurement. This second method consisted in an in-situ measurement of the optical density (OD) of the culture medium with a biomass probe (Model 652 cell growth monitor, Wedgewood Technology, Inc., San Carlos, USA). The biofilm dry weight was directly quantified by using the existing linear relations between OD and biofilm dry weight [START_REF] Joannis | Comparison of four methods for quantification of biofilms in biphasic culture[END_REF] . This method was only used for the liquid-liquid culture without air supply as air droplets led to interference with OD measurement.

Droplet size and biofilm thickness in the liquid-liquid reactor

In the stirred tank reactor, the hydrocarbon formed spherical droplets, so the surface (A) available for the micro-organisms could be calculated from:

d V A d ⋅ = 6
, where V d is the volume of dodecane, and d the diameter of the droplets.

The specific area (a), which is the ratio of the dodecane surface and the total volume of the dispersion expressed in m -1 , is calculated from: [START_REF] Michelsen | In-situ biological oxidation of hazardous organics[END_REF] , where ε is the volumic fraction of dodecane.

d a ε ⋅ =
The droplet diameter was determined from video pictures by comparing the droplet size with the diameter of a stainless rod (0.5 mm) immersed in the emulsion. The dodecane droplet diameter was found to be equal to the diameter predicted by the Calderbank's correlation [START_REF] Coulaloglou | Drop size distributions and coalescence frequencies of liquid-liquid dispersions in flow vessels[END_REF] , which was 200 µm for an agitation rate of 250 rpm. At the end of growth, the diameter of dodecane droplets and the thickness of the biofilm were estimated from video pictures of the reactor by comparing them to the diameter of various stainless steel rods (from 0.1 to 0.5 mm).

Other analytical methods

The dissolved oxygen in the liquid-liquid reactor was measured with an O 2 probe (PO 2 , Ingold, Inceltech, Toulouse, France) linked with an amplifier (Model 170, Ingold, Inceltech, Toulouse, France). The probe was standardized using an air-saturated solution (100% of dissolved oxygen).

The pH was measured with a pH probe (Ingold, Inceltech, Toulouse, France). A PID regulator (Inceltech, Toulouse, France) was used to maintain the pH at 7 with 1 mol.dm -3 NaOH.

The (NH 4 ) 2 SO 4 concentration in the aqueous phase was determined by the Nessler method [START_REF] Charlot | Azote et ses composés[END_REF] .

Proteins were extracted from the biofilm by hydrolysing 0.5 cm 3 of the sample at 100°C with 0.5 cm 3 of 0.25 mol.dm -3 H 3 PO 4 [START_REF] Joannis | Comparison of four methods for quantification of biofilms in biphasic culture[END_REF] . The protein concentration was estimated with Lowry's method [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF] using a solution of BSA (Bovine Serum Albumin) containing an equal volume of H 3 PO 4 as reference.

Statistics

A statistical analysis of the biofilm quantification methods was previously made [START_REF] Joannis | Comparison of four methods for quantification of biofilms in biphasic culture[END_REF] and the standard deviation (e%) was linked with biofilm dry weight. In this paper, results are presented on the graphs in terms of 95 % confidence intervals.

RESULTS AND DISCUSSION

Factors limiting growth in the liquid-liquid system

In this system, it could be observed that the biofilm growth took place on the dodecane surface (Figure 1-a). This surface, estimated from the size of the hydrocarbon droplets, decreased during the growth due to the consumption of the hydrocarbon. In the same time, the thickness of biofilm increased due to its development.

Theoretical considerations

Due to the thickness of the biofilm, diffusion limitations could occur during the biofilm development as reported by several authors for biofilms growing on solid supports [START_REF] Christensen | Physicals and chemicals properties of biofilms[END_REF][START_REF] Thibault | Reassessment of the estimation of dissolved oxygen concentration profile and KLa in solid-state fermentation[END_REF][START_REF] Emanuelsson | Overcoming oxygen limitations in membraneattached biofilms--investigation of flux and diffusivity in an anoxic biofilm[END_REF] . For biofilm growing on liquid hydrocarbon, diffusion limitation could concern molecules located on both sides of the biofilm: dodecane and the rate-limiting component of the aqueous phase.

Figure 2 explains this theoretical phenomenon for diffusion limitation of dodecane and dissolved oxygen. For thin biofilm (Figure 2-a) a gradient of both dissolved oxygen and dodecane could be observed, even if the hydrocarbon probably poorly diffused within the biofilm due to its non-miscibility with water. However, the two components are not exhausted in the biofilm, so the biofilm could develop. When the biofilm thickness increases (Figure 2b), both hydrocarbon and oxygen could be exhausted in some areas: oxygen near the dodecane surface and dodecane near the aqueous phase. This leads to the appearance of an active area and two inactive areas where growth can no longer occur. Beyond a critical thickness (Figure 2-c), the active zone should be reduced to one point. The biofilm development stops.

Growth kinetics

Figure 3-a presents the biofilm growth kinetics without air supply, in order to reproduce operating conditions encountered in the soil. Oxygen was transferred into the culture medium only through the liquid surface. The volumetric oxygen mass transfer coefficient (k L a) was 0.0008 s -1 . The biofilm growth shows 4 distinct phases. After a 10 h lag phase (1), the biofilm growth occurred at an average rate of 11.3 mg.dm -3 .h -1 between 10 and 60 h while the quantity of dissolved O 2 decreased (2). This confirms that the consumption of dodecane by Pseudomonas alkanolytica is aerobic, which is common for most of the bacteria growing on a hydrocarbon substrate [START_REF] Goswami | Different modes of hydrocarbon uptake by two Pseudomonas species[END_REF][START_REF] Rehm | Mechanisms and occurrence of microbial oxidation of long-chain alkanes[END_REF] . Between 60 and 225 h (3), the growth was linear (the observed rate was 5.4 mg. dm -3 .h -1 ). During this phase, dissolved oxygen was completely depleted and the growth rate should be controlled by the oxygen transfer from the surrounding air into the liquid by the impeller. After 225 h (4), the growth reached a stationary phase and the oxygen concentration increased up to 100%. During the growth, the droplet diameter reduced from 200 to 160 µm, which suggests a consumption of half the dodecane [START_REF] Joannis | Biofilms bactériens se développant sur un hydrocarbure en milieux liquide et poreux[END_REF] . At the end of the growth, biofilm thickness was 80 ± 20 µm, which is about half of the droplet size.

The arrest of the growth was probably caused by diffusion limitation of both dodecane and oxygen within the biofilm as it is described above. A series of experiments were carried out to verify that no other limiting factors were the cause of the growth stop, such as nutrient exhaustion, inhibition by low pH or product inhibition.

First, the final quantity of dodecane and nitrogen source was estimated. As already mentioned, about half of the dodecane was still present at the end of growth and the residual amount of (NH 4 ) 2 SO 4 , the only source of nitrogen, was 200 mg.dm -3 . For other components of the aqueous phase, an experiment was carried out by supplying 30 cm 3 of the mineral salt solution just before the stationary phase. No difference was observed with the previous experiment, demonstrating that the limitation was not due to exhaustion of a mineral salt.

Then, limitation due to low pH value was studied, since pH linearly decreased from 7 to 5.5

during the batch experiments (Figure 4) due to the production of acids [START_REF] Rehm | Mechanisms and occurrence of microbial oxidation of long-chain alkanes[END_REF] coupled with mineral salt consumption. Two experiments were performed with the same culture conditions except for the pH, which was either controlled or not (Figure 4). Even if the final quantity of biofilm was slightly higher when the pH was controlled, no significant difference was observed between the two cultures, and the growth reached a stationary phase in both cases.

At last, an experiment was made by supplying 30 cm 3 of dodecane to the culture medium when the growth reached the stationary phase. As can be seen on Figure 5, as soon as dodecane was supplied to the culture, the growth occurred on fresh hydrocarbon droplets at the same rate as the initial growth. Thus, no inhibiting products were present in the medium culture at the end of growth. This result supports the hypothesis of an arrest of growth caused by the diffusion limitation of both dodecane and oxygen within the biofilm and also evidences that the dodecane availability has an influence on the biofilm growth and thickness.

Influence of the dodecane surface

Experiments were carried out with several interfacial areas by changing the impeller rotation rate. For each stirring rate, the interfacial area was calculated from the droplet diameter estimated by Calderbank's correlation. The biofilm growth kinetics obtained for initial dodecane specific surfaces of 550 m -1 , 600 m -1 and 700 m -1 are shown on Figure 6. An improvement of the biofilm maximum growth rate with the specific surface was observed: 4.3 mg.dm -3 .h -1 for a = 500 m -1 , 11.4 mg.dm -3 .h -1 for a = 600 m -1 and 12.5 mg.dm -3 .h -1 for a = 700 m -1 . The acceleration of growth was probably due to the improvement of the oxygen transfer rate linked to the increase in the stirring rate [START_REF] Miura | Mechanism of liquid hydrocarbon uptake by microorganisms and growth kinetics[END_REF] . However, the amount of biofilm in the medium was almost the same at the end of growth (about 1.5 g.dm -3 ) in all three experiments. The greater the dodecane surface, the thinner the biofilm. The increase of the shear force on the biofilm surface consequent on the impeller rotation rate could disturb biofilm development.

Thus, modifying the impeller rotation rate did not clearly demonstrate the effect of the dodecane surface since it not only changed the dodecane surface area but also the surrounding conditions of growth.

Effect of oxygen supply

To improve the rate of oxygen dissolution into the culture medium, the medium was aerated at 8.6 cm 3 .s -1 . The k L a value increased up to 0.002 s -1 . The results are shown on Figure 3-b.

Aeration involved an amalgam between hydrocarbon, biofilm and air droplets which could explain the dry weight values dispersion on the graph. The average rate of biofilm growth was almost four times higher than for the non-aerated culture (2.8 mg.dm -3 .h -1 without aeration versus 11 mg.dm -3 .h -1 with aeration). Aeration prevented oxygen dissolution from limiting the reaction since the percentage of dissolved O 2 is almost always above zero. However, as for the non-aerated culture, growth severely slowed down as soon as the biofilm concentration reached 1.5 g.dm -3 , which appeared after 50h. At the same time, oxygen increased up to 100%, suggesting a non-consumption by the micro-organisms for the same reasons as explained above.

Bioremediation process

In this system, the dodecane was capillary-trapped. BATH (Bacterial Adherence To Hydrocarbon) experiments [START_REF] Rosenberg | Adherence of bacteria to hydrocarbons: a simple method measuring cell-surface hydrophobicity[END_REF] proved that Pseudomonas alkanolytica had a high adherence to dodecane [START_REF] Joannis | Biofilms bactériens se développant sur un hydrocarbure en milieux liquide et poreux[END_REF] . Moreover, it has been reported that bacteria are usually poorly absorbed onto sand [START_REF] Omar | Degradation of n-alkanes by Candida parapsilosis and penicillium frequentas immobilized on granular clay and aquifer sand[END_REF] . So, the hypothesis was made that during inoculation, the bacteria adhered to the hydrocarbon rather than on the sand, and that biofilm only grew at the surface of the dodecane (Figure 1-b).

Theoretical considerations

In comparison with the liquid-liquid system, the bioremediation system could generate additional limitation phenomena due to the presence of the porous matrix. Whereas nutrient distribution was spatially homogeneous in the liquid system, feeding the column from the top could lead to a concentration gradient through the sand. Oxygen exhaustion through the column has already been observed [START_REF] Martins | Aspects hydrodynamiques, physico-chimiques et biologiques du devenir des pesticides dans les sols[END_REF][START_REF] Song | Effects of jet fuel spills on the microbial community of soil[END_REF] . Moreover, the biofilm development could be limited by the size of the sand pores.

Effect of oxygen supply

Two experiments were carried out to study the effect of O 2 on biofilm growth. In the first test, no air was supplied in the aqueous phase which circulated through the column. Table 1 (line 1)

presents the dry weight of the biofilm produced and the biofilm distribution throughout the column at the end of the growth. It can be seen that 89.1 % of the biofilm developed in the upper 1/3 part of the sand. As dodecane was homogeneously distributed in the column and as a significant quantity of the nitrogen source was found in the outlet of the column, the biofilm spatial distribution seems to be due to a depletion of the dissolved oxygen in the upper third part of the column. A second test was undertaken with an aeration of the aqueous phase at a rate of 15 cm 3 .s -1 . The results in Table 1 (line 2) show that the total biofilm quantity was almost double than without aeration. The biofilm located in the middle of the column reached 42.3% of the total biofilm, which indicates a better distribution of the oxygen at this depth. No growth was observed at the bottom of the column due to the depletion of oxygen. Although oxygen distribution was improved by the aeration of the aqueous phase, it was still difficult to bring oxygen to the deepest parts of the sand. On large-scale in-situ systems, the oxygen limitation is one of the most frequently quoted reasons for bioremediation failures [START_REF] Downey | Performance of selected in-situ soil decontamination technologies: an Air Force perspective[END_REF][START_REF] Irvine | Periodic processes for in-situ and on-site bioremediation of leachates and soils[END_REF] .

Influence of the dodecane surface

The influence of the initial dodecane surface was investigated in the porous media. Contrary to the previous experiments, where pure dodecane was used to inoculate the columns, a waterdodecane emulsion (2% v/v) was introduced into the sand. In this case, 2 g of dodecane remained capillary-trapped in each column. Even if the surface of dodecane could not be precisely estimated, the specific surface should be higher for emulsified dodecane than for pure dodecane. The aqueous phase was aerated at 15 cm 3 .s -1 . The results are presented in Table 1 (lines 3). The total biofilm produced (0.48 g) was the same whether 15 g of pure dodecane or 2 g of emulsified dodecane were introduced into the sand. This was probably due to a lower dodecane surface availability for pure dodecane coupled with a lack of pore space for the biofilm development as 15 g of dodecane took about 2/3 of the empty volume of the sand. The comparison of the percentage of biofilm sloughed of the column (Table 1) for pure and emulsified dodecane consolidated this hypothesis as the percentage of detached biofilm was 3.5 times higher for pure dodecane.

These results confirmed the importance of the dodecane distribution in the polluted soil.

However, for dispersed dodecane, the bioremediation process was still limited by oxygen distribution in the column as only 16.3% of the growth occurred in the deepest third part of the sand.

Comparison of the growth kinetics in the liquid-liquid and the bioremediation systems

The biofilm growth kinetics in porous medium, with aeration of the aqueous phase and dispersed dodecane is presented on Figure 7. The development of biofilm in the liquid-liquid system at the room temperature [START_REF] Joannis | Biofilms bactériens se développant sur un hydrocarbure en milieux liquide et poreux[END_REF] is presented on the same graph. It can be seen that the biofilm development occurred at almost the same growth rate for both experiments. However, contrary to the liquid-liquid system, the growth continued after 300 h in the porous system.

This difference is probably the consequence of the difference of surrounding conditions for biofilms (shear stress, dodecane accessibility). The protein content of the biofilm was determined for each experiment: the percentage was 51% in the liquid-liquid system and 12% in the sand device. As proteins are partially responsible for the cohesion of the biofilm, this suggests that the operating conditions should be more favourable for the bacteria attachment in the porous medium: the cells should be more easily in contact with dodecane and the biofilm cohesion should not need to be as strong as in the stirred tank. Thus, the depth of nutrients diffusion should increase and the limitation due to diffusion transport of oxygen and nutrient would appear later than in the liquid system. However, beyond a critical thickness, the bioremediation process could fail because of the limitation of hydrocarbon and oxygen diffusion within the biofilm formed on the dodecane surface.

CONCLUSION

This study shows the importance of taking into account the biofilm formation on hydrocarbon's surface during bioremediation processes. A diffusion limitation of oxygen and dodecane within the biofilm was observed in the liquid-liquid reactor. This phenomenon adds to other difficulties encountered in the soil during bioremediation processes: oxygen distribution in the soil's depth and hydrocarbon surface availability. As diffusion limitations could not be removed without destroying the biofilm, future work should aim at improving substrate diffusion, for example by researching a consortium composed of bacteria growing on hydrocarbon and micro-organisms consuming the polymer of the biofilm. )

Bioremediation system liquid-liquid system

Figure 1 .

 1 Figure 1. Experimental devices and biofilm location in liquid-liquid system (a) and

Figure 2 .

 2 Figure 2. Theoretical schedule of diffusion of substrates in the biofilm in case of: thin

Figure 3 .

 3 Figure 3. Effect of aeration on the biofilm growth in the liquid-liquid system. The results

Figure 4 .

 4 Figure 4. Influence of pH on the biofilm growth in the liquid-liquid system with no

Figure 5 .

 5 Figure 5. Effect of dodecane supply on the biofilm growth kinetics in the liquid-liquid

Figure 6 .

 6 Figure 6. Influence of the surface of dodecane on the biofilm growth in the liquid-liquid

Figure 7 .

 7 Figure 7. Comparison of biofilm growth kinetics in the liquid-liquid system and in the

  The mineral salt solution composition was (NH 4 ) 2 SO 4 : 2 g, Na 2 HPO 4 : 3.61 g, KH 2 PO 4 : 1.75 g, MgSO 4 ,7H 2 O: 0.2 g, CaCl 2 : 50 mg, FeSO 4 ,7H 2 O: 1 mg, CuSO 4 ,5H 2 O: 50 mg, H 3 BO 3 : 10 mg, MnSO 4 ,5H 2 O: 10 mg, ZnSO 4 ,7H 2 O: 70 mg, (NH 4 ) 6 Mo 7 O 24 ,4H

  2

Table 1 Effect of the aqueous phase aeration and the dodecane distribution on the biofilm growth during bioremediation process.

 1

	Biofilm at the end of the growth
	Distribution in the column
	(% of dry weight)

b Between 6 and 12 cm below the surface c Between 12 and 18 cm below the surface