Dr R Caubet
email: r.caubet@istab.u-bordeaux1.fr

F Pedarros-Caubet

W J Ellison

The Quantitative Assessment of Antimicrobial Agents: The Case of Sub-Inhibitory Concentrations of Eucalyptus globulus Essential Oil Against Staphylococcus aureus

Keywords: antimicrobial, conductancemetry, essential oil, Eucalyptus globulus, growth parameters, Staphylococcus aureus

We have applied techniques that enable us to measure quantitatively the action of an antimicrobial agent upon the five characteristic growth parameters of a microbial culture. The methods were used to study the action of Eucalyptus globulus (Myrtaceae) essential oil upon a suspension of Staphylococcus aureus in a standard growth medium. We showed that the Eucalyptus oil produced a cultivable population reduction during both the adaptation phase and the exponential growth phase with the same specific reduction rate. This reduction rate was determined as an explicit function of the oil concentration. The oil does not affect the adaptation time. Our results suggest that the oil inactivates the bacteria via a "target" that is accessible to the oil whilst the internal cell transformation prepare the cell for its exponential growth period, but later, as the cells transform themselves into the stationary phase, this target ceases to be "accessible" to the oil. This process is reversible; the change from "accessible" to "inaccessible" occurs when the bacteria are transforming into the stationary phase.

Introduction

Many plant oils and extracts have been traditionally used as topical antiseptics or have been reported to have antimicrobial properties. With the resurgence of interest in natural therapies and increasing consumer demand for effective, safe, natural products quantitative data on the antimicrobial effects of plant extracts are required. This interest is reflected by the large number of articles concerned with the screening of potential antimicrobial agents published in this journal such as Da [START_REF] Da Silva | Antibacterial and antifungal activity of volatile oils from Zanthoxylum Rhoifolium leaves[END_REF], [START_REF] Dob | Composition and antimicrobial activity of the essential oil of Thymus fontanesii[END_REF], [START_REF] Dulger | Antimicrobial activity of some endemic Scrophulariaceae from Turkey[END_REF], Kuçuk et al. and Mahid (2006) to cite a few. These, and many similar articles, all use either the agar disc diffusion technique or the broth dilution method to test the possible antimicrobial activity via the determination of a minimum inhibitory concentration (MIC). The results obtained by the disc diffusion and dilution methods for the same microbe and antimicrobial agent may vary between assays and there is often only a weak correlation between the results obtained by the two methods [START_REF] Donaldson | Assessing of antimicrobial activity of fourteen essential oils when using dilution and diffusion methods[END_REF]). The usefulness of these methods is limited to the generation of preliminary qualitative data and they cannot give any more precise information than the estimation of a concentration threshold that delimits an "inhibition -no inhibition" effect. It is very important to screen a large number of potential antimicrobial agents before undertaking more sophisticated investigations.

In order to progress in our understanding of the action of an antimicrobial agent we must have quantitative information about how the agent modifies the principal parameters that characterize the growth of a microbial suspension in a medium containing the potential antimicrobial agent:

The adaptation time of the microbe to the medium.

A specific reduction rate of the cultivable cell population during the adaptation phase due to the action of the antimicrobial agent.

A specific growth rate of the cell population during the exponential growth phase.

If the antimicrobial agent does not totally inhibit growth, the maximum cell concentration attained at the beginning of the stationary growth phase.

A specific reduction rate of the cultivable cell population during the stationary phase due to the action of the antimicrobial agent. Certain agents, depending upon their concentration, might modify one or more parameters, others might only affect the adaptation time and yet others might only act upon dividing cells and so affect only the exponential growth phase. An antimicrobial agent is deemed to have an action if it modifies at least one of the above parameters.

In naturally occurring situations when antimicrobial action is required, the bacteria are in one of two forms: as a planktonic suspension or as a sessile biofilm. The study of antimicrobial action against biofilm bacteria is certainly very important, but it also requires very specific techniques. Biofilm bacteria proffer unexpected resistance to standard prophylaxis, due partly to the protection afforded by the extra-cellular polymeric substances that form the biofilm. See [START_REF] Caubet | A radio frequency electric current enhances antibiotic efficacy against biofilm bacterial biofilms[END_REF] for a technique using antimicrobial molecules combined with electric fields and for further references. Here, we restrict our attention uniquely to the investigation of the action of antimicrobial agents against microbes in suspension.

We have developed new original methods [START_REF] Ellison | Automatic and rapid measurement of microbial suspension growth parameters: application to the evaluation of effector agents[END_REF]), that can be used with automatic multicell conductance or multi-cell light absorbance techniques. Compared with the usual applications of these techniques our methods give precise quantitative information about the action of an antimicrobial agent upon the five suspension growth parameters as a function of the antimicrobial concentration. Our methods have a light work-load, avoid most of the traditional CFU plate-counts and can provide real-time specific growth rates. This latter possibility is useful when investigating the effects of physical and chemical stresses upon microbial growth.

As an illustration of these methods, we present a study of the antimicrobial activity of a commercially available Eucalyptus (Myrtaceae) essential oil against Staphylococcus aureus. Eucalyptus extracts are often used in oral hygiene products (mouth rinses, toothpastes, anti-plaque chewing gums), surface cleaning 'wipes' etc. Many screening reports, using disc diffusion and dilution techniques, have established an antimicrobial activity of Eucalyptus extracts from various species against a number of pathogens including [START_REF] Inouye | Screening of the antibacterial effects of essential oils on respiratory tract pathogens, using a modified dilution assay method[END_REF] (Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus), [START_REF] Farah | Composition chimique et activité antibactérienne et antifongique des huiles essentielles extraites des feuilles d'Eucalyptus camaldulensis et de son hybride naturel (clone 583)[END_REF] (Escherichia coli, Staphylococcus aureus, Aspergillus niger), [START_REF] Takarada | A comparison of the antibacterial efficacies of essential oils against oral pathogens[END_REF] (Porphyromonas gingivalis, Streptococcus sobrinus, Streptococcus mutans), [START_REF] Wilkinson | Antibacterial activity of essential oils from Australian native plants[END_REF] (Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa), [START_REF] Mounchid | The susceptibility of Escherichia coli to essential oils of Romarinus officinalis and Eucalyptus globulus[END_REF] (Escherichia coli) and [START_REF] Schelz | Antimicrobial and antiplasmid activities of essential oils[END_REF] (Escherichia coli, Staphylococcus epidermidis). To our knowledge, there is no published investigation of the effect of Eucalyptus oil on any of the parameters characterizing the growth phases of a microbial suspension.

Our growth paradigm is derived from the "three linear phase" model described by Buchannan et al. (1997), in which T Adapt (c) and µ Exp (c) are precisely defined. We have the following possible actions when Staphylococcus aureus cells are inoculated in a medium containing a concentration c of Eucalyptus oil:

The oil modifies the adaptation time, T Adapt (c), of the cells to the medium.

The oil causes a Cultivable Population Reduction (CPR) during the adaptation phase (CPR Adapt ). Thus, during the adaptation time, the cell concentration will decrease with a specific rate μ Adapt (c).

The oil modifies the specific exponential growth rate µ Exp (c) of the suspension.

The oil modifies the maximum cell concentration, CFU Max (c), of the suspension.

The oil causes a Cultivable Population Reduction during the stationary phase (CPR Stat ). Thus, during the stationary phase, the cell concentration will decrease with a specific rate μ Stat (c).

We shall determine T Adapt (c), µ Adapt (c), µ Exp (c), CFU Max (c), µ Stat (c) for a suspension of S. aureus in a standard growth medium containing a concentration c, 0 ≤ c ≤ 1600 ppm, of Eucalyptus oil and discuss the implications of this data and directions for further studies designed to elucidate the mode of action of Eucalyptus essential oil against this bacteria.

Material and Methods

Apparatus

The conductance data were obtained with the Rapid Automatic Bacterial Impedance Technique (RABIT), Don Whitley Scientific, Shipley, U.K.

Conductance growth medium

Whitley Impedance Broth (W.I.B.), a non-selective medium designed to have relatively large conductivity changes as a function of microbial growth, (Don Whitley Scientific).

Bacteria and temperature conditions

Staphylococcus aureus (C.I.P. 103429) was stored in a glycerol peptone medium at -80°C. Freshly thawed aliquots were used for each experiment. The growth temperature was 37°C.

Suspended bacteria culture

A pre-culture was made by inoculating frozen cells in 10 mL of Müller-Hinton broth. After incubation for 24 h at 37°C, 2 mL of the pre-culture was inoculated in 150 mL Müller-Hinton broth contained in a 250 mL Erlenmeyer flask and shaken at 120 rpm for 24 h in a water bath (type SW23, Julabo, Germany) at 37°C. Samples (0.1 mL) of different cell concentrations were then inoculated into 4 mL of W.I.B. medium at 37°C containing known a known concentration of antimicrobial agent.

CFU/ml counts

The CFU/ml counts were made using the spiral colony counting method (Whitley Automatic Spiral Plater, Don Whitley Scientific). The inoculated plates of Plate Count Agar (PCA), Difco, U.S.A., were incubated for 24 h at 37°C before counting the colonies.

The antimicrobial agent

The Eucalyptus (Eucalyptus globulus) was a commercial essential oil. No further refinement or chemical analysis was performed. It was stored at 4 °C. A 10% (volume-volume) mother solution was prepared using DMSO. Eucalyptus oil concentrations in the W.I.B. medium were in the range 0 to 1600 ppm.

The suspension growth parameters protocol

The protocols and the mathematical analysis behind our methods are fully described in [START_REF] Ellison | Automatic and rapid measurement of microbial suspension growth parameters: application to the evaluation of effector agents[END_REF]. We recall here the minimum explanation necessary to understand how the results are obtained. We use an automatic conductance method to monitor the microbial growth. With modern devices one can continuously monitor several hundred suspension samples simultaneously. The principle of the conductance method is that a growing microbial suspension, by the excretion of ionic metabolic substances into the surrounding liquid medium, modifies the conductivity of the growth medium. We have shown that, in the case under consideration, the conductance changes for S. aureus are proportional to the cell concentration. Thus, automatic monitoring of the conductance of the suspension gives real-time information about the microbial cell concentration. For very low cell concentrations the conductance change is not detectable, as growth continues the cell concentration increases to a certain value N Det , at which point the apparatus does detect a conductance change. The time, T Det , after inoculation of the microbes in the growth medium at which this occurs is called the 'detection time'. For suspension growing in a growth medium containing a concentration c of antimicrobial agent there is a relationship between the initial cell concentration C 0 , the adaptation time T Adapt (c), the CPR reduction rate µ Adapt (c), the specific exponential growth rate µ Exp (c) and the detection time:

Log C 0 = -µ Exp (c)T Det + β(c), where the adaptation factor β(c)

β(c) = (µ Exp (c) + µ Adapt (c))T Adapt (c) + Log N Det .
The quantities µ Exp (c), β(c), T Adapt (c), µ Adapt (c), CFU Max (c) and µ Stat (c) can be determined by a series of experimental protocols involving the automatic measurement of detection times. They are described in detail in [START_REF] Ellison | Automatic and rapid measurement of microbial suspension growth parameters: application to the evaluation of effector agents[END_REF]. The only plate-counting necessary is for the determination of the initial suspension cell concentrations. The first step is to determine µ Exp (c) and β(c) for a series of inhibitor concentrations. The second step is to determine the adaptation time T Adapt (c), for the same concentrations. Once the adaptation time is know one can determine µ Adapt (c) either from the above formula, since N Det is known. The penultimate step consists in the determination of CFU Max (c) and whether or not there is a CPR during the stationary phase for each concentration. If there is a CPR, then one must determine µ Stat (c).

Acquired microbial resistance protocol

We shall see that S. aureus cells in contact with Eucalyptus oil and grown until a stationary phase is reached are resistant to inactivation by the oil. The resistance may be due to a temporary adaptation or to a more permanent modification of the cells. To distinguish between these two possibilities we evaluate this resistance by growing suspensions of S. aureus in W.I.B. or W.I.B + Eucalyptus oil until the stationary phase is reached, then inoculating stationary phase bacteria into either pure W.I.B. or into W.I.B. + Eucalyptus oil and recording the conductance data. The cycle inoculation, growth to stationary phase is repeated several times. The degree of acquired microbial resistance to the Eucalyptus is reflected in the detection times.

Statistical analysis

Linear regressions were performed using a maximum likelihood analysis rather than a least squares method, as the latter is very sensitive to the occasional and inevitable outlaying points. Non-parametric significance tests for differences between mean values were used, since the necessary hypotheses required for using Student's t-test could not be verified.

Results

The relationship between the initial cell concentration C 0 and the conductance detection time T Det for a S. aureus suspension in pure W.I.B. is given by: Log C 0 = -µ Exp (0)T Det + β(0), where µ Exp (0) = 1.97, β(0) = 21.18, with r² = 0.997.

The adaptation time is given by the formula T Adapt (0) = (β(0) -LogN Det )/µ Exp (0), where N Det is the cell concentration that produces a conductance change detection. For this species of S. aureus N Det = 5.2x10 6 CFU/ml. This gives an adaptation time of T Adapt (0) = 2.9 h for stationary phase S. aureus, cultured in Müller-Hinton broth inoculated into pure W.I.B. medium.

Determination of µ Exp (c) and β(c)

The specific exponential phase growth rate µ Exp (c) and the factor β(c) for Eucalyptus oil concentrations in the range 0 ≤ c ≤ 1600 ppm are shown in Fig. 1. A maximum likelihood regression analysis gives µ Exp (c) = 1.97 -0.0007c, with a linear correlation coefficient r² = 0.98 and β(c) is constant and equal to 22.267, with sd = 0.392. Thus, β(c) is independent of the oil concentration. This is a special situation and, as shown in [START_REF] Ellison | Automatic and rapid measurement of microbial suspension growth parameters: application to the evaluation of effector agents[END_REF], suggests that for the oil concentration range 0 to 1600 ppm there is a CPR during both the adaptation and the exponential phases with the same specific reduction rate = µ Exp (0) -µ Exp (c) = 0.0007c and that the adaptation time T Adapt (c) of S. aureus from the stationary phase in the Müller-Hinton broth to the W.I.B. + Eucalyptus mixture does not depend upon the oil concentration. Thus T Adapt (c) should be equal to 2.9 h for all c up to 1600 ppm.

Adaptation phase results

In figure 2 we have the results of the adaptation phase experiments for 500, 1000 and 1500 ppm oil concentrations. After inoculation, the detection time differences between samples with and without oil decrease, this corresponds to the CPR during the adaptation phase. Once the bacteria have adapted to the medium the exponential growth phase begins with a linear relationship between contact time and detection time difference. The increasing straight line begins when the bacteria have been in contact with the Eucalyptus oil for slightly less than 3 h for each concentration. This represents the adaptation time, which is in very good agreement with the 2.9 h obtained from the previous experiments and confirms that the Eucalyptus oil concentration has no notable effect upon adaptation time.

As an independent check upon the estimation of T Adapt (c) we carried out CFU plate-counts every 30 min during the period from inoculation up to detection time for an oil concentration of 1000 ppm. This enabled us to determine independently the CPR during the adaptation phase and so determine T Adapt (1000). The results are shown in figure 3. Again, the slope change occurs slightly less than 3 h after the inoculation.

Stationary phase results.

The conductance changes as a function of time and Eucalyptus concentration are shown in Fig. 4. In our conditions the CFU/ml cell concentration is proportional to the conductance change and so the relative cell concentrations are the same as the relative conductance changes [START_REF] Ellison | Automatic and rapid measurement of microbial suspension growth parameters: application to the evaluation of effector agents[END_REF]). The maximum CFU concentration is always the same. The only variability is the time taken to reach this maximum value. Thus CFU Max (c) does not depend upon c.

The detection times of suspension samples taken during the first 24 h of the stationary phase and inoculated into pure W.I.B. are shown in figure 5. The detection times show no significant differences between all the oil concentrations and all stationary phase samples taken over the 24 h period. This means that the stationary phase cell concentrations are not significantly different. Hence, there is no CPR during the first 24 h of the stationary phase.

The microbial resistance persistence

The results for a Eucalyptus oil concentration of 1600 ppm are shown in table 1. The significance of the inoculation growth cycle notation is as follows: For example, "W+E → W → W+E" means:

Samples In all samples, there was no significant difference in the microbial growth characteristics from the detection time up to the resulting stationary phase. This means that once the exponential growth phase begins, the past history of the cells does not influence the subsequent microbial growth.

Discussion

It is obviously important to have some information about the Eucalyptus oils' composition, but large variations exist, both in extraction methods and in the plants themselves. For example, Della Port et al. (1999) analyzed the composition of Eucalyptus globulus. They found that with one type of extraction method the principal components were 1,8-cineole 62.6%, α-pinene 10.5%, aromadendrene 8%, p-mentha-1,3,5-triene 1.6%. A different extraction method, using the same source of Eucalyptus leaves, gave the components: 1,8-cineole 48.2%, α-pinene 6.9%, p-mentha-1,3,5-triene 3.8% and aromadendrene 13.7%. Other published composition analyses naturally vary in the percentages of each component. This illustrates one of the major difficulties working with essential oils, namely the difficulty of obtaining reproducible samples. In our case this is not a problem.

The principal component is not necessarily the active antimicrobial component. One cannot draw hasty conclusions about the active component of the oil uniquely from a standard chromatography analysis. The antimicrobial effect of Eucalyptus essential oils has sometimes been ascribed to the presence of 1,8-cineole, its principal component. This is not necessarily true. [START_REF] Lis-Balchin | Bioactivity of selected plant essential oils against Listeria monocytogenes[END_REF] tested Eucalyptus globulus, Eucalyptus radiata, and Eucalyptus citriodora against 20 different strains of Listeria monocytogenes. The major component of Eucalyptus globulus was 1,8-cineole (90.8%), in Eucalyptus radiata it was present at 84% and in Eucalyptus citriodora it was only present at 0.005%. However, the Listeria strains used in the study did not correlate with the 1,8-cineole concentration. The Eucalyptus globulus inhibited 6 out of 20 strains, the E. radiata and E. citriodora inhibited all 20 strains. The above results indicate that 1,8-cineole is not necessarily the principal active agent, at least for Listeria strains. [START_REF] Belaiche | Application of a two levels factorial design to the study of the antimicrobial activity of three terpenes[END_REF] studied the antimicrobial effects of three components of Eucalyptus oil: 1,8-cineole, linalool and eugenol upon Staphylococcus aureus. They found that 1,8-cineole had no effect, but the other two terpenes, used individually, had significant effects, but used at the same time had antagonistic effects. [START_REF] Farah | Composition chimique et activité antibactérienne et antifongique des huiles essentielles extraites des feuilles d'Eucalyptus camaldulensis et de son hybride naturel (clone 583)[END_REF][START_REF] Farah | The essential oils of Eucalyptus camaldulensis and its natural hybrid (clone 583) from Morocco[END_REF] also confirmed the lack of antimicrobial activity of 1,8-cineole against Staphylococcus aureus, Escherichia coli and Aspergillius niger.

In this report we are concerned with the general effect of the oil upon the growth parameters of a S. aureus suspension. The detailed study of each of the principal components of Eucalyptus essential oil will be the object of a separate study.

The adaptation and exponential phase results

For Eucalyptus globulus oil concentrations in the range 0 to 1600 ppm there is a CPR during the adaptation phase, with a specific reduction rate µ Adapt (c) proportional to the oil concentration. During the exponential growth phase the observed specific growth rate is the difference between the specific growth rate in pure W.I.B. and µ Adapt (c). Thus, the reduction in the specific growth rate during the exponential phase is the same as the CPR during the adaptation phase. This suggests that the action of the Eucalyptus globulus oil does not depend upon the cell states during the adaptation and exponential phases.

The adaptation time of S. aureus to the W.I.B. + oil growth medium over the concentration range is practically constant and corresponds to the adaptation time of S. aureus pre-cultured in Müller-Hinton broth and inoculated into W.I.B. This suggests again that the presence of the Eucalyptus oil in the growth medium does not affect in a substantial way the internal metabolic cell transformations involved in the adaptation process.

The stationary phase results

During the adaptation and exponential phases Eucalyptus globulus produces a CPR, with a specific reduction rate µ(c) that depends upon the oil concentration c. If this specific reduction rate is less than the cell division rate, then it is to be expected that, once the stationary phase is reached, CFU Max (c) = CFU Max (0). In fact, we do observe that there is no effect upon CFU Max (Figure 4). Whatever the initial concentration in cells or concentration in Eucalyptus, the final cell concentration in the medium is always the same. The only factor that varies is the time necessary to reach this maximum cell concentration.

During the stationary phase there is no CPR. This is slightly surprising. The hypothesis that there is some component of the oil that has a non specific destructive effect upon the integrity of the cell membrane is frequently evoked, often with little or no convincing experimental evidence. If there is some such agent in Eucalyptus oil, then one would also expect it to continue acting during the stationary phase, i.e. one should observe a CPR and this is not the case. A possible explanation is that this hypothetical non specific inactivating agent is depleted as the suspension grows. If this is the case then once the inactivating agent has been reduced to a concentration below a threshold level, the instantaneous specific growth rate µ(t,c) in the medium containing a concentration c of Eucalyptus, should become equal to µ(t,0) at some point before the stationary phase is reached. The time at which this occurs corresponds to the moment at which all the 'inactivating factor' in the Eucalyptus oil has been reduced to below its threshold level. The instantaneous specific growth rates as a function of time and as a function of instantaneous cell suspension concentration are shown in figures 6 and 7. We observe that µ(t,c) does not converge to µ(t,0) at some time before the beginning of the stationary phase and that in figure 7,µ(t,c) is equal to µ(t,0) for all Eucalyptus oil concentrations only when the cell suspension concentration is close to the stationary phase cell concentration. This indicates that the lack of CPR during the stationary phase is not due to some inhibiting factor in the Eucalyptus that has been depleted. This leaves us with the possibility that it is the bacteria themselves have changed in some way so as to be resistant to the Eucalyptus oil in the medium. The second representation of the specific growth rates (figure 7) shows that this adaptation is achieved, for all the Eucalyptus oil concentrations used, when the cell concentration has reached about 70% of its final value. This is very near to the final cell concentration and the microbes are either in, or close to, a stationary phase physiological state.

The resistance of the stationary phase S. aureus to the Eucalyptus oil suggests the following phenomenological scenario: Suppose that S. aureus cells possess a "target" via which some component of the Eucalyptus oil inactivates the cell.

When the target is "accessible" the cell is inactivated. If, due to physiological transformations within the cell, the target becomes "inaccessible", then the oil does not inactivate the cell.

When such cells are inoculated into a medium containing Eucalyptus there is a CPR during the initial adaptation phase and during the exponential growth phase, so the target is accessible.

As the stationary phase is reached, internal physiological transformations make the target inaccessible and so the cells are not inactivated by the Eucalyptus.

This scenario is, of course, very simplified, but it has the merit of being testable experimentally. We tested the existence of this phenomenon.

The results indicate that there is no residual effect of the acquired resistance to inactivation of the stationary phase cells by Eucalyptus oil when these cells are re-inoculated into the growth medium containing Eucalyptus oils and that successive growth cycles of cells in a medium containing Eucalyptus oils do not modify the acquired resistance of the stationary phase cells. These observations suggest that the resistance to the Eucalyptus is not due to some mutation effect but that the accessibility -inaccessibility of the Eucalyptus target is the result of reversible physico-biochemical transformations within the cells as they enter the stationary state.

Conclusion

We have applied techniques that enable us to measure quantitatively the action of an antimicrobial agent upon the five characteristic growth parameters of a microbial culture. The methods were used to study the action of Eucalyptus globulus essential oil upon a suspension of Staphylococcus aureus in a standard growth medium. We showed that the Eucalyptus oil produced a cultivable population reduction during both the adaptation phase and the exponential growth phase with the same specific reduction rate. This reduction rate was determined as an explicit function of the oil concentration. The oil does not affect the adaptation time. Our results suggest that the oil inactivates the bacteria via a "target" that is accessible to the oil whilst the internal cell transformation prepare the cell for its exponential growth period, but later, as the cells transform themselves into the stationary phase, this target ceases to be "accessible" to the oil. This process is reversible; the change from "accessible" to "inaccessible" occurs when the bacteria are transforming into the stationary phase. The next step in the elucidation of the action of Eucalyptus oil against S. aureus would be to test individually, and in combination, different fractional components of the oil in order to determine those that do have an effect upon the bacteria.

FIGURE CAPTIONS

  are inoculated in W.I.B. containing a 1600 ppm Eucalyptus concentration and allowed to grow to a stationary phase. Samples are then inoculated in pure W.I.B. and allowed to grow to a stationary phase. Samples are inoculated in W.I.B. containing a 1600 ppm Eucalyptus concentration and allowed to grow until a conductance change is detected. The detection time of the final suspension is recorded. Each of the cycle experiments was performed with n = 8 samples. The detection time for S. aureus growing in pure W.I.B. is 1.4 h. When the cells reach a stationary phase and are re-inoculated into W.I.B. containing 1600 ppm of Eucalyptus oil the detection time is 6.4 h. Cells cultivated in W.I.B. + 1600 ppm Eucalyptus until they reach a stationary phase and then re-inoculated into pure W.I.B. again have a detection time of 1.4 h. Thus Eucalyptus resistant cells, when inoculated into pure W.I.B. have a normal growth pattern. S. aureus cells that have undergone two or three growth cycles in W.I.B. + 1600 ppm Eucalyptus oil do not show any change in the corresponding detection times. Thus, the acquired resistance of the stationary phase cells is not modified by successive contacts with Eucalyptus oil.

Fig. 1 .

 1 Fig. 1. Specific growth rate µ Exp (c) (♦) and β(c) (■) as functions of Eucalyptus oil concentration.

Fig. 3 .

 3 Fig. 3. CFU/ml counts v Contact time for a concentration of 1000 ppm Eucalyptus oil.

Fig. 4 .

 4 Fig. 4. The conductance changes of the S. aureus suspension as a function of the contact time for each Eucalyptus oil concentration.

Fig. 5 .

 5 Fig. 5. Detection times of samples taken during the stationary phase and inoculated into pure growth medium.Fig. 6. Instantaneous specific growth rate, as function of time, for different oil concentrations.

Fig. 7 .Fig. 1 .Fig. 2 .

 712 Fig. 7. Instantaneous specific growth rate as function of the maximum cell concentration percentage for various oil concentrations.

Fig. 4 .Fig. 5 .Fig. 6 .Fig. 7 .

 4567 Fig. 3. CFU/ml counts v Contact time for a concentration of 1000 ppm Eucalyptus oil.

TABLETable 1 :

 1 Detection times for the various growth to stationary phase cycles (W = pure W.I.B., W + E = pure W.I.B. + 1600 ppm Eucalyptus oil).

	Cycle	Mean Detection time (h) sd
	W	1.4	0
	W → W+E	6.4	1.7
	W+E→W	1.4	0
	W+E→W→ W+E	6.3	1.3
	W+E→W+E→W+E	6.3	0.8

Acknowledgments

We would like to thank the 'Région d'Aquitaine, France' for partial financial support in the acquisition of the conductance device and Spiral Plater apparatus.


V Laur

R Benzerga
email: r.benzerga@univ-rennes1.fr

R Lebullenger

L Le Gendre

G Lanoë

A Sharaiha

P Queffelec

Green foams for microwave absorbing applications: synthesis and characterization

Keywords: Dielectric properties, Glasses, Inorganic compounds, Glass foams, Electromagnetic absorption

Highlights

 Carbon-filled CRT-based glass foams have been studied for microwave application  The dielectric loss of a glass foam made with 1.5 %wt. C are tan=1.4 @ 9 GHz.

A combination of C, MnO2, AlN and TiO2 as foaming agent reduces the foam density Glass foams properties depend both on their carbon content and their densities Residual C in foam made with 1.5%C induces a shielding efficiency of 18dB/cm @ 12GHz  Simulated reflection coefficient of pyramidal absorbers are lower than -30dB

Abstract: This study deals with the dielectric properties of recycled glass foams and their use as microwave absorbers. Glass wastes from Cathode Ray Tube have been used to produce foams by heating a mixture consisting of glass cullet and foaming agents (carbon or a combination of carbon, TiO2, MnO2 and AlN). As only part of the carbon introduced into the initial load oxidizes and contributes to the foaming process, the residual fraction acts as an electromagnetic absorber. The effect of the carbon load rate on the foams density and microwave properties was studied. These materials present a low permittivity and high dielectric losses in X-band. Foams density mainly controls the dielectric properties. Carbon-based glass foams show a great potential as microwave absorbers with a shielding effectiveness of 20 dB/cm at 12 GHz. Moreover, a tradeoff between the density and shielding efficiency can be achieved by controlling the load rate of carbon.

Introduction

Recycling materials is a burning issue in our modern societies. Indeed, with the continuous improvement of people's living standard, the needs in raw materials increase whereas resources drastically decrease. Moreover the reintroduction of end-of-life materials in the production chain also reduces global energy consumption, which directly reduces greenhouse gas emissions. In this context, since early seventies, recycling chains of glass wastes were developed with a great success and glass containers (bottles) are now widely recycled. That way, the cullet resulting from the collection of used liquid-food packaging in the form of bottles and jars covers 70% of the glass industry needs in terms of raw materials. As the melting temperature of the cullet (1000°C) is lower than that of the raw materials (1500°C), the glass industry's carbon balance is significantly improved [START_REF] Pasqualino | The carbon footprint and energy consumption of beverage packaging selection and disposal[END_REF][START_REF] Schmitz | Energy consumption and CO2 emissions of the European glass industry[END_REF]. Other types of glass compositions, often polluted with heavy metals, pose a tougher problem. Indeed, as hazardous materials, glass wastes from electronic industry (e-wastes) should be treated in specific recycling process [START_REF] Singh | Global responses for recycling waste CRTs in e-waste[END_REF]. This is particularly the case of cathode ray tubes (CRT) found in older style TV screens and monitors that can be neither recycled in a closed loop production process due to the termination of this technology nor reintroduced as a cullet in the production line of conventional hollow glasses due to their high heavy metal content (Pb, Ba, Sr) [START_REF] Yot | Characterization of lead, barium and strontium leachability from foam glasses elaborated using waste cathode ray-tube glasses[END_REF]. Foam glass manufacture is a promising mode for re-using CRT glasses: depending on the foaming process, this cellular material combines low density, low thermal conductivity, excellent thermal stability and high rigidity [START_REF] Pasqualino | The carbon footprint and energy consumption of beverage packaging selection and disposal[END_REF][START_REF] Schmitz | Energy consumption and CO2 emissions of the European glass industry[END_REF][START_REF] Singh | Global responses for recycling waste CRTs in e-waste[END_REF][START_REF] Yot | Characterization of lead, barium and strontium leachability from foam glasses elaborated using waste cathode ray-tube glasses[END_REF][START_REF] Yatsenko | Foamed slag glass -eco-friendly insulating material based on slag waste[END_REF][START_REF] Petersen | The mechanism of foaming and thermal conductivity of glasses foamed with MnO2[END_REF][START_REF] Benzerga | Waste-glass recycling: A step toward microwave applications[END_REF][START_REF] Lebullenger | Glass foam for environmental applications[END_REF]. As it has been demonstrated by leaching tests that the fraction of lead released, in end-use conditions, from foam glass was lower than the statutory limit [START_REF] Yot | Characterization of lead, barium and strontium leachability from foam glasses elaborated using waste cathode ray-tube glasses[END_REF], this material can be inserted in a commercial device. As the primary function of those leaded glasses was to block electromagnetic (EM) radiation in CRTs, we focused our attention on EM absorptive properties of those glass foams based on cathode ray tube cullet. Inorganic additives (powders or fibers) can be used to reinforce the shielding properties in a specific spectral range [START_REF] Singh | Global responses for recycling waste CRTs in e-waste[END_REF][START_REF] Kazmina | Microwave absorption properties of foam glass material modified by adding ilmenite concentrate[END_REF][START_REF] Suslyaev | Electrophysical characteristics of a foam glass crystal material[END_REF][START_REF] Legendre | New radar absorbent material based of carboned foam glass application to pyramidal radar absorber[END_REF]. Indeed, when Aluminum Nitride (AlN) or Silicon Carbide (SiC) are used to initiate the foaming process, part of those adds do not chemically react with the glassy matrix and, as a result, glass foams present a low permittivity together with low dielectric losses [START_REF] Benzerga | Waste-glass recycling: A step toward microwave applications[END_REF]. In the same way, when Carbon (C) is used as the foaming agent, high dielectric losses are observed making of these foams a good candidate for microwave absorbing applications [START_REF] Benzerga | Waste-glass recycling: A step toward microwave applications[END_REF][START_REF] Legendre | New radar absorbent material based of carboned foam glass application to pyramidal radar absorber[END_REF].

In this paper, we studied the microwave behavior of glass foams for different load rates of carbon.

In a first part, the process of elaboration will be presented. We especially focused on the impact of the load rate and nature of the foaming agent on the density of the foams. Indeed, an original combination of foaming agent based on the results of previous studies [START_REF] Lebullenger | Glass foam for environmental applications[END_REF][START_REF] Tasserie | New expanded material based on industrial glass. II. Role and effect of the amount of an expansive additive on the characteristics of the material[END_REF][START_REF] Tasserie | A new expanded material: optimization of the fabrication process[END_REF] has been tested to reach a low-density C-doped foam glass. Then, the microwave properties of these foams will be presented and their potentiality for microwave absorbing applications will be discussed.

Materials and foam manufacturing

The elaboration process of the foams is illustrated in Fig. 1 and the synthesis parameters are summarized in Table 1. As a first step, the cullet and the foaming agent (C for example) are In this study, the CRT cullet is a mixture of panel (front part) and funnel (rear part) glasses. The weight ratio of the mixture is near 2/3 (panel) and1/3 (funnel). Front glass is typically a SiO2 based glass containing barium and strontium oxide, while funnel glass is also a SiO2 based glass but containing around 20% in lead oxide. So, the CRT cullet is a mixture of silicate glasses containing between 5 to 7 wt% in lead oxide. This CRT glass cullet is supplied by Environment Recycling, a French company expert in WEEE dismantlement. A crushing step is achieved on CRT mixture with a Gabrielli Fast-Mill planetar mortar with alumina balls, to obtain a fine powder whose granulometry is lower than 125 µm. The CRT cullet pre-dried at 150°C for 12 hours is milled together with the carbon foaming agent (from Ventron Alfa Produdkte with a granulometry of 325 meshes / 44 µm) or a combination of Carbon (C), Manganese dioxide (MnO2), Aluminum nitride (AlN) and Titanium dioxide (TiO2)). Then, the resulting mixture is placed into a heat resistant steel mold and heated at 850°C during 30 min. At this temperature, bubbles occur as the carbon is oxidized to CO2 or as the nitride ions are oxidized in dinitrogen or as dioxygen results from the MnO2 to Mn2O3 reduction [START_REF] Petersen | The mechanism of foaming and thermal conductivity of glasses foamed with MnO2[END_REF]. Note that in the combination of foaming agents, TiO2 acts as a sacrificial oxidant in order to promote the gas-releasing reaction.

Methods of characterization

For the microstructure observation, and as given the millimeter-sized porosities of the foam glasses, the structure of the samples was observed by a Leica camera (DFC295) coupled with a stereomicroscope Leica MS5. The density of the glass foams was calculated using the measured mass and volume of each sample.

For the dielectric characterization, the properties of the foams were extracted by a reflectiontransmission method between 8 and 12 GHz (X-band). This technique is based on a quasi-static analysis of a rectangular waveguide loaded by a sample to be characterized [START_REF] Arbaoui | Full 3D printed microwave termination: a simple and low-cost solution[END_REF]. After a Thru-Reflect-Line calibration [START_REF] Arbaoui | Full 3D printed microwave termination: a simple and low-cost solution[END_REF], S-parameters of the empty waveguide are measured with a vector network analyzer (VNA Agilent E8364A) in order to take into account metallic losses. The glass foam sample is cut to the dimension of the waveguide (22.86 mm x 10.16 mm x 20 mm) and inserted for measurements. The modifications of S-parameters (both amplitude and phase) are analyzed by a Nicolson-Ross-Weir procedure [START_REF] Nicolson | Measurement of the Intrisic Properties of Materials by Timedomain Techniques[END_REF][START_REF] Weir | Automatic Measurement of Complex Dielectric Constant and Permeability at Microwave Frequencies[END_REF] in order to extract the complex permittivity (ε* = ε' -jε'') of the samples.

Results and discussion

Effect of the foaming agent content

To check the influence of materials density on the electromagnetic properties we prepared samples with different amount of carbon [0.5 wt% -5 wt%] on the one hand, and with a mixture of carbon, TiO2, AlN and MnO2 on the other. Fig. 2 shows the influence of the carbon foaming agent content on the apparent density of the foams. As previously observed [START_REF] König | Fabrication of highly insulating foam glass made from CRT panel glass[END_REF][START_REF] Zhang | Preparation of high strength glass ceramic foams from waste cathode ray tube and germanium tailings[END_REF], this curve presents a minimum density for a carbon load rate between 2 and 3 wt%. Indeed, a low C concentration results in a moderate amount of gas release that leads to weakly expanded parts (d0.5 wt% C = 0.58 g/cm 3 ).

On the other hand, a carbon overload generates an internal gas pressure that the viscous liquid phase cannot retain. In that case, too many bubbles grow and coalesce leading to the collapse of the foam (d5% C = 1.76 g/cm 3 ). Fig. 3 presents images of the foams obtained with different carbon content which confirm this bubbles growth process. Up to 1.5 C wt%, one can see an homogeneous pore size distribution resulting from the trapping of near-spherical bubbles whose diameters range from 0.5 to 2 mm. The move to higher C load, 2 wt% and 3 wt%, leads to larger pores resulting from gas bubbles collapse and interconnection. That way, for a glass foam prepared with 3 wt% of carbon, the pore size lies within a range of 1 to 5 mm approximately. For excessive amount of carbon (5 wt%), the growth in the number and size of the CO2 gas bubbles reduces the cell wall thickness until the collapse of the foam. In that case, after cooling down to room temperature, one can see (Fig. 3) deformed bubbles residues trapped in the collapsed glassy matrix.

The density of the foam can be reduced by using a combination of foaming agents. Indeed, in addition to 1.5 wt% of carbon, a combination of MnO2 (4 wt%), AlN (2 wt%) and TiO2 (2 wt%) makes it possible to half the density of the resulting foam. Micrographs illustrate the increase of the pore diameters in this case (Fig. 4). This increase in pore size is logically accompanied by a reduction in the thickness of the cell walls to a minimum of 20 m which is still above the critical thickness of rupture (1-20 nm) [START_REF] Petersen | The mechanism of foaming and thermal conductivity of glasses foamed with MnO2[END_REF].

Microwave characterizations and applications

Fig. 5.a presents the evolution of the real part of the permittivity, as a function of the frequency, for load rates of carbon between 1 and 3 wt%. From a general point of view, C-based glass foams present a dispersive behavior. For example, when a 1wt% load rate is considered, the permittivity decreases from 4.5 at 8 GHz to 3.7 at 12 GHz. Dielectric losses (tanδ = ε'' / ε') of the samples are very high (Fig. 5.b) with a maximum value of 1.4 at 9 GHz for a 1.5 wt% load rate of carbon.

However, it appears difficult to directly link the dielectric properties of the samples to the load rate of carbon because of the differences in density between the expanded foams.

Moreover, the very high losses observed on the samples associated with their porous structure suggest that the losses may be due to moisture absorption. Indeed, the high level of porosity of these materials can be accompanied by some water molecules adsorption which may thus be contributing to the microwave absorption. In order to address this point, all samples were oven-dried at 60°C for 18 hours. Measurements before and after drying at 60°C show that microwave absorption is not due to moisture absorption on the foam surface but to its intrinsic high dielectric losses. Fig. 6 presents an example of these measurements: S-parameters of the rectangular waveguide loaded with a 1 wt% carbon-loaded glass foam (sample length = 10.06 mm). This figure presents exactly the same S-parameters for this sample before and after drying step. A very low level of transmission (S21), lower than -19 dB, is observed over the frequency band of measurement. Fig. 7 shows the evolution of the dielectric properties of the glass foams as a function of the density. It appears that dielectric properties are mainly correlated with the density of the resulting foams. Permittivity increases proportionately with density whereas losses show a maximum value for a 1.5 wt% C load rate (d1.5 wt% C = 0.30 g/cm 3 ). A low value of permittivity and high dielectric losses are generally required to get highly efficient microwave absorber materials. As a consequence, the optimum carbon load rate could be chosen around 1.5 wt%.

Lossy materials with a low permittivity are of interest for shielding applications. The shielding efficiency (SE) is defined by the following equation:

in dB/cm ( 1
)
where S21 is the amplitude of the transmission parameters and l the length of the sample.

The shielding efficiency as a function of frequency for different load rate of carbon is presented in Fig. 8. SE is higher than 13 dB/cm for all the foams. A 1 wt%-carbon loaded foam provides the best shielding efficiency with a value higher than 19 dB/cm over the whole frequency band. Now, if we take into account the density of the materials, one could define a new quantity, called here Shielding Efficiency Relative to Mass (SERM):

in dB/g ( 2
)
where d is the density of the foam and S the section of the rectangular waveguide. and 2 wt% give the best results with a SERM value of about 25 dB/g at 10 GHz. These foams seem to be the most efficient, tailored for systems in which the mass is a critical issue.

We pointed out earlier that using a combination of foaming agents (C, AlN, MnO2 and TiO2) helps to reduce the glass foams density while keeping an efficient electromagnetic absorbent C-based fraction. Fig. 10.a compares dielectric losses of the samples prepared using only carbon or a combination of foaming agents. The same carbon amount of 1.5 wt% was used for these two samples. Quite similar dielectric losses are observed over the frequency band between 8 and 12

GHz. Indeed, it appears possible to keep high dielectric losses while strongly reducing the density of the foam by using such a combination of foaming agents. One should note that, due to the pairing of high dielectric losses and low density, a combination of foaming agents (C, AlN, MnO2 and TiO2) enables to increase the SERM. SERM values of 25 dB/g and 29.5 dB/g are obtained at 10 GHz (Fig. 10.b) for, respectively, foam achieved with only carbon as foaming agent (1.5 wt%) and the foam achieved with the combination of foaming agents (1.5 wt% C, 4 wt% MnO2, 2 wt% AlN and 2 wt% TiO2).

Finally, we simulated the performances of a microwave termination consisting of a pyramidal sample of foam inserted in a short-circuited X-band rectangular waveguide. Microwave terminations are devices that are usually integrated in telecommunications systems to protect the other devices from impedance mismatches or undesired electromagnetic radiations received by the antenna. These devices are usually realized by using pyramidal samples of polymer composites or lossy ceramics for low and high power applications respectively.

We performed electromagnetic simulations of a 7 cm long pyramidal foam (Fig. 11). Amplitude of the electric field of the EM wave, inside the pyramidal absorber (Fig. 11.a), shows that the entire EM wave is absorbed before reaching the base of the pyramid. Simulated reflection coefficients are less than -30 dB over the whole frequency band whatever the composition of the foam is (Fig.

11.b). These simulations demonstrate that carbon-based glass foams can be a good compromise between polymer composites which suffer from thermal limitations (i.e. low power handling capability) and lossy ceramics for which the weight and cost remain high.

Conclusion

We synthesized glass foams by using recycled cullet of cathode ray tube (CRT) and particles of carbon as foaming agent. As expected, the carbon load rate impacts the foaming process leading to a large range of foams density [0.23 -0.58 g/cm 3 ]. One can note that an excessive amount of carbon (i.e. 5 wt%) results in a complete collapse of the foam structure (d = 1.76 g/cm 3 ). We demonstrated that a combination of foaming agents (C, AlN, MnO2 and TiO2) leads to highly porous glass foams with a density of 0.16 g/cm 3 .

The microwave properties of these foams were extracted by using a rectangular waveguide method between 8 and 12 GHz. Carbon-based glass foams present dispersive dielectric properties at X-band with low permittivity values and high dielectric losses. Dielectric properties are mainly correlated to the density of the foams. That way, permittivity tends to increase with increasing density whereas dielectric loss reaches a maximum for a 1.5 wt% carbon loaded foam.

These materials present very effective properties for shielding applications. Indeed, shielding effectiveness reaches a value of 20 dB/cm for a 1 wt% carbon load rate. For applications in which mass is a critical issue, 1.5 wt% or 2 wt% load rate seems to be the best compromise between mass and shielding efficiency.

Carbon-based glass foams are also a good candidate for the realization of cost-effective microwave terminations. Whatever the composition is, simulations of microwave terminations that integrate carbon-based glass foams demonstrated a return loss lower than -30 dB over the X-band.

List of

  thoroughly mixed and grinded to produce an homogeneous batch (Fig 1.a). When this mixture reaches a temperature close to the softening temperature of the glass, the foaming agent reacts to release gaseous products that is the key factor of the expansion (Fig 1.b). A short chemical reaction time results in a partial consumption of the foaming agent [8] and the unreacted part (Fig 1.c), after cooling down to room temperature, acts as EM-absorbent dispersed in a light matrix.

Fig. 1 .

 1 Fig. 1. Schematic illustration of the elaboration process: (a) Glass cullet and foaming agent mixture, (b) Foaming process at 850°C and (c) Resulting glass foam / C particles composite.

Fig. 2 .Fig. 3 .

 23 Fig. 2. Evolution of the foam density as a function of carbon amount

Fig. 4 .

 4 Fig. 4. Microscope images of glass foams made with (a) 1.5 wt% of carbon only and (b) a mixing of 1.5 wt% of of C, 4 wt% of MnO 2 , 2 wt% of AlN and 2 wt% of TiO 2 as foaming agents.

Fig. 5 .Fig. 6 .

 56 Fig. 5. (a) Real part of the permittivity and (b) loss tangent of glass foams loaded with different rates of carbon as a function of frequency

Fig. 7 .

 7 Fig. 7. (a) Real part of the permittivity and (b) Loss tangent of glass foams with different rates of carbon as a function of density at different frequencies.

Fig. 8 .

 8 Fig. 8. Shielding Efficiency (SE) as a function of frequency for different carbon load rates.

Fig. 9 .

 9 Fig. 9. Shielding Efficiency Relative to Mass (SERM) as a function of frequency for different carbon load rates.

Fig. 10 .

 10 Fig. 10. (a) Loss tangent and (b) Shielding Efficiency Relative to Mass (SERM) as a function of frequency of glass foams realized with C and a mixing of C, MnO2, AlN and TiO2 as foaming agents (amount of carbon is 1.5 wt% for both samples).

Fig. 11 .

 11 Fig. 11. (a) Electromagnetic electrical field amplitude in a microwave rectangular waveguide termination made of a 7 cm-long pyramidal 1% wt-loaded carbon glass foam and (b) Simulated reflection coefficient (S11) of the device for different loaded glass foams.

Fig. 1

 1 Fig. 1

Fig 2 Fig

 2 Fig 2

  Fig 6

Fig. 8 .

 8 Fig. 8.

Fig. 11

 11 Fig. 9


Maxime Guillerm

Annabelle Couvert

Abdeltif Amrane

Edith Norrant

Audrey Breton

Éric Dumont
email: eric.dumont@mines-nantes.fr

Energy, resources and environmental technology Toluene degradation by a water/silicone oil mixture for the design of Two Phase Partitioning Bioreactors

Keywords: Degradation, Toluene, Silicone oil, Multiphase reactor, Biomass

Highlights

-Toluene degradation was studied in a Two-Phase Partitioning Bioreactor (TPPB) -The liquid phase consisted of 75 % water and 25 % silicone oil (PDMS 50) -Biodegradation rates reached 104 g m -3 h -1 for 100 % toluene removal efficiency -A TPPB for toluene removal can therefore be designed for large-scale applications

Abstract

Toluene degradation performances were studied in a 10 L Two-Phase Partitioning Bioreactor (TPPB). The liquid phase consisted of a mixture of water and PDMS 50 (PolyDiMethylSiloxane, i.e. silicone oil, viscosity of 46 mPa•s) in the volume ratio of 75 %/25 %. Two series of experiments were carried out: in the first, the reactor was sequentially supplied with toluene whereas in the second, toluene was continuously supplied. Activated sludge from the wastewater treatment plant of Beaurade (Rennes, France) was used at an initial concentration of 0.5 dry mass g• (mixture L) -1 . The Elimination Capacity (EC) was investigated as well as the change in biomass concentration over time. Toluene biodegradation was very efficient (Removal Efficiency, RE = 100 %) for toluene flows ranging from 0.2 to 1.2 ml• h -1 , corresponding to elimination capacities of up to 104 g•m -3 •h -1 . For a toluene flow of 1.2 ml• h -1 , the biomass concentration measured at the end of the experiment was 4.7 dry mass g• (mixture L) -1 . The oxygen concentration in the liquid phase was clearly not a limiting factor in these operating conditions. Based on these results, an extrapolation leading to the design of an large-scale pilot TPPB can now be considered to study toluene degradation performances in industrial conditions.

A C C E P T E D

M A N U S C R I P T

Introduction

Atmospheric emissions of Volatile Organic Compounds (VOCs) represent an important environmental and human health issue. Biological treatment of VOC industrial emissions is particularly interesting due to its good performances obtained at low cost. However, traditional bioprocesses can be inadequate for removing hydrophobic VOCs as not only are such compounds usually poorly soluble in water, they can also be toxic for microorganisms. Toluene is a favorite hydrophobic model VOC among researchers because it is considered as an environmental priority pollutant and human carcinogen. Using a Two-Phase Partitioning Bioreactor (TPPB) could be an attractive alternative to remove such pollutants. A TPPB involves two immiscible liquid phases: an aqueous phase containing microorganisms and nutrients, and a Non-Aqueous Phase Liquid (NAPL) able to solubilize large amounts of hydrophobic VOCs. The targeted VOC is gradually transferred from the NAPL to the aqueous phase to be degraded by the microorganisms present in the TPPB, which thus enables the NAPL to be regenerated [START_REF] Darracq | Silicone oil: An effective absorbent for the removal of hydrophobic volatile organic compounds[END_REF] while avoiding toxicity effects on the microorganisms. The VOCs can be removed from the air flux by absorption in a separate gas-liquid contactor before entering the TPPB (two-stage unit) or by direct blowing into the TPPB (one-stage unit). A hybrid system can also be considered [START_REF] Dumont | Hydrophobic VOC absorption in two-phase partitioning bioreactors; influence of silicone oil volume fraction on absorber diameter[END_REF]. Several review papers have highlighted the pros and cons of this technology [START_REF] Daugulis | Two-phase partitioning bioreactors: a new technology platform for destroying xenobiotics[END_REF][START_REF] Muñoz | Two-phase partitioning bioreactors for treatment of volatile organic compounds[END_REF][START_REF] Quijano | Two-phase partitioning bioreactors in environmental biotechnology[END_REF][START_REF] Kennes | Bioprocesses for air pollution control[END_REF][START_REF] Muñoz | Recent advances in two-phase partitioning bioreactors for the treatment of volatile organic compounds[END_REF]. Since the 2000s, some key scientific and technical limitations have gradually been solved. Thus, the selection of the most appropriate NAPL (i.e. immiscible with water, not biodegradable, not toxic for microorganisms and showing high affinity for hydrophobic VOCs) has been extensively studied [START_REF] Daugulis | Two-phase partitioning bioreactors: a new technology platform for destroying xenobiotics[END_REF][START_REF] Muñoz | Recent advances in two-phase partitioning bioreactors for the treatment of volatile organic compounds[END_REF][START_REF] Béchohra | Absorption and biodegradation of toluene: Optimization of its initial concentration and the biodegradable non-aqueous phase liquid volume fraction[END_REF]. To date, silicone oils (polydimethylsiloxane, PDMS) with a viscosity ranging from 20 to 200 mPa•s, appear to be the only NAPLs with the desired characteristics [START_REF] Muñoz | Recent advances in two-phase partitioning bioreactors for the treatment of volatile organic compounds[END_REF]. Once the best NAPL had been selected, efforts were focused on the determination of the gas/liquid partition coefficients between the target VOCs and silicone oils [START_REF] Darracq | Silicone oil: An effective absorbent for the removal of hydrophobic volatile organic compounds[END_REF][START_REF] Muñoz | A systematic selection of the non-aqueous phase in a bacterial two liquid phase bioreactor treating α-pinene[END_REF][START_REF] Bailón | Removal of dichloromethane from waste gases in one-and two-liquid-phase stirred tank bioreactors and biotrickling filters[END_REF][START_REF] Hernández | A comparative study of solid and liquid non-aqueous phases for the biodegradation of hexane in two-phase partitioning bioreactors[END_REF][START_REF] Dumont | Determination of partition coefficients of three volatile organic compounds (dimethylsulphide, dimethyldisulphide and toluene) in water/silicone oil mixtures[END_REF][START_REF] Rocha-Rios | Methane biodegradation in a two-phase partition internal loop airlift reactor with gas recirculation[END_REF][START_REF] Guillerm | Characterization and selection of PDMS solvents for the absorption and biodegradation of hydrophobic VOCs[END_REF][START_REF] Tourani | Prediction of Henry's constant in polymer solutions using PCOR equation of state coupled with an activity coefficient model[END_REF]. Moreover, optimization of the volume fraction of silicone oil needed for an efficient mass transfer in the gas-liquid contactor was considered [START_REF] Dumont | VOC absorption in a countercurrent packed-bed column using water/silicone oil mixtures: Influence of silicone oil volume fraction[END_REF][START_REF] Zhang | Mass Transfer Enhancement of Gas Absorption by Adding the Dispersed Organic Phases[END_REF] as well as the contactor hydrodynamics [START_REF] Dumont | Hydrophobic VOC absorption in two-phase partitioning bioreactors; influence of silicone oil volume fraction on absorber diameter[END_REF][START_REF] Guillerm | Absorption of toluene in silicone oil: Effect of the solvent viscosity on hydrodynamics and mass transfer[END_REF]. Simultaneously, much research was centered on mathematical modeling to determine the most important parameters governing VOC mass transfer and kinetic biodegradation [START_REF] Fazaelipoor | A model for treating polluted air streams in a continuous two liquid phase stirred tank bioreactor[END_REF][START_REF] Nielsen | Dynamic simulation of benzene vapor treatment by a twophase partitioning bioscrubber: Part I: Model development, parameter estimation, and parametric sensitivity[END_REF][START_REF] Nielsen | Dynamic simulation of benzene vapor treatment by a twophase partitioning bioscrubber: Part II: Model calibration, validation, and predictions[END_REF][START_REF] Hernández | Modeling of VOC mass transfer in two-liquid phase stirred tank, biotrickling filter and airlift reactors[END_REF][START_REF] Dorado | A novel mathematical approach for the understanding and optimization of two-phase partitioning bioreactors devoted to air pollution control[END_REF][START_REF] Shen | An Unsteady Heterogeneous Mass Transfer Model for Gas Absorption Enhanced by Dispersed Third Phase Droplets[END_REF][START_REF] Shen | Mass Transfer Enhancement of Propane Absorption into Dodecane-Water Emulsions[END_REF]. Nevertheless, although significant data reported in the literature seem to demonstrate that TPPBs could be satisfactorily used at large-scale, no test in situ in real conditions has yet been carried out because some design issues remain to be solved. Indeed, the design of a large-scale TPPB is still not possible because there is no sufficient reliable data concerning the VOCs degradation performances in the presence of silicone oil. The results reported in the literature are extremely varied. Data can differ by one or two order of magnitude for the same VOC. For instance, studying the treatment of toluene as representative VOC in presence of silicone oil (PDMS 5 with a viscosity of 5 mPa• s), Darracq et al. [START_REF] Darracq | Optimization of the volume fraction of the NAPL, silicone oil, and biodegradation kinetics of toluene and DMDS in a TPPB[END_REF] reported an elimination capacity (EC) of 0.95 g• m -3 •h -1 (25 % v/v of PDMS 5 in the mixture) whereas Littlejohns and Daugulis [START_REF] Littlejohns | A two-phase partitioning airlift bioreactor for the treatment of BTEX contaminated gases[END_REF] reported an EC of 52 g• m -3 •h -1 (10 % v/v of PDMS 5 in the mixture). A toluene elimination capacity of 75 g •m -3 •h -1 (Removal Efficiency (RE) of 75 %) was obtained by Volckaert et al. [START_REF] Volckaert | SIFT-MS analysis of the removal of dimethyl sulphide, n-hexane and toluene from waste air by a two phase partitioning bioreactor[END_REF] in the case of the treatment of a mixture of dimethylsulfide, hexane and toluene by a mixture of water/PDMS 20 (viscosity of 20 mPa• s) with a ratio of (75/25 v/v). The use of PDMS in stirred TPPBs has also been studied for the biodegradation of hexane, identified as a very hydrophobic pollutant.

Elimination capacities of 21 g •m -3 •h -1 [START_REF] Muñoz | Hexane biodegradation in two-liquid phase bioreactors: High-performance operation based on the use of hydrophobic biomass[END_REF] and 60 g• m -3 •h -1 [START_REF] Hernández | Key Role of Microbial Characteristics on the Performance of VOC Biodegradation in Two-Liquid Phase Bioreactors[END_REF] were obtained using mixtures of water/PDMS 200 (viscosity of 200 mPa• s) with ratios of (90/10 v/v) and (80/20 v/v), respectively. Using a (75/25 v/v)

A C C E P T E D M A N U S C R I P T

mixture of water/PDMS 20 for the treatment of hexane, Volckaert et al. [START_REF] Volckaert | SIFT-MS analysis of the removal of dimethyl sulphide, n-hexane and toluene from waste air by a two phase partitioning bioreactor[END_REF] obtained elimination capacities up to 242 g• m -3 •h -1 (RE = 69 %). Such performances are lower than those reported by Montes et al. [START_REF] Montes | Effect of oil concentration and residence time on the biodegradation of α-pinene vapours in two-liquid phase suspended-growth bioreactors[END_REF] for the biodegradation of -pinene (a moderately hydrophobic VOC). Thus, using water/PDMS 200 mixtures with ratios of (98/2 v/v) and (95/5 v/v), these authors obtained RE = 100 % for loading rates up to around 100 g •m -3

•h -1 and reported a maximum elimination capacity of around 650 g •m -3 •h -1 (RE = 55 %). This short overview of data in the recent literature highlights that comparing results obtained for different VOCs is not really relevant.

Even if the comparison is informative, the hydrophobicity of the VOC as well as its toxicity towards microorganisms and the mass transfer limitations have to be taken into account. Consequently, with the final objective to implement a full TPPB for the treatment of air polluted with toluene under industrial conditions, there is a need to determine the ability of microorganisms contained in a water/PDMS mixture to degrade this VOC. The purpose of this study was therefore to obtain useful data from the determination of toluene degradation performances by activated sludge in order to design an industrial TPPB. On the basis of -dimensional analysis‖, the experiments carried out in a semi-continuous stirred tank reactor at laboratory-scale will be used to design and build a large-scale apparatus. In this study, toluene was selected as targeted VOC because it is largely used and emitted by many industries. Moreover, toluene is considered by the Total Company, partner of this project, as a compound of interest for the development of TPPBs.

Material and methods

Chemicals

Toluene (C 7 H 8 ; CAS number: 108-88-3; purity ≥ 99.5 %; molecular weight: 92.14 10 -3 kg• mol -1 ; density: 867 kg• m -3 ; Sigma Aldrich) was selected as the target VOC because it is widely used in various industries and is highly hydrophobic. The silicone oil Rhodorsil 47V50 (PDMS 50; dynamic viscosity 46 mPa• s; density 959 kg• m -3 ), provided by the Bluestar Silicones Company, was selected based on characteristics such as its affinity for toluene (partition coefficient at 25 °C: 2.9 ± 0.3 Pa• m 3 •mol -1 ; [START_REF] Guillerm | Characterization and selection of PDMS solvents for the absorption and biodegradation of hydrophobic VOCs[END_REF], non-biodegradability, biocompatibility and low aqueous solubility [START_REF] Darracq | Silicone oil: An effective absorbent for the removal of hydrophobic volatile organic compounds[END_REF]. As the partition coefficient of toluene between water and air is 680 Pa• m 3 •mol -1 at 25 °C [START_REF] Mackay | Handbook of Physical-Chemical Properties and Environmental Fate for Organic Chemicals[END_REF], it can be calculated that the affinity of toluene is 234 times higher for PDMS 50 than for water.

Experiments

Two sets of experiments were carried out in a semi-continuous stirred tank reactor (Fig. 1). The tank was aerated by a gas sparger and stirred by a Rushton turbine (300 r•min -1 ). In the first set of experiments, the reactor was sequentially supplied with toluene (10 toluene injections) whereas in the second series, toluene was supplied continuously. Liquid toluene was injected into the mixture of water/PDMS 50 using a syringe driver. The operating conditions for all experiments are detailed in Table 1. Liquid temperature and pH were regulated at 25 °C and 7, respectively. The liquid phase (10 L) consisted of 75 % water and 25 % silicone oil (PDMS 50) in volume corresponding to an optimum ratio for biodegradation performances [START_REF] Darracq | Optimization of the volume fraction of the NAPL, silicone oil, and biodegradation kinetics of toluene and DMDS in a TPPB[END_REF]. The volume of the gas phase (head-space) was 2.3 L. Taking the stirring rate and the bubbling due to the aeration system into account, both the liquid and gas phases could be reasonably considered to be perfectly mixed. Activated sludge from the wastewater treatment plant of Beaurade (Rennes, France) was used at an initial concentration of 0.5 dry mass g• (mixture L) -1 (i.e. 0.38 dry mass g• (water L) -1 .). Nutrients were added to the reactor at the beginning of ). Consequently, the amount of oxygen required to biodegrade 1 mole of toluene corresponds to 6.5 moles (i.e. 4.5 moles for energy production and 2 moles for biomass production). Moreover, it was recently evidenced that the presence of silicone oil has no significant influence on the microbial community in terms of richness and diversity [START_REF] Lebrero | Abatement of odorant compounds in one-and two-phase biotrickling filters under steady and transient conditions[END_REF].

A C C E P T E D M A N U S C

Table 1 Operating conditions for the two sets of experiments

Analytical methods

Biomass concentration was measured by extracting suspended solids from samples of the water/PDMS mixture by centrifugation (at 4000 rpm for 20 min) and weighing the dry matter (dried in an oven at 105 °C for 16 hours). However, this measurement was difficult to make accurately because after centrifugation, on the one hand, a small part of the sludge was removed with the supernatant and, on the other hand, some traces of silicone oil remained -stuck‖ to the dry matter, distorting the weight values obtained (error of ± 20 %). This issue is inherent to the use of water/NAPL mixtures. In fact, Ascon-Cabrera and Lebeault [START_REF] Ascon-Cabrera | Selection of Xenobiotic-Degrading Microorganisms in a Biphasic Aqueous-Organic System[END_REF] observed that approximately half of the total biomass adhered to the water/NAPL interface. For the sequential experiments, biomass measurements were carried out daily, before toluene injection. For the continuous experiments, biomass

A C C E P T E D

M A N U S C R I P T measurements were carried out at the end of each experiment.

The oxygen and carbon dioxide in the output gas were monitored simultaneously and continuously using an IPOS analyzer (Abiss, France). The dissolved oxygen in the aqueous phase was also monitored using a standard electrode SZ10T-PB (Consort, Belgium).

The toluene phase concentration in the gas phase was measured using a gas chromatograph (GC) coupled with a flame ionization detector (FID) from Thermo Scientific (USA) as described by Darracq et al. [START_REF] Darracq | Removal of Hydrophobic Volatile Organic Compounds in an Integrated Process Coupling Absorption and Biodegradation-Selection of an Organic Liquid Phase[END_REF]. Assuming that gas-liquid equilibrium was reached in the reactor, the toluene concentration in the water/PDMS 50 mixture was then deduced from the partition coefficient value determined from the calculation procedure developed by Dumont et al. [START_REF] Dumont | Determination of partition coefficients of three volatile organic compounds (dimethylsulphide, dimethyldisulphide and toluene) in water/silicone oil mixtures[END_REF]. For a liquid phase consisting of 75 % water and 25 % PDMS 50 in volume, the partition coefficient value is 11.5 Pa •m 3 •mol -1 , corresponding to a dimensionless value of 0.0046 (= C gas /C mixture ).

Biodegradation rate

For the sequential experiments, the toluene degradation rate (EC in g m -3 h -1 ) was calculated using Eq. ( 1):

(1)

The volume of the mixture (V mixture ) was 10 L and the amount of injected toluene was 10 ml. The overall toluene stripping in the gas output during the whole experiment was deduced by monitoring the toluene concentration in the gas phase over time. Moreover, the degradation time was calculated from normalized curves (n (t) /n (t=0) )

describing the decrease in the toluene content in the liquid phase over time, as well as from the oxygen concentration curves in the liquid and gas phases monitored during the course of experiments, respectively.

For the continuous experiments, the toluene biodegradation rate was calculated based on the mass balance between the toluene flow rate, toluene stripping, toluene degradation and toluene accumulation, as described in Eq. ( 2) and Fig. 2.

(

) 2
The toluene flow rate (Q toluene in g• h -1 ) was a controlled parameter in the experiment and toluene stripping (Q air C gas ) was monitored over time. The overall toluene stripping in the gas output during the whole experiment was thus obtained by the cumulative addition of the stripping measured between two time intervals (Eq. 3).

(

) 3
In the same way, the accumulation term was deduced from Eq. ( 4), where C mixture over time was calculated assuming that gas-liquid equilibrium was reached in the reactor (which can be reasonably assumed because the air residence time in the reactor was long, 13 min, and the toluene flow rate was very small compared to the volume of the mixture; Table 1).

(4)

A C C E P T E D M A N U S C R I P T

Results and discussion

Sequential experiments

Sequential experiment was carried out in duplicate in order to assess the reproducibility of degradation rates and biomass measurements. After the first toluene injection, corresponding to the first day of experiment (day 1), a lag phase due to an acclimation period of about 20 h was observed. After the acclimation period, biomass activity started immediately after each toluene injection. An example of the time-course of the normalized amount of toluene in the liquid phase (n (t) /n (t=0) ) recorded after the toluene injection (t=0) is shown in Fig. 3. The beginning and the end of the degradation can be directly determined from the simultaneous and dramatic changes in the O 2 and CO 2 concentration curves in the gas phase, as well as O 2 dissolved in the aqueous phase. It should be noted that the real CO 2 level reached during experiments cannot be known due to the saturation of the analyzer (plateau at 3 % CO 2 ). As observed in Fig. 3, the amount of oxygen dissolved in the liquid phase could not be considered a limiting factor. After each toluene injection, the biodegradation rate (accuracy ± 10 %) was determined using the curves reported in Fig. 3 and Eq. ( 1). It should be noted that for all experiments, the stripping of toluene in the air output was less than 10 % of the total amount of toluene injected into the mixture.

The ten biodegradation rates determined during the sequential experiments are displayed in Fig. 4, which also

shows the biomass concentration measured before each toluene addition. After the acclimation period, the biodegradation rate was roughly constant (until day 7) at around 1 g• h -1 , i.e. 100 g• m -3 •h -1 . The amount of biomass increased daily. However, after day 7, the biodegradation rates dropped to 0.5 g h -1 (i.e. 50 g• m -3 •h -1 ) while the biomass continued to increase. According to this figure, the biomass production can be correlated with the amount of degraded toluene. Taking into account the relative accuracy of the biomass concentration measurements due to the presence of PDMS, the yield was 0.055 ± 0.011 biomass g•(toluene g) -1 , which corresponds to data reported in the literature [START_REF] Alvarez | Kinetics of Toluene Degradation by Denitrifying Aquifer Microorganisms[END_REF]. The drop in the biodegradation rate is difficult to explain because (1) the dissolved oxygen concentration never became nil. As a result, the availability of O 2 also did not limit the degradation rate (a test carried out without aeration (not shown) highlighted that the concentration of dissolved O 2 could be nil, which limited biodegradation); (2) since the biomass increased continuously, the

A C C E P T E D M A N U S C R I P T

amount of available biomass did not limit the biodegradation; (3) since the nutrients were added in excess at the beginning of each experiment, they could not be considered as a limiting factor. As the supply of toluene was sequential, the drop in the biodegradation rate could be due to an irregular availability of toluene, which should not be observed in the series of continuous experiments. Moreover, the occurrence of inhibitory metabolites due to toluene degradation could be contemplated and should be investigated in future works. Compared with the literature data, it appears that the biodegradation rates obtained are of the same order of magnitude as performances usually reported for conventional bioreactors for air treatment, from 10 to 70 g• m -3 •h -1 [START_REF] Kennes | Bioprocesses for air pollution control[END_REF][START_REF] Kennes | Waste gas biotreatment technology[END_REF][START_REF] Kennes | Bioreactors for Waste Gas Treatment[END_REF].

Studying toluene removal in laboratory-scale peat biofilters, Alvarez-Hornos et al. [START_REF] Álvarez-Hornos | Biofiltration of toluene in the absence and the presence of ethyl acetate under continuous and intermittent loading[END_REF] reported an elimination capacity of 93 g• m -3 •h -1 at an Empty Bed Residence Time (EBRT) of 57 s. Moreover, it seems that higher performances could be reached using fungal strains Paecilomyces variotti and Exophiala oligosperma.

Elimination capacity values as high as 164 g• m -3 •h -1 have been reported [START_REF] Estévez | Biofiltration of waste gases with the fungi Exophiala oligosperma and Paecilomyces variotii[END_REF]. Clearly, although the selection of a pure culture for VOC degradation leads to better removal efficiency, the use of a mixed culture, such as activated sludge, for industrial applications is preferable, owing to its robustness.

A C C E P T E D M A N U S C R I P T

Continuous experiments

An example of the time-course of toluene changes during a continuous experiment is presented in Fig. 5. This figure shows, in the upper part, the mass balance of the amount of injected toluene between stripping, biodegradation and accumulation in the water/PDMS 50 mixture (Eq. 2) and, in the bottom part, the derivative curves corresponding to the stripping and biodegradation rates, respectively. As for sequential experiments, a lag phase was observed at the beginning of the experiment, due to the acclimation of the microorganisms to toluene.

Hence, no degradation was observed during the first 20 h of culture and consequently toluene was predominantly accumulated in the liquid phase reactor. At the same time, a part of toluene was stripped from the reactor (between 5 and 10 %). The greatest stripping rate was monitored for the maximum amount of toluene accumulated in the liquid phase. After the initial lag phase, the degradation began and a peak in the toluene removal rate was observed after 25 h of treatment (13.4 mmol• h -1 ). Once the toluene reserve was depleted, the microorganisms degraded toluene as soon as it was injected into the reactor, which is illustrated by the negligible residual gas-phase concentration from less than 45 h until the end of the experiment. Consequently, from this time, the toluene removal rate was equal to the injection rate (Fig. Toluene degradation was efficient (RE = 100 %) for toluene inputs ranging from 0.2 to 1.2 ml h -1 , (i.e. up to 11.3 mmol •h -1 ) which correspond to an elimination capacity of 104 g •m -3 •h -1 . It should be noted that this performance, which is consistent with the results recorded during the sequential experiments, corresponds to the degradation rate obtained at the end of the experiment, i.e. at steady-state (the biodegradation rate peak may be much higher as highlighted in Fig. 5). This result is two times higher than data reported by [START_REF] Littlejohns | A two-phase partitioning airlift bioreactor for the treatment of BTEX contaminated gases[END_REF] using an airlift TPPB (water/PDMS 5, 90/10 v/v) to treat a mixture of BTEX (EC of 52 g• m -3 •h -1 corresponding to a toluene removal efficiency of 87.2 % for a loading rate of 60 g• m -3 •h -1 ). Using the oxygen measurements in the gas phase between the beginning and end of the experiments, it was possible to calculate the amount of oxygen transferred during the degradation of toluene at steady-state for these operating conditions. Results are ranged from 5.0 to 6.5 moles of oxygen per mole of toluene. Taken into account the part of toluene stripped during the experiment, such results are in agreement with the expected value. Indeed, as described in Section 2.2., it is usually assumed that half of the organic compound is converted to cellular mass and half oxidized for energy [START_REF] Wang | Advances in Hazardous Industrial Waste Treatment[END_REF]. With this assumption, the amount of oxygen required to biodegrade 1 mole of toluene is 6.5 moles (i.e. 4.5 moles for energy production and 2 moles for biomass production). The biomass production determined during the continuous experiments corresponded to that measured during the sequential experiments. Thus, at the end of the experiment displayed in Fig. 5, the biomass concentration was 1.5 dry mass g• (mixture L) -1 (toluene injection: 0.4 ml• h -1 ), and values of 4.7 dry mass g• (mixture L) -1 were recorded for a toluene injection of 1.2 ml• h -1 . The trend of a linear increase in biomass concentration with elimination capacity is in agreement with the result reported by Littlejohns and Daugulis [START_REF] Littlejohns | A two-phase partitioning airlift bioreactor for the treatment of BTEX contaminated gases[END_REF]. Using an airlift TPPB to treat a mixture of BTEX (Benzene, Toluene, Ethylbenzene, o-Xylene), these authors obtained a linear correlation between the average EC and biomass concentration. However, this result was obtained using silicone rubber beads (10 % v/v) as the nonaqueous phase.

Conclusions

Experiments were carried out in a semi-continuous stirred tank reactor to determine the ability of a mixture of water/silicone oil PDMS 50 (75/25 v/v) to degrade toluene. The performances of biodegradation obtained from sequential and continuous experiments, up to 104 g• m -3 •h -1 (RE = 100 %), are thus of primary importance in designing the stirred tank reactor for large-scale applications. Based on this laboratory result, a pilot device coupling the absorption step of toluene by PDMS in a separate column with the biodegradation step in a TPPB can now be designed and tested on an industrial site to study the biodegradation performances on a real effluent loaded with toluene. The next work is to confirm over a long period the ability of a TPPB to degrade toluene and to study the impact of the possible accumulation of inhibitory metabolites due to biomass activity. The biodegradation performances will be studied for sequential and continuous operating conditions encountered in industrial companies. The transient-state conditions and shock-loads will be also investigated.

2 Figure 1

 21 Figure 1 Semi-continuous 10 L stirred tank reactor for toluene biodegradation

Figure 2

 2 Figure 2 Calculation of the toluene degradation rate

Figure 3 Figure 4

 34 Figure 3 Sequential experiments: example of toluene biodegradation and parameters monitored in the gas phase and in the aqueous phase

  5(b)). Oxygen concentrations in the liquid and gas phases were monitored during the course of experiments (insert in Fig.5(b)). Concentrations in both phases followed the same trend. Roughly constant during the lag phase, the oxygen concentration dropped dramatically when toluene degradation started. The dramatic decrease in the oxygen concentration in both liquid and gas phases, down to values close to 50 % for the former and 15 % for the latter, corresponded to the high removal rate of toluene observed at the same time. It is noteworthy that even during this peak of consumption, oxygen toluene injection (10 mL)A C C E P T E D M A N U S C R I P Tremained not limiting. During toluene degradation at a constant rate (after 50 h), oxygen concentrations stabilized at values close to 70 % and 16.5 % for dissolved and gas phase oxygen, respectively.

AFigure 5

 5 Figure 5 Continuous experiments: example of the determination of toluene biodegradation (toluene injection: 0.4 ml• h -1 , i.e. 3.8 mmol •h -1 )

  R I P T experiments (all concentrations in g• (water L) -1 : KH 2 PO 4 : 3.5; K 2 HPO 4 ,3H 2 O: 8; NH 4 Cl: 5.5; MgSO 4 ,7H 2 O: 0.25; CaCl 2 ,2H 2 O: 0.07; ZnSO 4 ,7H 2 O: 0.02; CuSO 4 ,5H 2 O: 0.005; (NH 4 ) 2 Mo 7 O 24 ,6H 2 O: 0.004; FeSO 4 ,7H 2 O: 0.1). Since it was not regulated, biomass accumulated in the reactor during experiments. According to [33], half of the toluene degraded by the biomass is converted to cellular mass (C 7 H 8 + 4O 2 + NH 4 + → C 5 H 7 O 2 N + 2CO 2 + 2H 2 O + H + ) and half is oxidized for energy (C 7 H 8 + 9O 2 → 7CO 2

Acknowledgements

The authors would like to thank the French Agency for the Environment and the Control of Energy (ADEME) for their support through a PhD fellowship for M. Guillerm.