Asiah Nusaibah Masri

M I Abdul Mutalib

Wan Zaireen

Nisa Yahya

Noor Fathanah

Jean Marc Leveque
email: jean-marc.leveque@univ-smb.fr

Rapid Esterification of Fatty Acid using Dicationic Acidic Ionic Liquid Catalyst via Ultrasonic-assisted Method

Keywords: Biodiesel, ultrasound, pulse, continuous, ionic liquids, dicationic

Biodiesel production via esterification/transesterification reactions can be catalyzed by homogenous or heterogeneous catalysts. Development of heterogeneous catalysts for biodiesel production is highly advantageous due to the ease of product purification and of catalyst recyclability. In this current work, a novel acidic [DABCODBS][CF3SO3]2 dicationic ionic liquid (DIL) was used as heterogeneous catalyst to produce biodiesel using oleic acid as model oil. The esterification was conducted under ultrasonic irradiation (20 kHz) using a 14 mm ultrasonic horn transducer operated at various duty cycles at ambient temperature. It was observed that the duty cycle, amplitude, methanol to oil molar ratio, catalyst amount and reaction temperature were the major factors that greatly impact the necessary reaction time to lead to a high yield of biodiesel. The reaction conditions were optimized with the aid of Response Surface Methodology (RSM) designed according to the Quadratic model of the Box Behnken method. The optimum conditions were found to be at catalyst amount of 0.64 mol%, methanol to oil ratio of 14.3:1, temperature of 59ºC, reaction time of 83 min and amplitude of 60% in continuous mode. The results showed that the oleic acid was successfully converted into esters with conversion value of 93.20% together with significant reduction of reaction time from 7 h (using mechanical stirring) to 83 min (using ultrasonication) . The results also showed that the acidic DIL catalyst we designed purposely was efficient to catalyze the ultrasonic-assisted esterification yielding high conversion of oleic acid to methyl oleate on short times. The DIL was also recycled and reused for at least five times without significant reduction in performance. Overall, the procedure offers advantages including short reaction time, good yield, operational simplicity and environmentally benign characteristics.

Introduction

The future exhaustion of petroleum reserves and concern over global warming issue has led to the urgent requirements for exploring alternative renewable energy sources that are environmentally more acceptable. Biodiesel derived renewable resources such as vegetable oils and animal fats have been known to be among the alternatives to the highly polluting petroleum-based fuels [START_REF] Lam | Homogeneous, heterogeneous and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: a review[END_REF]. Typically, biodiesel feedstock can be divided into several classes comprising of edible and non-edible oils, animal fats, waste oil, micro-algal oil and fungi oil [START_REF] Kumar | Assessment of potential of oils for biodiesel production[END_REF]. Up to recently, the main feedstock for biodiesel production were mainly from highly available edible oils, such as from palm, soybean, and sunflower oil as the biodiesel produced showed comparable properties to diesel. Nevertheless, their large scale production would impact the supply balance between food and fuel which potentially may lead to a crisis or at least to ethical issues [START_REF] Atabani | A comprehensive review on biodiesel as an alternative energy resource and its characteristics[END_REF][START_REF] Yan | Biotechnological preparation of biodiesel and its high-valued derivatives: A review[END_REF]. Use of non-edible oils, such as Jatropha curcas, Pongamia pinnata, and more recently Calophyllum inophyllum, could overcome the problem as they are not suitable for human consumption and their crops could be grown in wastelands unsuitable for food crops. However, they produce low biodiesel yield and contain high free fatty acids, thus requiring multiple reaction steps to produce biodiesel, leading to an increase in operating cost [START_REF] Tiwari | Biodiesel production from jatropha oil (Jatropha curcas) with high free fatty acids: An optimized process[END_REF][START_REF] Sahoo | Process optimization for biodiesel production from Jatropha, Karanja and Polanga oils[END_REF][START_REF] Patil | Optimization of biodiesel production from edible and non-edible vegetable oils[END_REF]. Waste cooking oil (WCO) is a more environmental friendly and potentially better sustainable feedstock but the biodiesel produced contain many undesired impurities and the properties are closely dependent on the fatty acids contents of the source cooking oil. The collection infrastructure and logistics also pose significant problems given that the sources are scattered widely [START_REF] Singh | Biodiesel production through the use of different sources and characterization of oils and their esters as the substitute of diesel: A review[END_REF][START_REF] Gui | Feasibility of edible oil vs. non-edible oil vs. waste edible oil as biodiesel feedstock[END_REF][START_REF] Leung | Transesterification of neat and used frying oil: Optimization for biodiesel production[END_REF]. Consequently, micro-algal and fungi oils do emerge as potentially best alternative feedstock as they can afford up to 30 times more oil product than any other feedstock sources while requiring a shorter period for harvesting as compared to other oil crops [START_REF] Kumar | Status of biofuel production from microalgae in India[END_REF]. Nevertheless, the application also requires a high production cost due to the high yielding of algae strains needing effective large-scale bioreactors [START_REF] Atabani | A comprehensive review on biodiesel as an alternative energy resource and its characteristics[END_REF].

Biodiesel feedstocks are mainly composed of a wide range of fatty acids such as myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2). Among them, oleic acid is found to be the highest content in most feedstock which explains the reason for it being chosen as the model oil by researchers for lab-scale biodiesel production study.

Biodiesel is commonly produced through esterification or transesterification reactions due to their simplicity and economic viability by reacting free fatty acids or triglycerides with alcohol. However, the immiscibility between the two main reactants has proven to be one of the key reason behind their low conversion. Typical production requires significant reaction time and is often energy consuming. The immiscibility problem can be potentially overcomed by utilizing low-frequency ultrasonic irradiation which causes formation of micro bubbles, hence maximising the interfacial surface area of the molecules.

Using ultrasound for chemical reaction within the frequency range of 20 kHz to 2 MHz can improve the mixing efficiency between reactants in a liquid medium through the generation of micro-bubbles caused by cavitation effect. This unique physical phenomenon subsequently leads to several physical and/or chemical effects depending mainly on the incident frequency [START_REF] Badday | Intensification of biodiesel production via ultrasonic-assisted process: A critical review on fundamentals and recent development[END_REF]. At low frequency, shock waves allow for harsh stirring of the medium together with jetstreams leading to an enhanced mass transfer, whilst, at high frequency, the bubble collapsing time is shorter leading to the formation of free radicals [START_REF] Timothy | Applied sonochemistry -The Uses Of Power Ultrasound in Chemistry and Processing[END_REF]. Since the reaction mixture in the esterification/transesterification process is heterogeneous in reason of the immiscibility between alcohol and oil, the reaction mixture could be considered as suitable for low-frequency ultrasound irradiation. During the process, the asymmetric collapse of the bubble disrupts the interface between the alcohol and oil with extreme energy release (heat and pressure), creating local hotspots and microjets. This results in an enormous increase in the interfacial area between the immiscible reactants, leading to more intimate mixing and hence faster reaction kinetics [START_REF] Salamatinia | Quality evaluation of biodiesel produced through ultrasound-assisted heterogeneous catalytic system[END_REF][START_REF] Sarve | Ultrasound assisted biodiesel production from sesame (Sesamum indicum L.) oil using barium hydroxide as a heterogeneous catalyst: Comparative assessment of prediction abilities between response surface methodology (RSM) and artificial neural network (ANN)[END_REF].

Ultrasound reaction can be operated on either continuous or pulse mode as shown in Figure 1. Generally, continuous operating mode is the standard method used as it may energize the reaction by offering thermal energy suitable for reactions that require thermal excitation.

Besides that, it can induce good emulsion between alcohol and oil phases within a short time.

However, the mode delivers acoustic radiation continuously hence preventing time for relaxation among reactants. Thus, it increases the thermal energy of the reaction medium. The phenomenon is possibly caused by the partial loss of the applied energy converted to vibrational or cavitational energy due to absorption or scattering effect [START_REF] Martinez-Guerra | Continuous and pulse sonication effects on transesterification of used vegetable oil[END_REF]. The long continuous mode also often results in difficulty in product separation [START_REF] Gude | Biodiesel from waste cooking oils via direct sonication[END_REF]. On the other hand, pulse mode allows for relaxation intervals providing excitation gap to the reaction mixture to form good emulsions without increasing thermal energy. It also results in smaller sized bubbles thus avoiding energy absorption or scattering. Hence, pulse mode offers better energy distribution and efficiency and enhances mass transfer at the same time even with heterogeneous catalytic esterification or transesterification reaction [START_REF] Weilin Guo | Ionic liquids-catalyzed transesterification of soybean oil under ultrasound irradiation[END_REF][START_REF] Martinez-Guerra | Transesterification of used vegetable oil catalyzed by barium oxide under simultaneous microwave and ultrasound irradiations[END_REF]. However, the pulse mode may result in lower excitation compared to the continuous mode and may therefore requires longer sonication times. So, it is important to screen the best pulse mode that could lead to comparable results (in this case is the conversion of oil to biodiesel) on continuous mode. The pulse mode is often represented by the period of On : Off ratio where the On period represents the time which ultrasound operates while the Off period refers to the interruption or interval time where the ultrasound does not operate. Many studies reported on the effect of pulse sonication on the transesterification of fresh and used oils for both direct and indirect (ultrasonic bath) irradiation modes [START_REF] Kumar | Ultrasonic-assisted transesterification of Jatropha curcus oil using solid catalyst, Na/SiO2[END_REF][START_REF] Thanh Le | Ultrasound-assisted production of biodiesel fuel from vegetable oils in a small scale circulation process[END_REF][START_REF] Choudhury | Ultrasonic biodiesel synthesis from crude Jatropha curcas oil with heterogeneous base catalyst: Mechanistic insight and statistical optimization[END_REF][START_REF] Pal | Biodiesel production of waste cooking oil through ultrasonic cavitation[END_REF]. Indirect mode was shown to have much lower efficiency reflected by prolonged reaction times compared to direct ultrasound application [START_REF] Gude | Biodiesel from waste cooking oils via direct sonication[END_REF]. Several studies have reported on the direct and continuous application of ultrasonic processing of biodiesel production [START_REF] Gude | Biodiesel from waste cooking oils via direct sonication[END_REF][START_REF] Choudhury | Ultrasonic biodiesel synthesis from crude Jatropha curcas oil with heterogeneous base catalyst: Mechanistic insight and statistical optimization[END_REF][START_REF] Gharat | Ultrasound assisted enzyme catalyzed transesterification of waste cooking oil with dimethyl carbonate[END_REF][START_REF] Hingu | Synthesis of biodiesel from waste cooking oil using sonochemical reactors[END_REF]. Nevertheless, none of the studies compared the continuous and pulse effects of direct ultrasound application for acid-catalyzed biodiesel production. In the production of biodiesel, the catalyst plays evidently an important role. Currently, ionic liquid technology has been explored as new prospect for catalyst in biodiesel synthesis [START_REF] Mohammad Fauzi | An overview of ionic liquids as solvents in biodiesel synthesis[END_REF]. Their exceptional properties such as high thermal stability, simple recovery process and negligible vapour pressure offer great alternative to volatile and hazardous materials which has attracted the attention of many researchers for utilizing them in a wide range of chemical reaction applications [START_REF] Mohammad Fauzi | Optimization of oleic acid esterification catalyzed by ionic liquid for green biodiesel synthesis[END_REF][START_REF] Man | A Brønsted ammonium ionic liquid-KOH two-stage catalyst for biodiesel synthesis from crude palm oil[END_REF]. Recently, the introduction of a new class of dicationic ionic liquids (DILs) has attracted wide attention as they represent an interesting variation of the monocationic analogues in terms of properties. DILs show indubitable and several benefits as compared to typical monocationic ionic liquids in term of volatility and thermal stability, as well as tenability of chemical and physical properties [START_REF] Payagala | Unsymmetrical Dicationic Ionic Liquids: Manipulation of Physicochemical Properties Using Specific Structural Architectures[END_REF] exhibiting promising potential to be used in many applications [START_REF] Fang | Dicationic Ionic Liquids as Environmentally Benign Catalysts for Biodiesel Synthesis[END_REF][START_REF] Chinnappan | Environmentally benign catalyst: Synthesis, characterization, and properties of pyridinium dicationic molten salts (ionic liquids) and use of application in esterification[END_REF][START_REF] Zhao | Synthesis, characterization, and properties of imidazole dicationic ionic liquids and their application esterification[END_REF][START_REF] Chinnappan | Hydrogen generation from the hydrolysis of sodium borohydride with new pyridinium dicationic salts containing transition metal complexes[END_REF][START_REF] Liu | Cycloaddition of CO2 and epoxides catalyzed by dicationic ionic liquids mediated metal halide Influence of the dication on catalytic activity[END_REF][START_REF] Zhang | Asymmetrical dicationic ionic liquids based on both imidazolium and aliphatic ammonium as potential electrolyte additives applied to lithium secondary batteries[END_REF].

In the field of biodiesel production, acid catalysts are highly favourable as they can simultaneously conduct esterification and transesterification reaction while avoiding soap formation. The biodiesel can be directly produced from feedstock with high free fatty acid content such as non-edible oil including Calophyllum inophyllum oil and cheap feedstock including grease and waste cooking oils. In acid-catalyzed esterification and transesterification reactions, added protons activate the carbonyl group of the free fatty acids or triglyceride molecules whose then undergo a nucleophilic attack of the alcohol to produce water and fatty acid alkyl esters. Several researchers highlighted that sulphonate-based catalysts either on heterogeneous [START_REF] Dawodu | Effective conversion of non-edible oil with high free fatty acid into biodiesel by sulphonated carbon catalyst[END_REF][START_REF] Dacquin | Interdependent lateral interactions, hydrophobicity and acid strength and their influence on the catalytic activity of nanoporous sulfonic acid silicas[END_REF] or homogenous conditions [START_REF] Zhang | A Brønsted acidic ionic liquid as an efficient and environmentally benign catalyst for biodiesel synthesis from free fatty acids and alcohols[END_REF][START_REF] Guo | One-step production of biodiesel from Jatropha oil with high-acid value in ionic liquids[END_REF] increased the acidity of the catalyst and resembles sulfuric acid in term of acidity and activity [START_REF] Xing | The sulfonic acid-functionalized ionic liquids with pyridinium cations: Acidities and their aciditycatalytic activity relationships[END_REF][START_REF] Konwar | Reaction kinetics with catalyst deactivation in simultaneous esterification and transesterification of acid oils to biodiesel (FAME) over a mesoporous sulphonated carbon catalyst[END_REF].

Combining the aforementioned properties, this present study aims at enhancing the reaction through the utilization and reuse of sulfonic group-functionalized ionic liquids as an acid catalyst with a combination of low-frequency ultrasound cavitation.

In the present work, a SO3H-functionalized dicationic ionic liquid as depicted in Figure 2 is prepared and characterized. Its performance as catalyst for ultrasound-assisted esterification reaction is evaluated and compared with conventional mechanical stirring approach. The effect of different mode (pulse and continuous) and amplitude are also investigated. The reaction condition is then optimized using Response Surface Methodology (RSM) and compared with literature. The recyclability and reusability of the green IL are also studied.

Experimental

Reagents and materials

All chemicals (analytical reagent grade) used are commercially sourced and used as received without further purification unless otherwise stated. 1 H NMR spectra are done in deuterated water (D2O) at 298 K, recorded on a Bruker Advance 500MHz spectrometer, and processed using the Top-Spin software.

Synthesis of ionic liquids

1,4-diazabicyclo[2.2.2]octane (DABCO) is dissolved in ethanol.
A double molar equivalent of 1,4-butane sultone is added in a three-neck round-bottom flask. The mixture is reacted at 55 °C for 3 days. As the reaction progresses, a white precipitate began to form. After completion of the reaction, the precipitate is filtered off and washed with cyclohexane and diethyl ether several times and dried using a rotary evaporator to remove unreacted materials.

The zwitterion precursor is obtained at a 96% yield as a white powder. The double equivalent of trifluoromethanesulfonic acids is reacted on the zwitterion precursor to produce IL having trifluoromethane sulfonate as anions. The mixture is reacted under vigorous stirring at 70 °C for 4h. The resulting product is then dried under vacuum, affording SO3H-functionalized dicationic acidic IL as solids at room temperature. Di-(4-sulfonic acid) dibutyltriethylene [START_REF] Atabani | A comprehensive review on biodiesel as an alternative energy resource and its characteristics[END_REF].627 (t, 2×2H), and 4.009 (s, 6×2H).

diammonium di(trifluoromethanesulfonate), ([DABCODBS][CF3SO3]2): 1 H NMR: δ 1.830 (m, 2×2H), 2.009 (m, 2×2H), 2.985 (t, 2×2H),

Ultrasonic-assisted esterification reaction

The esterification reaction is conducted under ultrasonic irradiation and conventional mechanical stirring separately under the same experimental conditions for comparison purpose. The schematic diagram of the process flow for ultrasonic-assisted esterification reaction is shown in Figure 3. The reaction is performed in a 30ml double-jacketed glass flask in order to finely control the reaction temperature using cooling-fluid circulation. The reaction solution containing the feed materials is ultrasonically irradiated using a 14mm horn system connected to a Sonic Vibracell generator (20 kHz, VCX 750W, Newton, USA). The ultrasonic power received by the reaction solution is determined using a calorimetric method and set at 30 acoustic power (W) [START_REF] Kimura | Standardization of ultrasonic power for sonochemical reaction[END_REF]. After completion of the reaction, the resulting mixture is centrifuged for 30 minutes at 4000 rpm and consequently three layers are formed within the liquid solution. Desired fatty acid methyl esters is located in the upper phase and is simply separated from the bottom layers containing a mixture of catalyst, methanol and water.

The catalyst is then recovered by removal of methanol and water using rotary evaporator and then recycled for another reaction experiment. The conversion yield is calculated through titration method using the following formula: where Y is the conversion yield, AV0 is the acid value of oleic acid and AV is the acid value of the esterification product.

Power calculation

The ultrasonic energy dissipated into the reaction solution is measured using a calorimetric measurement since most of the ultrasonic energy is converted to several forms of mechanical energy that ultimately produces heat [START_REF] Romdhane | Experimental study of the ultrasound attenuation in chemical reactors[END_REF]. The ultrasonic power is calculated using the equation below:

= …………………………………….………..( 2
)
Where m is the mass of the irradiated solution, Cp is the heat capacity per unit mass of solution (J/kg K) and dT/dt is the slope of the temperature rise versus time (K/s).

Experimental design

In the present investigation, a Box-Behnken method is used as the tool to optimize the four Experimental data from the Box-Behnken method is analyzed using regression (Design Expert™ 8.0) and fitted to a second-order polynomial model in order to identify all possible interactions between the selected factors using a response function as follows:

= + ∑ + ∑ + ∑ ∑ …………….……(3)
where, Y is a response (conversion, %), β0 is a constant and βi, βii and βij are the regression coefficients, xi and xj are independent variables. The fitness and adequacy of the quadratic regression model are investigated through analysis of variance (ANOVA). The coefficients of the fitted model is tested for their significance using Fisher's F-test F-value and p-value [START_REF] Brahmkhatri | 12-Tungstophosphoric acid anchored to SBA-15: An efficient, environmentally benign reusable catalysts for biodiesel production by esterification of free fatty acids[END_REF][START_REF] Montgomery | Design and Analysis of Experiments[END_REF]. Also, the F-value for each terms namely linear (A, B, C, D), interactive

(Prob > F)

Results and Discussion

Comparison of ultrasonic-assisted reaction with the conventional method

The performance of ultrasonic-assisted reaction is firstly compared to a conventional mechanical stirring method with and without the presence of a catalyst. The reaction is done using oleic acid as a model oil. The reaction is conducted at 60 °C in a 1-hr duration with 0.4 mol% of [DABCODBS][CF3SO3]2 and a methanol to oleic acid molar ratio of 9. Results, depicted in Figure 4, show that ultrasonic irradiation produced at least 16% conversion of oleic acid to methyl oleate even in the absence of catalyst in comparison to the conventional mechanical stirring which only attained 7%. This can be explained by the emulsification thus interaction between the immiscible oil and alcohol phase through the physical effects brought up by cavitational bubbles formation issued from ultrasonic irradiation [START_REF] Hingu | Synthesis of biodiesel from waste cooking oil using sonochemical reactors[END_REF].

When the IL catalyst is used, the amount of oleic acid converted to methyl oleate significantly increase to 89% and 59% respectively for ultrasonic-assisted and mechanical stirring reaction. This phenomenon can be explained by the fact that the IL catalyst provides more proton to accelerate the reaction. The simultaneous effect of the catalyst and ultrasonic cavitation synergized the reaction.

Effect of pulse and continuous modes of ultrasonication

To investigate the impact of continuous and pulsed ultrasonic irradiation modes on the biodiesel conversion, experiments with pre-determined operating conditions were conducted on both modes. Figure 5 displays the results of biodiesel conversion and respective powers consumed under both modes. The result shows that the reaction conducted under continuous mode leads to a better conversion as than under pulse mode. Under pulse mode, by reducing the On : Off ratio, the conversion value also significantly decreased. This result is in agreement with Ji et al [START_REF] Ji | Preparation of biodiesel with the help of ultrasonic and hydrodynamic cavitation[END_REF] where they found that at lower pulse On : Off ratio, less power is supplied by ultrasound to the reaction and hence less mixing effects occurred preventing a good and rapid conversion. Therefore, continuous mode is chosen for the next optimization of the amplitude.

Effect of amplitude

Since this amplitude represents the power applied to the system, therefore in theory, the higher the amplitude, the higher the amount of energy supplied and hence favoring the reactions. Higher amplitude causes higher mixing due to fast bubble formation and explosion, overcoming the mass transfer barrier between the immiscible oil and alcohol phased.

However, a very high amplitude may also result in ineffective cloud bubbles. In this case, ultrasonic waves are attenuated by the absorption and scattering effect of too numerous bubbles due to high turbulent conditions. Therefore, energy supplied to the system is wasted and will not improve the reaction. Hence, the esterification reaction is investigated at various amplitude levels within the range of 40% to 70%. The calorimetric power is calculated based on Equation (2) as discussed in section 2.4. The results on the effect of amplitude against power and conversion of biodiesel are depicted in Figure 6. From the figure, it is shown that the calorimetric power increased from 20.8 W to 28.5 W when changed from 40% to 60% amplitude setting, respectively. But, increasing the amplitude to 70% reduces the calorimetric power to 26.2 W. This power trend is in line to the conversion of biodiesel studied for the same range of amplitude. As for 40% to 60% amplitude ultrasound system, the conversion of oleic acid to methyl oleate increased from 71.9% to 93.2%. This can be attributed to the improved mixing and emulsification of oleic acid and methanol layers at higher power dissipation. However, increasing the amplitude of the ultrasound to 70% onwards, the conversion reduces to 82.8% of methyl oleate highlighting the already mentioned scattering effect. Similar trend was also observed by Ji and his co-workers [START_REF] Ji | Preparation of biodiesel with the help of ultrasonic and hydrodynamic cavitation[END_REF]. Therefore, for further optimization process through RSM, the ultrasonic amplitude is fixed at 60% amplitude under continuous mode.

Experimental results and statistical analysis

Response Surface Methodology

A set of experimental designs is executed using Box-Behnken method to study the effect of all process variables and at the same time, determine the optimum condition for the esterification of oleic acid using dicationic [DABCODBS][CF3SO3]2 IL as a catalyst. For ease of evaluation, the relation between predicted and actual conversion values of the biodiesel produced is represented in good correlation between the predicted and actual values of the oleic acid conversion to methyl oleate for all the experimental data. The final equation in term of actual factors is as follows:

= -33.1540 + 0.6236# + 2.0087@ + 58.3828 -0.5204A + 0.0007@# -0.1646 # + 0.0195A# -0.1311@ + 0.0611@A + 2.0135 A -0.0032# -0.0233@ -25.5532 -0.1977A . ..…… [START_REF] Yan | Biotechnological preparation of biodiesel and its high-valued derivatives: A review[END_REF] where Y is conversion, T is temperature, t is time, M is methanol to oil molar ratio and C is amount of catalyst.

Figure 7. The actual and predicted conversion for optimization of esterification reaction using dicationic ILs as a catalyst.

The ANOVA table is shown in Table 1. The ANOVA result shows that a high Fisher's F-test F-value of 64.01 and very low p-value of < 0.0001 are achieved. It implies that the model is highly significant [START_REF] Prakash Maran | Modeling and optimization of supercritical fluid extraction of anthocyanin and phenolic compounds from Syzygium cumini fruit pulp[END_REF]. Besides that, the F-value for each terms namely linear (A, B, C, D), interactive (AC, AD, BD, CD), and 15uadratic (A 2 , B 2 , C 2 , D 2 ) terms indicates high influence towards the methyl oleate conversion. Influence of process variables in linear, interactive and quadratic terms towards methyl oleate conversion with respect to the sum of squares (SS) of all factors in ANOVA is shown in Figure 8. The figure shows that the linear term possesses the predominant effect of 77.72% towards the conversion, followed by the quadratic term that has a marginal effect of 16.37%, and interactive term that has the least effect of 5.91%. The trend agrees with those found in past literature. Maran and co-worker used central composite rotatable design (CCRD) to optimize the reaction conditions for biodiesel production. They obtained the highest linear effect of 54.32%, followed by the quadratic effect of 39.24% and lastly the interactive effect of 6.22% [START_REF] Maran | Comparison of response surface methodology and artificial neural network approach towards efficient ultrasound-assisted biodiesel production from muskmelon oil[END_REF]. In another study by Maran and co-worker, investigation of the biodiesel production from neem (Azadirachta indica) oil through the same two-step esterification-transesterification reaction, using CCRD design, they also achieved the highest linear effect of 54.93%, followed by the quadratic effect of 36.53% and interactive effect of 7.56% [START_REF] Prakash Maran | Modeling of ultrasound assisted intensification of biodiesel production from neem (Azadirachta indica) oil using response surface methodology and artificial neural network[END_REF]. the biodiesel conversion independently and there is a high interaction between mechanisms of effects of the factors. In addition, the p-value for lack-of-fit of 0.0790 implies that it is not significant relative to the pure error. This essentially means that the model is fit and there is only a 7.9% chance that a Lack of Fit value (F-value = 4.54) this large could occur due to noise. The value of R 2 is calculated to be 0.985. The high value of adjusted R 2 of 0.969 which is very close to R 2 value of 0.985 indicates that the sample factors and the sample size are large enough to represent the model and the model is highly significant. The low coefficient of variation (CV) of 4.13% does not only indicate a high degree of precision but also a good reliability in the experiments conducted [START_REF] Maran | Box-Behnken design based multi-response analysis and optimization of supercritical carbon dioxide extraction of bioactive flavonoid compounds from tea (Camellia sinensis L.) leaves[END_REF]. The adequate precision reflex the measure of signal to noise ratio. A value greater than 4 is desirable to support the fitness of the model [START_REF] Balasubramani | Microencapsulation of garlic oleoresin using maltodextrin as wall material by spray drying technology[END_REF]. For the present study, the adequate precision is found to be 26.851 which shows it supports the fitness of the quadratic model.

The interaction between the variables are further studied using the three dimensional response surface plots. The plots are constructed for various combinations of two variables change whilst keeping the other two variables constant and are shown Figure 9 where the value of conversion decreases in the following colors: red > orange > yellow > green > blue.

Based on previous ANOVA result, the trending for the influence of all parameters independently decreases in the following order: reaction time > catalyst amount > temperature > methanol to oil molar ratio.

Figure 9. Response surface plot for conversion (%) for optimization of esterification reaction using dicationic Ils as a catalyst.

Effect of reaction time

The effect of reaction time on the conversion of oleic acid was investigated within the range of 15 to 120 min. The influence of reaction time interactively with other factors comprising of temperature, catalyst amount and methanol to oleic acid molar ratio can be observed in Figure 9a, b andd, respectively. In Figure 9a, even at a low temperature, the conversion increases with time from approximately 40% to 70%. With the addition of heat, at the highest setting temperature which is 60°C, the conversion increases significantly from 58.1% to 90.3%. In Figure 9b, at low catalyst amount, the reaction increases from 37.1% to 75.0% with time. With high amount of catalyst, the reaction speed up and the conversion increases from 67.4% to 91.4% with time until reaching the 78 th minute where it has reached maximum value. In Figure 9d, at low methanol to oil molar ratio, the conversion increases from 49.1% to 67.8% with time whilst at high methanol to oil molar ratio, the conversion increases from 49.3% to 92.5%. In all cases, by increasing the time, more oleic acid is converted to methyl ester until it reaches equilibrium.

Kouzu et al stated that as the reaction started with high oil concentration, the reaction accelerated to produce methyl ester, following zero-order kinetic at the early phase. By the time, the oil concentration reduced, the esterification reaction became slower. Hence the speed in the conversion of oleic acid to methyl oleate also reduced [START_REF] Kouzu | Calcium oxide as a solid base catalyst for transesterification of soybean oil and its application to biodiesel production[END_REF]. According to Martinez-Guerra and co-workers [START_REF] Martinez-Guerra | Transesterification of waste vegetable oil under pulse sonication using ethanol, methanol and ethanolmethanol mixtures[END_REF], biodiesel production may require a long time due to heterogeneity and poor solubility of methanol in the oil phase requiring harsh mixing to form a homogeneous suspension to allow the proton taking part to the esterification reaction. This harsh and necessary mixing effect can be brought obviously by low-frequency ultrasound as long as key operating parameters (frequency, amplitude, temperature, etc.) have been correctly optimized.

Effect of catalyst amount

The use of catalyst can speed up the reaction remarkably and reduced the alcohol requirement. Hence, it will improve the esterification process economically and environmentally [START_REF] Sabudak | Biodiesel production from waste frying oils and its quality control[END_REF]. In this study, the use of dicationic Ils having twice the amount of SO3H-functionalized group compared to the monocationic Ils is to enhance the reaction by providing a double amount of proton to the reaction. The study on the catalyst impact is conducted within the range of 0.2 to 1.0mol% (based on the mole of oleic acid). The results are shown in Figure 9 (d,e,f), which demonstrate the interactive effect of the catalyst amount with time, temperature and methanol to oil molar ratio, respectively. In all cases, initially, a low amount of catalyst results in low conversion of oleic acid to methyl oleate. As the amount of catalyst is increased, even at low reaction time of 15 min, temperature of 25°C and methanol to oil molar ratio of 3, the conversion of oleic acid to methyl oleate increases. The same trend is observed for the effect of the catalyst at high reaction time (120 min), temperature (60°C) and methanol to oil molar ratio [START_REF] Gunstone | Fatty acid and lipid chemistry[END_REF]. This can be explained by the mechanism of acid-catalyzed based reaction through the mechanism shown in Figure 10.

Increasing the number of catalyst molecules will increase the acidity and active sites of the catalyst hence increasing the number of available proton from the sulphonate functional group needed for the protonation of oleic acid to form a carbocation. Hence, the conversion increases [START_REF] Vlada | Barium Hydroxide as a catalyst in the sunflower oil methanolysis reaction[END_REF][START_REF] Liang | Highly efficient procedure for the synthesis of biodiesel from soybean oil using chloroaluminate ionic liquid as catalyst[END_REF][START_REF] Han | Preparation of biodiesel from waste oils catalyzed by a Brønsted acidic ionic liquid[END_REF]. With the aid of ultrasound, the catalyst is also well dispersed in the reaction system thus enhancing the acid-catalyzed esterification process.

Effect of temperature

The effect of operating temperature is investigated within the range of 25°C to 60°C to ensure that the reaction proceeds below the boiling point methanol (64.7 °C). The trend showing the effect of temperature can be observed in Figure 9 (a,c,e) which represented the interactive effect between the operating temperature with the reaction time, the methanol to oil molar ratio and the catalyst amount, respectively. Generally, at lower temperature, the conversion achieved is understandably low. As the temperature increases, the solubility of methanol in oil is enhanced thereby allowing better mass transfer between the reactants, hence improving catalytic activity and improved reaction conversion. In addition, by increasing the temperature, the reaction rate also increases and consequently increases the conversion of oleic acid to methyl ester [START_REF] Mahamuni | Application of Taguchi Method to Investigate the Effects of Process Parameters on the Transesterification of Soybean Oil Using High Frequency Ultrasound[END_REF]. The same trend was observed by Gronroos et al who also studied the effect of operating temperature on ultrasonic-assisted esterification reaction [START_REF] Grönroos | Ultrasound accelerated esterification of bile acids[END_REF].

Effect of methanol to oil molar ratio

The methanol to oil molar ratio is one of the most influential factor in determining the extent of conversion. Commonly, a significance and excess amount of methanol is required to drive the reaction forwards to the product direction [START_REF] Leung | Transesterification of neat and used frying oil: Optimization for biodiesel production[END_REF]. The effect of methanol to oil molar ratio is investigated within the range of 3 to 15 molar ratio. Theoretically, only a minimum molar ratio of 1:1 is required to achieve a full reactivity. However, as the reaction is reversible, higher methanol to oil molar ratio is required to shift the reaction equilibrium forwards and to speed up the reaction [START_REF] Stavarache | Aspects of ultrasonically assisted transesterification of various vegetable oils with methanol[END_REF][START_REF] Soriano | Biodiesel synthesis via homogeneous Lewis acid-catalyzed transesterification[END_REF][START_REF] Ghesti | Synthesis, characterization and reactivity of Lewis acid/surfactant cerium trisdodecylsulfate catalyst for transesterification and esterification reactions[END_REF]. The trend showing the effect of the methanol to oil molar ratio can be observed in Figure 9 (b,c,f) which represents the interactive effect of the methanol to oil molar ratio with the reaction time, the operating temperature and the catalyst amount, respectively. At a lowest reaction time of 15 min, there was no significant effect of methanol to oil molar ratio towards the conversion of oleic acid to methyl oleate.

This indicates that a longer reaction time is required to ensure better mixing of the methanol and oil hence producing higher conversion. At the lowest operating temperature of 25°C and lowest catalyst amount of 0.2 mol%, a similar trend was observed. This can be due to the insufficient energy of the reaction system and lack of catalyst to provide the proton in order to initiate the esterification reaction. However, with the aid of other parameters at the highest level, the increase of the methanol to oil molar ratio increases the cavitational intensity hence enhancing the reaction conversion. The same trend was observed by Stavarache and co-workers who investigated the effect of the methanol to oil molar ratio on the ultrasonic-assisted the transesterification of vegetable oil [START_REF] Stavarache | Ultrasonically driven continuous process for vegetable oil transesterification[END_REF].

Optimization process and data validation

The model equation ( 4) is used to find the optimum values of parameters to get the highest reaction conversion and 55 solutions are found. The selected optimum values of all variables to get a predicted conversion of 95.43% are found to be the temperature of 59 °C, the time of 83 min, the methanol to oil molar ratio of 14.3 and the catalyst amount of 0.64 mol%. Table 2shows the reaction conversion achieved and the relative errors as compared to the predicted value. A minimum average relative error of 2.23% is obtained hence the model can be considered as accurate. The optimized conditions and conversions are compared with the past literatures that also used sulfonated ionic liquid catalysts and ultrasound as tabulated in Table 3 [START_REF] Guo | Ultrasound-assisted production of biodiesel from soybean oil using Bronsted acidic ionic liquid as catalyst[END_REF][START_REF] Guo | Ionic liquids-catalyzed transesterification of soybean oil under ultrasound irradiation[END_REF][START_REF] Bi | Orthogonal Optimization of Ultrasound-Assisted Ionic Liquid Transesterification of Corn Oil Biodiesel[END_REF][START_REF] Bi | Preparation of Biodiesel from Soybean Oil by Ultrasound-Assisted Transesterification[END_REF]. Our proposed ionic liquid is able to catalyze the biodiesel production at a comparable conversion with the literature that used higher amount of catalyst, temperature and reaction time. This could be explained by the higher number of sulphonate group in the catalyst that increases the number of the proton that can be donated during the esterification reaction. Therefore, the production cost and the energy consumption can possibly be reduced. The power for the current study is acoustic power while other literatures refer to electrical power

Catalyst recyclability & reusability

High cost is one of the main restriction for the vast usage of IL in many applications.

Therefore, to tackle this issue, the recyclability and the reusability study of the IL in the system are very essential. In this study, five successive runs were performed using the recovered IL to determine its recyclability and reusability. The result of oleic acid conversion to methyl oleate using the recovered IL is shown in Figure 11. As shown in Figure 11 the conversion reduces from 93.11% to 89.03% after the fifth cycles which is considered as insignificant. The reduced conversion might be attributed to the loss of IL during the phase separation process, in which subsequently effect the amount of IL for the next reaction [START_REF] Han | Preparation of biodiesel from waste oils catalyzed by a Brønsted acidic ionic liquid[END_REF].

Conclusion and Future Work

In conclusion, the continuous ultrasonic irradiation affords a better conversion than pulse mode. The SO3H-functionalized dicationic IL is found to be a potential catalyst to replace conventional catalyst to produce biodiesel. At the optimized conditions were found to be at temperature of 59 °C, 83 minutes of reaction with methanol to oil molar of 14.3 and 0.64 mol% catalyst, a high conversion of 93.2% is attained. The separation of the biodiesel and IL is successfully achieved by simple liquid-liquid extraction followed by centrifugation.

The ability of the IL to be reused as an esterification catalyst for at least five times without significant reduction in performance indicates its potential for cost saving and sustainable used.

Acknowledgement

The authors acknowledged the Yayasan UTP grant, Centre of Research in Ionic Liquids (CORIL) and Chemical Engineering Department of Universiti Teknologi PETRONAS for their support in conducting this study.

Figure 1 .

 1 Figure 1. Ultrasonic continuous and pulse mode reaction.

Figure 2

 2 Figure 2 Structure of SO3H-functionalized dicationic ionic liquid, X = CF3SO3.

=

  × 100%...................................................................(1)

Figure 3 .

 3 Figure 3. Schematic diagram of process flow for esterification reaction by ultrasonic cavitation.

  independent variables namely time (A), temperature (B), catalyst amount (C), and methanol to oil molar ratio (D). The range of variables is decided based on literature studies and initial experimentations conducted. The range of experimental values used are as follows: 25-60 °C, 15-120 min, 3-15 methanol to oil molar ratio and 0.2 to 1.0 mol % catalyst amount.

Figure 4 .

 4 Figure 4. Esterification reaction by ultrasonic irradiation and conventional mechanical stirring with and without IL catalyst.

Figure 5 .

 5 Figure 5. Conversion of biodiesel and power consumption at continuous (cont) and various pulse (on:off, i.e 11:1, 5:1, 1:1) modes.

Figure 6 .

 6 Figure 6. Conversion of biodiesel at a various amplitude in continuous mode.

Figure 7 .

 7 The quadratic regression model is fitted with the actual experimental results obtained using the Box-Behnken method. The figure showed a

Figure 8 .

 8 Figure 8. Influence of process variables (parameters/factors) in Linear (Independent), interactive and quadratic terms on FAME conversion (t = reaction time, T = temperature, C = catalyst amount and M = methanol to oil molar ratio).

Figure 10 .

 10 Figure 10. Mechanism of acid-catalyzed esterification reaction.

Figure 11 .

 11 Figure 11. Reusability of [DABCODBS][CF3SO3]2. IL catalyst for the esterification reaction.

  (AB, AC, AD, BC, BD, CD) and quadratic (A 2 , B 2 , C 2 , D 2 ) terms are evaluated. A high F-value and p-value < 0.05 indicate that the model is significant. Besides that, if the p-value for lack-of-fit of the model > 0.05, it means that the model fits well. Additionally, influence of process variables in linear, interactive and quadratic terms towards methyl oleic conversion are studied using the sum of squares of all factors generated in ANOVA table.After the model is derived from the Box Behnken method, a confirmation reaction is conducted to test the validity of the model. The procedure is repeated for three times to evaluate the relative error. The relative error of the model was calculated using the equation

	as follows:
	!"#$%	&&'& (%) =	|+, +-.+/ 01 2013+ 4+5-+ 601 2013+| 4+5-+ 601 2013+

× 100%........................(4)

Table 1

 1 ANOVA for quadratic regression model for optimization of esterification reaction using dicationic Ils as a catalyst

	Source	Sum of Squares df	Mean Square	F Value p-value Prob > F
	Model	7380.12 14	527.15	64.01	< 0.0001	significant
	A-Time	2875.21	1	2875.21	349.11	< 0.0001
	B-Temperature	1084.57	1	1084.57	131.69	< 0.0001
	C-Catalyst amount	1632.41	1	1632.41	198.21	< 0.0001
	D-Methanol to oil molar ratio	467.17	1	467.17	56.72	< 0.0001
	AB	1.54	1	1.54	0.19	0.6722
	AC	47.81	1	47.81	5.81	0.0303
	AD	150.20	1	150.20	18.24	0.0008
	BC	3.37	1	3.37	0.41	0.5329
	BD	164.67	1	164.67	19.99	0.0005
	CD	93.41	1	93.41	11.34	0.0046
	A^2	508.61	1	508.61	61.76	< 0.0001
	B^2	330.88	1	330.88	40.18	< 0.0001
	C^2	108.43	1	108.43	13.17	0.0027
	D^2	328.44	1	328.44	39.88	< 0.0001
	Residual	115.30 14	8.24
	Lack of Fit	105.96 10	10.60	4.54	0.0790	not significant
	Pure Error	9.34	4	2.33
	Cor Total	7495.42 28
	Std. Dev.	2.87		R-Squared	0.985
	Mean	69.41		Adj R-Squared	0.969
	C.V. %	4.13		Pred R-Squared	0.917
	PRESS	624.94		Adeq Precision	26.851

Table 2

 2 Corresponding measured and predicted data for the statistical model validation.

			Conversion (%)
	No.	Condition			Relative error (%)
			Predicted	Measured
	1	Temperature: 59 °C, time: 83 min, methanol		93.20	2.34
	2	to oil molar ratio: 14.3, and catalyst	95.43	93.11	2.43
	3	amount: 0.64 mol%		93.59	1.93
			Average relative error (%)	2.23

Table 3

 3 Ultrasound-assisted biodiesel production using various types of SO3H-functionalized ionic liquids.

	Reference	This study	[68]	[69]	[70]	[71]
		DABCODBS	TMAPS	TEAPS	BMIM	BMIM
	Ionic Liquid
		CF3SO3	HSO4 a	HSO4 b	HSO4 c	HSO4 c
	Feedstock	Oleic acid	Soybean oil	Soybean oil	Corn oil	Soybean oil
	Temperature (°C)	59	60	60	90	100
	Time (min)	83	60	20	50	50
	Methanol to oil molar
		14.3	9	14	11	10
	ratio
		0.64 mol%			40.7 mol%	37.2 mol%
	Catalyst amount		3.2 mol%	2.7 mol%
		(~1.55 wt%)			(11 wt%)	(10 wt%)
	Ultrasound frequency
		20	24	24	-	40
	(kHz)
	Power (W) d	12.4	200	80	360	280
	Conversion/yield (%)	93.3±0.29%	93.2%	96%	91.85%	92.5%

a Trimethylamino-propanesulfonic hydrogen sulfate b Triethylamine-propanesulfonic hydrogen sulfate c hydrogen sulfate


Hannah Luise Bruck

Frank Delvigne

Pascal Dhulster

Philippe Jacques

Dr François Coutte
email: francois.coutte@univ-lille.fr

Hannah Luise Brück

Molecular strategies for adapting Bacillus subtilis 168 biosurfactant production to biofilm cultivation mode

Keywords: Bacillus subtilis, biofilm bioreactor, filamentation, cell adhesion, surfactin

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

Introduction

The gram-positive soil bacterium Bacillus subtilis produces naturally different classes of lipopeptides as secondary metabolites [START_REF] Jacques | Surfactin and Other Lipopeptides from Bacillus spp[END_REF]. These lipopeptides combine remarkable physicochemical properties and biological activities and thus have a wide range of applications in various fields [START_REF] Jacques | Surfactin and Other Lipopeptides from Bacillus spp[END_REF]. Since lipopeptides are very powerful biosurfactants, the bioreactor design and operating conditions have to be chosen properly in order to control or to avoid foam formation [START_REF] Coutte | Microbial lipopeptide production and purification bioprocesses, current progress and future challenges[END_REF].

Innovative lipopeptide production processes avoiding foam formation based on an air/liquid membrane contactor [START_REF] Coutte | New integrated bioprocess for the continuous production, extraction and purification of lipopeptides produced by Bacillus subtilis in membrane bioreactor[END_REF](Coutte et al., , 2010b) and on a trickle-bed biofilm reactor [START_REF] Zune | Influence of liquid phase hydrodynamics on biofilm formation on structured packing : Optimization of surfactin production from Bacillus amyloliquefaciens[END_REF][START_REF] Zune | Highenergy X-ray tomography analysis of a metal packing biofilm reactor for the production of lipopeptides by Bacillus subtilis[END_REF] have been developed in previous works. Both systems have shown to promote biofilm formation. In the first system, a thin surfactin producing biofilm has been developed by B. subtilis 168 derivative strains on the air/liquid membrane contactor [START_REF] Coutte | New integrated bioprocess for the continuous production, extraction and purification of lipopeptides produced by Bacillus subtilis in membrane bioreactor[END_REF]. In the second system, the reactor contains a metal structured packing that provides a high specific surface area for the cell adhesion and biofilm development [START_REF] Zune | Highenergy X-ray tomography analysis of a metal packing biofilm reactor for the production of lipopeptides by Bacillus subtilis[END_REF]. In this tricklebed biofilm reactor, natural filamentous microorganism such as the fungi Aspergillus oryzae and Tricoderma reesei have shown to have much better cell adhesion capacities than the natural non-filamentous and lipopeptide producing bacterial strain Bacillus amyloliquefaciens [START_REF] Khalesi | Fungal biofilm reactor improves the productivity of hydrophobin HFBII[END_REF][START_REF] Zune | A fungal biofilm reactor based on metal structured packing improves the quality of a Gla::GFP fusion protein produced by Aspergillus oryzae[END_REF][START_REF] Zune | Highenergy X-ray tomography analysis of a metal packing biofilm reactor for the production of lipopeptides by Bacillus subtilis[END_REF]. Other interesting biofilm-based processes consisting of a rotating disc reactor [START_REF] Chtioui | Rotating discs bioreactor, a new tool for lipopeptides production[END_REF] or an inverse fluidized bed bioreactor [START_REF] Fahim | Oxygen transfer in three phase inverse fluidized bed bioreactor during biosurfactant production by Bacillus subtilis[END_REF] have shown that the lipopeptide productivity could be increased through cell immobilization.

Biofilm bioreactors provide increased productivity and process stability through the generation of a highly active attached biomass with a high resistance to external influences and toxic compounds [START_REF] Ercan | Current and future trends for biofilm reactors for fermentation processes[END_REF]. Especially for surfactin production, biofilm bioreactors can be conducive, since surfactin is linked to the biofilm regulation mechanism as a trigger molecule for the expression of matrix genes (Mielich-Süss and Lopez, 2015).

The B. subtilis wild-type strain NCIB3610 forms robust and highly structured biofilms on solid surfaces and air/liquid interfaces [START_REF] Kearns | A master regulator for biofilm formation by Bacillus subtilis[END_REF], whereas the widely used laboratory strain B. subtilis 168 forms only thin and relatively undifferentiated biofilms [START_REF] Branda | Genes Involved in Formation of Structured Multicellular Communities by Bacillus subtilis[END_REF]. [START_REF] Mcloon | Tracing the domestication of a biofilm-forming bacterium[END_REF] have shown that several genetic mutations in B. subtilis 168, which have accumulated during the domestication process, contribute to impaired biofilm formation.

Especially, a deficiency in exopolysaccharide (EPS) production, due to a point mutation in the epsC gene, is responsible for a strongly reduced matrix production [START_REF] Mcloon | Tracing the domestication of a biofilm-forming bacterium[END_REF].

Another known alteration is the defective sfp gene [START_REF] Mcloon | Tracing the domestication of a biofilm-forming bacterium[END_REF]. The gene sfp codes for a phosphopantetheine-transferase, which is essential for the non-ribosomal peptide synthesis of lipopeptides such as surfactin (Coutte et al., 2010a;[START_REF] Mcloon | Tracing the domestication of a biofilm-forming bacterium[END_REF]. The defective biofilm formation is a limiting factor for a robust colonization of the biofilm bioreactor support by B. subtilis 168 derivatives strains. For a good bioreactor performance, enhanced support colonization capacities are necessary. In wild-type strains of B. subtilis, architecturally complex biofilm structures are associated with the growth in chains of cells that are bound together in bundles via exopolysaccharides [START_REF] Kearns | A master regulator for biofilm formation by Bacillus subtilis[END_REF]. Focusing on the spatial organization of the cells in the biofilm, it might be possible to improve the support colonization through the engineering of cell shapes.

Numerous metabolic engineering strategies have been already developed to design more efficient cell factories [START_REF] Volke | Getting Bacteria in Shape: Synthetic Morphology Approaches for the Design of Efficient Microbial Cell Factories[END_REF]. The manipulation of cell shapes has been rarely exploited to optimize bioprocesses [START_REF] Volke | Getting Bacteria in Shape: Synthetic Morphology Approaches for the Design of Efficient Microbial Cell Factories[END_REF]. Gene deletions affecting the cell division induce morphological changes in cells. In B. subtilis, the cell septation protein SepF has shown to be involved in the septum formation and is required for a later step in cell division but does not represent an essential gene [START_REF] Hamoen | SepF, a novel FtsZ-interacting protein required for a late step in cell division[END_REF]. The deletion of SepF perturbates the division septum assembly in the cells and thus provokes filamentous growth due to a deficiency in cell division [START_REF] Hamoen | SepF, a novel FtsZ-interacting protein required for a late step in cell division[END_REF]. Recently, [START_REF] Zhao | Engineering peptidoglycan degradation related genes of Bacillus subtilis for better fermentation processes[END_REF] have deleted several genes related to peptidoglycan hydrolases in a B. subtilis strain leading to elongated bacterial cells with increased specific growth rates and improved enzyme production capacities.

In this work, we investigate different possibilities of engineering B. subtilis 168 strains to improve the cell adhesion capacities through the change of cell shape and enhanced biofilm matrix production. The goal is to be able to produce surfactin in a continuous bioprocess with immobilized cells on a reactor support through the formation of a structural organized biofilm.

In the first step, the engineered strains are characterized at single cell level with a time lapse microscope to evaluate their growth dynamic. Then, the colonization and adhesion capacities of the engineered strains are tested under more real conditions in a drip-flow reactor (DFR) with continuous flow. Images with a live camera are taken to establish a cell colonization and biofilm formation model. Moreover, the surfactin production capacity of the adhered cells is analyzed. Based on the results, we discuss the impact of filamentous growth, surfactin production and biofilm formation on the performance of biofilm-based bioprocesses.

Materials and methods

Strains and strain construction

All genetically engineered strains that were used in this study are derived from the laboratory strain B. subtilis 168 (trpC2). The strains have been selected and/or modified focusing on three genetic modifications: the introduction or respectively the restoration of the genes sfp and epsC as well as the deletion of sepF. For a complete list of the strains and their corresponding genotype as well as the plasmid used in this work see Table 1.

For the transformation, B. subtilis strains have been grown in natural competence medium (14 g/L K2HPO4•3H2O, 5.3 g/L KH2PO4, 20 g/L Glucose, 8.8 g/L Tri-Na Citrate, 0.22 g/L Ferric-NH4-citrate, 1g casein hydrolysate, 2 g K glutamate, 1 M MgSO4, 1.6 mg/L tryptophan) at 37°C and 160 rpm to favor the DNA uptake and integration. Selective media were prepared by adding various antibiotics to lysogeny broth (LB) (10 g/L tryptone, 5g/L yeast extract, 10 g/L NaCl) or LB containing 1.7% agar: chloramphenicol (Cm) 5 µg/mL, neomycin (Neo) 5 µg/mL, erythromycin (Erm) 1 µg/mL, spectinomycin (Spc) 100 µg/mL.

In sfp+ B. subtilis 168 mutants, a functional sfp gene has been inserted into the amyE locus through homologous recombination of the plasmid pBG129, as previously described (Coutte et al., 2010a). Positive clones, showing a chloramphenicol-resistance and spectinomycin sensibility due to a double cross-over homologous recombination of pBG129, were selected. A correct sfp gene transformation was further confirmed by a positive hemolytic test due to the presence of surfactin and negative amylase activity test as a result of the successful insertion of sfp into the amyE locus. Moreover, surfactin production of the Sfp+ strains was verified in planktonic cultures using reversed-phase UPLC-MS analysis (see section 2.7).

The gene deletion of sepF was performed by using the gene deletion strategy "Pop in -pop out", previously described by [START_REF] Tanaka | Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate large-scale metabolic model[END_REF]. Based on this technique, a master strain was constructed by replacing the upp gene with a neomycin resistance gene under the control of the Lambda Pr promoter (λPr-Neo) through homologous recombination of the plasmid pBG402.

Positive clones with a neomycin-resistance were selected. In the following, the gene deletions were introduced in the master strain through homologous recombination of the targeted gene sequence sepF with the gene deletion cassette. The gene deletion cassette was synthesized by polymerase chain reaction (PCR) through the assemblage of different components: the up and down stream element of the gene to be deleted (sepF) and the element containing a phleomycin resistance gene, the repressor gene of the Lambda promoter cI which is necessary for counterselection. Positive clones, showing a phleomycin resistance and neomycin sensitivity as a result of the cassette insertion, were selected. All genetic manipulations have been verified by PCR-based assays and the sequencing of the manipulated gene segment. Figure 1 summarizes the different genetic modification strategies and their corresponding outcome for adapting B. subtilis 168 surfactin production to biofilm cultivation mode.

Time-lapse microscopy analysis of single B. subtilis cells

The cell morphology and growth behavior at single cell level was analyzed using an inverted phase-contrast time lapse microscope system (Eclipse Ti2, Nikon Instruments Europe BV, Amsterdam, Netherlands). The B. subtilis pre-cultures and agar pads were exactly prepared as described in the article of [START_REF] Jong | Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy[END_REF]. The pre-cultures were diluted to an OD600nm of 0.03 and the cells of the mutants were deposited on the solid agar surface. The microscope slide with the agar pad and the loaded cells was incubated at 37°C during 1h prior to the microscope analysis. The prepared microscope slide was then placed on the pre-heated (37°C) microscope table and 100x oil immersion objective. The cell development of selected single cells was then followed in real-time during 8h. Images were taken each 12 minutes.

Drip-flow reactor composition and growth conditions

For the cell adhesion capacity analysis, biofilms were grown on silicone coupons in six parallel flow chambers per DFR (six-chamber Drip Flow Biofilm Reactor ® , Biosurface Technologies Corporation, Montana, USA). The DFR facilitates the observation of biofilm initiation and spreading on a solid surface (called coupon) under low shear stress conditions. In our case, we used silicone coupons with a rough surface to increase the specific surface area that will be available for the initial cell adhesion and biofilm formation. The surface structure image of the silicone coupon was recorded with a 3D high resolution digital microscope VHX-6000 (KEYENCE International Belgium NV/SA, Mechelen, Belgium).

The strains were cultivated in Landy MOPS medium at pH 7.0 (20 g/L glucose, 5 g/L glutamic acid, 1 g/L yeast extract, 0.5 g/L MgSO4, 1 g/L K2HPO4, 0.5 g/L KCl, 1.6 mg/L CuSO4, 1.2 mg/L MnSO4, 0.4 mg/L FeSO4, 21 g/L MOPS, 1.6 mg/L tryptophan). The DFR was placed in a cell culture room kept at 37°C. For the inoculation, overnight cultures of the engineered strains grown in Landy MOPS medium at 37° and 160 rpm were diluted with Landy MOPS medium to an OD600nm of 1. The reactor was kept horizontally and 20 mL of the diluted culture was injected per chamber with a syringe. The inoculation has been followed by a 6h batch phase permitting the cells to settle down and adhere on the support. After the batch phase, the reactor was inclined and the continuous phase with the delivery of fresh medium was launched with a flow rate of ~13 mL/h per chamber during 42h, resulting in a total incubation time of 48h. For each mutant the cell adhesion capacity has been analyzed with 1 to 3 technical replicates per experiment that has been repeated at least 3 times (biological replicates).

Cell counting after initial adhesion on the drip-flow reactor support

To determine the initial adhesion capacities of the mutants, the strains were cultivated and inoculated in the DFR as previously described (cf. 2.3). After 6h of batch phase, a continuous flow ( ~13 mL/h) was launched during 1h to flush gently non-attached cells from the coupons.

Then, the coupons were taken out of the chambers and put into a 50 mL Falcon tube containing 10 mL of phosphate-buffered saline (PBS). After vigorous vortexing, ten-fold dilution series from10 0 to 10 -6 were performed with the cell solutions. From each dilution, 100 µL of the cell solution was dropped and plated on LB agar Petri dishes. The Petri dishes were incubated overnight at 37°C. The developed colony were counted to estimate the number of viable adhered cells on the coupon surface. The cell counting of each mutant was performed in triplicates.

Cell dry weight analysis of the adhered cells after 48h

After 48h, the silicone coupons with the developed biofilm on the surface have been taken out of the DFR and put into a 50 mL Falcon tube containing 10 mL of PBS. The biofilm was dissolved into the liquid through vigorous vortexing. Then, the dissolved biofilm has been gently sonicated (1 to 3 times for 40 sec with 30% of amplitude) to extract the surfactin molecules trapped in the biofilm matrix and dissolve the exopolysaccharides attached to the cells. After the sonication, the samples have been centrifuged. The supernatant was collected and the surfactin concentration was determined as described below (cf. 2.7). The cell pellets were washed by resuspending them in distilled water followed by centrifugation in order to eliminate the dissolved exopolysaccharides. The supernatant was discarded and the remaining cell pellet was re-dissolved in water and filtered (0.2 µm). The filter with the retained cells has been dried in the oven at 105°C and weighted to determine the corresponding cell dry weight.

Real-time observation of biofilm formation dynamics in the drip-flow reactor

For a better understanding of the support colonization by the mutants, the biofilm development in the DFR has been visualized by a real-time camera. For this purpose, the plastic cover of the chamber was replaced by a purpose-made cover composed of an integrated fully transparent glass window for growth observation. Images were taken with a live camera every 15 min for the whole incubation time of 48h. The image sequence has been used to build a general colonization model.

Surfactin production analysis

Cell culture samples were taken after a total incubation time of 48h from the whole liquid phase that has passed and has been collected at each DFR chamber exit (~575 mL per chamber).

Besides, the surfactin concentration has been determined in the sonicated biofilm samples (cf. section 2.5). The culture samples were centrifuged and the supernatant was filtered (0.2 µm) prior to the surfactin analysis by reversed-phase UPLC-MS (AQUITY UPLC H-Class, Waters, Zellik, Belgium) with an AQUITY UPLC BEH C-18 1.7 µm, 2.1x50mm, column (Waters, Zellik, Belgium) coupled to a single quadrupole MS (AQUITY SQ Detector, Waters, Zellik, Belgium). For sample ionization, the source temperature was set at 130°C with a desolvation temperature of 400°C, a nitrogen flow of 1000 L/h and a cone voltage of 120V. The UPLC analysis method was based on an acetonitrile/water gradient containing 0.1% formic acid with a flow rate of 0.6 mL/min and an analysis time of 7 min per sample. The elution was started at 30% of acetonitrile. After 2.43 min acetonitrile was brought up to 95% and then again reduced representing the surfactin isomers C-12 to C-16 respectively. The overall surfactin concentration was calculated on the basis of the calibration curve.

Statistical analysis

Comparison of the cell dry weight and colony forming unit results between groups of B. subtilis mutants were performed using a pairwise two-tailed Student's t test. The differences between groups were considered as significant when p<0.05.

Results and discussion

Single cell phenotypic characterization of filamentous B. subtilis strains

In the first part of this work, we looked at the dynamics of cell growth and spatial organization of the genetically engineered B. subtilis strains on agarose pads by time-lapse microscopy.

Three main genetic targets have been selected, i.e. the introduction of a functional sfp gene necessary for lipopeptide synthesis, the restoration of the epsC gene required for the extracellular biofilm matrix production, and the deletion of the sepF gene involved in cell septation. This last mutation is known to impair cell septation leading to cell filamentation [START_REF] Gündoğdu | Large ring polymers align FtsZ polymers for normal septum formation[END_REF][START_REF] Hamoen | SepF, a novel FtsZ-interacting protein required for a late step in cell division[END_REF]. The growth of isolated B. subtilis cells on agarose pads and the resulting microcolonies (single layer) have been tracked with a time-lapse microscope until the stationary growth phase was reached. As expected and already described by [START_REF] Hamoen | SepF, a novel FtsZ-interacting protein required for a late step in cell division[END_REF], the deletion of sepF led to filamentous growth due to less efficient cell division. However, this deletion also had a considerable impact on the colony formation and colonization behavior. Cells with functional sepF (i.e. B. subtilis 168, BBG111 and RL5260) exhibited normal cell division dynamics which led to more packed colonies containing small cells that were easily distinguishable from each other with mean cell lengths comprised between 3 to 6 µm. For the filamentous strains containing the sepF deletion (i.e. B. subtilis TB92, BBG270 and BBG512), a less efficient cell division could be clearly observed in the exponential growth phase (~4h), leading to elongated cells that developed in length. After the exponential growth phase, the filamentous cells also tended to separate. In the stationary phase at ~8h, maximum cell lengths of up to 26 µm were observed with mean cell lengths comprised between 8 and 12 µm. The strains with sepF deletion (TB92, BBG270 and BBG512) developed rather loosely packed micro-colonies with large spaces that were devoid of cells due to the filamentous cell growth. Consequently, they explored a larger area on the agarose surface by comparison with the sepF+ strains. The increased colonization capacity was also observed for the filamentous surfactin producing strains BBG270 and BBG512 during macroscopic colony development on 0.7% agar LB plates. Hence, filamentous growth might be advantageous for a broader colonization of the bioreactor support material.

Evaluation of colonization and biofilm formation capacity in a continuous drip-flow reactor

As a second characterization step, the engineered B. subtilis strains have been cultivated in a drip-flow reactor (DFR) in order to investigate the biofilm formation capacity on a solid inert support and under continuous nutrient supply.

Initial cell adhesion capacity

Firstly, it was checked to what extend filamentous growth and EPS production is beneficial for the initial cell adhesion of surfactin producing B. subtilis strains on the DFR support. For this purpose, the bacterial cells present on the DFR support after 6h of batch phase followed by 1h of continuous flow have been counted. Therefore, the adhered cells have been detached and quantified by plate counting (Figure 2).

The initial cell adhesion capacities of the surfactin producing EPS+ strains (i.e. RL5260 (sfp+, epsC+) and BBG512 (sfp+, epsC+, ΔsepF)) were up to ten-fold increased by comparison with the surfactin producing EPS deficient strains (i.e. BBG111 (sfp+) and BBG270 (sfp+, ΔsepF)).

EPS are natural polymers composed of sticky sugar substances that help the cells to adhere to a surface and to each other in the case of biofilm formation [START_REF] Flemming | Biofilms : an emergent form of bacterial life[END_REF][START_REF] Vlamakis | Sticking together: building a biofilm the Bacillus subtilis way[END_REF]. However, no significant differences have been observed inside the groups (i.e., neither EPS+ nor EPS-strains), suggesting that cell filamentation upon deletion of sepF has no significant impact on the cell's initial adhesion in the surfactin producing strains.

Regarding the non surfactin producing strains BS168 and TB92 (ΔsepF), the initial cell adhesion of the non-filamentous strain BS168 was slightly increased compared to the filamentous strain TB92. This negative impact of cell filamentation is probably linked to the less efficient cell division of TB92 which lead to coherent, not properly separated cells.

Consequently, it is difficult to spread and plate single cells on the agar plate for a correct counting of the single colony forming units.

Biofilm formation capacity

In the next step, the engineered B. subtilis strains were incubated for 48h, including a 6h batch phase and 42h phase with continuous nutrient supply, until the development of a biofilm on the DFR coupon was observed. A schematic view of the used device is presented in Figure 3A.

Figure 3B shows the coupons colonized by the different B. subtilis strains after 48h in the DFR.

The corresponding amounts of cell dry weight that were measured in g per m 2 of coupon area are presented in Figure 3C. The surface structure of the silicone coupons used as support for the biofilm development in the DFR is presented in Figure 3D.

The induction of filamentous growth in the surfactin negative strain TB92 (ΔsepF) resulted in no significant increase in cell adhesion on the support compared to B. subtilis 168 (control), the cell adhesion capacities were similar. Since the silicone coupons possess a hydrophobic surface and these strains do not produce surfactin to decrease the surface tension, it is more difficult for the cells to spread. In this case, filamentous growth seemed to be neither advantageous nor unfavorable for the support colonization. The presence of surfactin showed a clear impact on the cell distribution on the coupon surfaces.

The biofilm of the surfactin negative strains 168 and TB92 (ΔsepF) showed a clear front line on the coupon surface whereas the border regions of the surfactin producing strains BBG111 (sfp+) and BBG270 (sfp+, ΔsepF) were smooth, an indicator for swarming motility due to the presence of surfactin [START_REF] Kearns | Swarming motility in undomesticated Bacillus subtilis[END_REF]. The increased spreading capacity of BBG111 and BBG270 due to the presence of surfactin led to the colonization of larger zones with a lesser cell density. Hence, the surfactin producing strains BBG111 (sfp+) and BBG270 (sfp+, ΔsepF) were able to cover more homogenously the coupon surface by developing more smooth and better dispersed biofilms than the non surfactin producing strain BS168 or respectively TB92 (ΔsepF).

However, the cell adhesion capacity of BBG111 (sfp+) decreased two to three times compared to BS168. This occurred probably due to cell detachment and the washing out of cells through the presence of surfactin. But the cell adhesion capacity was recovered upon induction of filamentous growth (strain BBG270 (sfp+, ΔsepF)). The cell adhesion capacities of BBG270 were up to three times higher than the ones of the strain BBG111 (sfp+) and thus similar to the cell adhesion capacities of B. subtilis 168.

Regarding the initial cell adhesion after the batch phase (6h), the number of cells present on the coupons were similar for the filamentous strain BBG270 (sfp+, ΔsepF) and non-filamentous strain BBG111 (sfp+). Though, after 48h of incubation, the results have shown that provoked filamentous growth in the surfactin producing strain BBG270 permitted to increase up to three times the cell adhesion capacity resulting in a higher biomass adhered to the support material.

Möller et al. (2013) have already demonstrated that the colonization of heterogeneous surfaces

under physiological flow conditions is accelerated in filamentous E. coli cells. The bacterial cell shape adaption resulted in an improved ability of bridging non-adhesive distances [START_REF] Möller | Bacterial filamentation accelerates colonization of adhesive spots embedded in biopassive surfaces[END_REF]. As the coupon surface analysis with the digital microscope has revealed, the silicone coupons consist of a rough surface with height differences of up to 42.5 µm (cf. Figure 3D) that have an impact on the cell distribution and colonization. Probably, filamentous cells overcome more easily structural irregularities than small cells and consequently possess better colonization capacities. Furthermore, the formed cell aggregates of the filamentous cells seemed to have a better cohesion than the ones formed by small cells making the detaching and washing out of single cells more difficult, especially in the presence of surfactin. The advantages of the increased cohesion of filamentous cells is an interesting feature for biosurfactant production in biofilm reactors with B. subtilis strains to obtain a more efficient and stable colonization of the support materials and to reduce cell detachment from the biofilm.

The adhesion capacities of the strains with restored EPS production (RL5260 (sfp+, epsC+) and BBG512 (sfp+, epsC+, ΔsepF)) increased 10 to 50 times compared to the strains displaying reduced EPS production (168, TB92, BBG111, BBG270). Moreover, the EPS+ mutants (RL5260 and BBG512) developed exceptional wrinkled biofilm structures on the DFR coupons.

The provoked cell filamentation in BBG512 (sfp+, epsC+, ΔsepF) showed no significant improvement in initial cell adhesion and biofilm formation after 48h of incubation compared to RL5260 (sfp+, epsC+).

As expected, the presence of EPS was a key factor for initial cell adhesion and biofilm formation on the drip-flow biofilm reactor support. These natural sticky compounds that are produced by the cells are involved in surface-cell and cell-to-cell interactions [START_REF] Flemming | Biofilms : an emergent form of bacterial life[END_REF][START_REF] Marvasi | Exopolymeric substances (EPS) from Bacillus subtilis: polymers and genes encoding their synthesis[END_REF][START_REF] Vlamakis | Sticking together: building a biofilm the Bacillus subtilis way[END_REF]. Hence, the presence of EPS was found to increase the cell adhesion to a surprisingly high extent of up to 50-fold. No additional increase in cell adhesion was observed in EPS+ mutants with induced filamentous growth (BBG512), neither at the initial cell adhesion after 6h of incubation nor after 48h of incubation. However, it has to be considered that the cell adhesion was analyzed using a simple coupon surface. Biofilm bioreactors such as the previously mentioned trickle-bed biofilm reactor [START_REF] Zune | Highenergy X-ray tomography analysis of a metal packing biofilm reactor for the production of lipopeptides by Bacillus subtilis[END_REF] contain a highly structured packing with a very high specific surface area. In this case, probably, the cell adhesion capacities can be boosted much more through filamentous growth, even in EPS+ mutants. Obviously, the presence of EPS outcompeted the advantage of filamentous cells to colonize the silicone coupons due to an improved adhesion. [START_REF] Seminara | Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix[END_REF] investigated the role of EPS in B. subtilis biofilm expansion. They found out that matrix production indeed contributes to biofilm spreading due to osmotic forces, probably to increase nutrient uptake. In this case, cell filamentation seemed to have a minor effect on biofilm formation than the EPS production.

In the EPS+ mutants (RL5260 and BBG512), the biofilm developed very complex wrinkled structures, characteristic of mature B. subtilis biofilms [START_REF] Vlamakis | Sticking together: building a biofilm the Bacillus subtilis way[END_REF]. Moreover, a hydrophobic layer on the top of the biofilm was observed. The surface hydrophobicity of this protection layer is demonstrated by the colored water droplet staying at the top of the biofilm of RL5260 in Figure 3B. This hydrophobic layer is composed of the protein BslA, a hydrophobin that is synthesized in the last stages of biofilm maturation, as already described by several researchers [START_REF] Arnaouteli | Just in case it rains: building a hydrophobic biofilm the Bacillus subtilis way[END_REF][START_REF] Kobayashi | BslA(YuaB) forms a hydrophobic layer on the surface of Bacillus subtilis biofilms[END_REF]; Mielich-Süss and Lopez, 2015).

Cell colonization and biofilm development mode in the drip-flow reactor

As reported in the previous section (cf. 3.2.2), the EPS+ B. subtilis mutants (RL5260, BBG512) were able to develop remarkable wrinkled biofilm structures within 48h. Moreover, they were able to colonize the whole DFR coupon surface whereas the EPS deficient strains colonized only a part of the DFR coupons after 48h. Since the cell colonization and structural biofilm development on the DFR support seemed to be rather a heterogeneous phenomenon, the dynamics of biofilm formation has been studied. For this purpose, the biofilm formation has been tracked in real time with a camera placed in front of a window integrated in the chamber cover (cf. Figure 3A). A schematic representation of the biofilm development is presented in for the EPS+ mutants. Biofilm formation displayed by mutants with no EPS production stopped during the second development phase since there is neither a structural complex biofilm development nor a maturation phase. Mutants with restored EPS production reached the last phase showing a structurally complex and mature biofilm covering the whole coupon. The biofilm formation took place according to the generally recognized biofilm developing steps: attachment -growth of micro-and macro-colonies -biofilm maturation -cell detachment and dispersion [START_REF] Vlamakis | Sticking together: building a biofilm the Bacillus subtilis way[END_REF]. However, in the beginning, the surface conditioning and nutrient delivery was crucial for cell development. The cells only started to develop where the bulk medium was passing on the coupon. Since the medium had a quite low flow rate of ~13 mL/h, it entered only dropwise into the cultivation chamber and then flowed down randomly on the coupon surface. This means that not the complete coupon surface was continuously delivered by fresh medium. Consequently, the coupon became only partly colonized by a biofilm. The development of this first biofilm until its complete maturation required 18-20h of incubation in the continuous mode preceding 6h of batch phase. Due to the maturation, a hydrophobic protein layer covered the biofilm. This special feature of B. subtilis biofilms has already been mentioned previously in the upper part and demonstrated through the colored water droplet staying on the biofilm surface in Figure 3B. Since the hydrophobic surface became impervious to the bulk medium, the latter one bypassed to uncolonized surfaces on the coupon. This gave the starting point for a new biofilm development of dispersed cells until the whole coupon was colonized by multiple biofilms. Actually, the mature biofilm at the end of the cultivation (~ 40h) was composed of several associated biofilms with different ages and maturations stages.

In EPS+ mutants, a mature biofilm with complex wrinkled structures could be observed after 20h of continuous nutrient supply in the DFR, a complete colonization of the DFR coupon was achieved after around 40h, whereas EPS deficient mutants were neither able to develop an architecturally complex biofilm structure nor to colonize completely the DFR coupon. Besides, it has been demonstrated that EPS gave structural integrity to the biofilm and triggered its maturation through the formation of a hydrophobic protection layer. Although the biofilm matrix provides advantages in biofilm-based processes like increased adhesion capacities and protection from external forces such as shear forces or pH changes, there are also some drawbacks. The hydrophobic protection layer which is formed by B. subtilis at the final maturation stage through the secretion of the hydrophobin BslA represents an effective barrier that prevents the penetration of gas and liquids [START_REF] Arnaouteli | Just in case it rains: building a hydrophobic biofilm the Bacillus subtilis way[END_REF]. This may provoke undesirable nutrient limitations during fermentations in biofilm bioreactors with B. subtilis.

Enhanced biofilm formation leads to higher surfactin production

After characterizing the cell adhesion and colonization of the support, the resulting surfactin production has been analyzed using UPLC-MS as described in section 2.7. Hence, after 48h of incubation, the surfactin concentration was measured in the biofilm as well as in the supernatant of the liquid passing the reactor chamber with a total volume of ~575 mL. The measured amounts of surfactin are presented in Table 2. Surfactin was mainly present in the liquid phase and only in small amounts in the biofilm.

Apparently, the surfactin molecules released by the cells were effectively flushed out by the passing medium, only a low amount stayed trapped in the biofilm. BBG111 (sfp+) and BBG270 (sfp+, ΔsepF) produced comparable amounts of surfactin, as well as RL5260 (sfp+, epsC+) and BBG512 (sfp+, epsC+, ΔsepF), suggesting that the deletion of sepF has no detrimental impact on surfactin production. Globally, the surfactin production in the EPS+ strains was 8 to 10 times higher than in the EPS deficient strains as the number of adhered cells was also increased (10 to 50 times) compared to the EPS deficient strains.

Conclusions

In this work, genetic engineering strategies to improve support colonization in biofilm cultivations with B. subtilis 168 are presented. The support colonization capacity was three times increased in surfactin producing mutants through the induction of cell filamentation. The presence of EPS improved up to 50 times the support colonization whereby cell filamentation had a minor impact. EPS were essential for the initial cell adhesion and for giving structural integrity to the cells in the biofilm. The B. subtilis mutants are potential candidates for the future use in biofilm bioreactors to achieve an enhanced support colonization for an increased lipopeptide productivity. E-supplementary data for this work can be found in e-version of this paper online.

Figure captions

Tables

  to 30% at 5.1 min until the end. Purified surfactin samples (>98%) (Lipofabrik, Villeneuve d'Ascq, France) were used to determine the retention time of the surfactin molecules and a calibration curve. Surfactin isomers were further identified through the recorded mass spectra. Specific m/z peaks were observed at 994, 1008, 1022, 1036, 1050 [M+H] + and 1016, 1030, 1044, 1058, 1072 [M+Na] +

  [START_REF] Leclère | The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties[END_REF] have already demonstrated that it is necessary to reduce the surface friction to increase the surface colonization capacity of B. subtilis 168. Surfactin is a surface-active agent that reduces the surface tension and thus permits the cells to spread more easily, as already shown by several authors(Coutte et al., 2010a;[START_REF] Deleu | Interfacial and emulsifying properties of lipopeptides from Bacillus subtilis[END_REF][START_REF] Julkowska | Comparative Analysis of the Development of Swarming Communities of Bacillus subtilis 168 and a Natural Wild Type : Critical Effects of Surfactin and the Composition of the Medium[END_REF][START_REF] Julkowska | Branched swarming patterns on a synthetic medium formed by wild-type Bacillus subtilis strain 3610 : detection of different cellular morphologies and constellations of cells as the complex architecture develops[END_REF][START_REF] Kearns | Swarming motility in undomesticated Bacillus subtilis[END_REF][START_REF] Leclère | The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties[END_REF].

Figure 4 .

 4 Figure 4. Several biofilm development stages on the DFR coupon (I-VI) have been identified

Figure 1 :

 1 Figure 1: Molecular strategies to obtain a lipopeptide producing B. subtilis 168 strain adapted

Figure 2 :

 2 Figure 2: Initial cell adhesion capacity of the B. subtilis strains on the DFR coupons. Samples

Figure 3 :

 3 Figure 3: (A) Schematic view of the drip-flow cultivation device with six parallel growth

Figure 4 :

 4 Figure 4: Scheme displaying cell colonization and biofilm development over time on the

Figure 1 BBG111

 1 Figure 1

Table 1 : Bacterial strains and plasmids used in this study. Strains or plasmids Genotype or plasmid composition Source Bacterial strains

 1

	Echerichia coli JM109	endA1, recA1, gyrA96, thi, hsdR17 (rk,	Promega Corporation
		mk + ), relA1, supE44, Δ(lac-proAB),
		[F´traD36, proAB, laqI q ZΔM15]
	Bacillus subtilis 168	trpC2	Lab stock
	TB92	trpC2, ΔsepF::Spc R (derived from 168) (Hamoen et al., 2006)
	BBG111	trpC2, amyE::sfp-Cm R (derived from	(Coutte et al., 2010a)
		168)
	BBG270	trpC2, ΔsepF::Spc R , amyE:: sfp-Cm R	This study
		(derived from TB92)
	RL5260	trpC2, epsC+, sfp+, Erm R	(McLoon et al., 2011)
	Master strain BBG501	trpC2, epsC+, sfp+, Erm R , Δupp::Pλ-	This study
		Neo R (derived from RL5260)
	BBG512	trpC2, epsC+, sfp+, Erm R , Δupp::Pλ-	This study
		Neo R , ΔsepF::Phleo R -upp-cI (derived
		from BBG501)
	Plasmids
	pGEM®-T Easy	Cloning vector	Promega Corporation
	pBG129	amyE-sfp-Cm R -amyE-Spec R cloned into	(Coutte et al., 2010a)
		pGEM®-T Easy
	pBG402	upp UP -λPr-Neo R -upp DOWN cloned into	This study
		pGEM®-T Easy

Table 2 : Surfactin production and productivity of the engineered strains grown in continuous DFR biofilm cultures after 48h of cultivation with the corresponding standard deviation.

 2

		BBG111 (sfp+)	BBG270 (sfp+, ΔsepF)	RL5260 (sfp+, epsC+)	BBG512 (sfp+, epsC+, ΔsepF)
	Surfactin production
	in the liquid phase	7.42 ± 2.26	7.20 ± 2.56	70.64 ± 28.05	56.23 ± 22.80
	after 48h [mg/L]
	Amount of surfactin
	present in the biofilm	0.02 ± 0.02	0.06 ± 0.04	0.66 ± 0.20	0.60 ± 0.38
	after 48h [mg]
	Surfactin productivity
	per DFR chamber	0.09 ± 0.03	0.09 ± 0.03	0.85 ± 0.34	0.67 ± 0.27
	[mg/h]

Acknowledgement

This research received funding from the European INTERREG Va SmartBioControl / BioProd project and the ALIBIOTECH program funding administered by the Hauts-de-France Region.

Met steun van het europees fonds voor regionale ontwikkeling. Avec le soutien du fonds européen de développement régional.

HB is supported by an international PhD grant co-funded by the Université de Liège and the Hauts-de-France Region. The authors are grateful to Prof. Leendert Hamoen from the University of Amsterdam for donation of the B. subtilis TB92 strain and Prof. Richard Losick from Harvard University for donation of the B. subtilis RL5260 strain. Furthermore, we would like to thank Jean-François Willaert (Gembloux Agro-Bio Tech -Université de Liège) for his technical assistance for the purpose-made DFR covers used for the biofilm growth observation as well as Andrew Zicler (Gembloux Agro-Bio Tech -Université de Liège) for his help with some of the figures.

Conflict of interest

The authors have declared no conflict of interest.