Effect of Novel Quercetin Titanium Dioxide-Decorated Multi-Walled Carbon Nanotubes Nanocomposite on Bacillus subtilis Biofilm Development

The present work was targeted to design a surface against cell seeding and adhering of bacteria, Bacillus subtilis. A multi-walled carbon nanotube/titanium dioxide nano-power was produced via simple mixing of carbon nanotube and titanium dioxide nanoparticles during the sol-gel process followed by heat treatment. Successfully, quercetin was immobilized on the nanocomposite via physical adsorption to form a quercetin/multi-walled carbon nanotube/titanium dioxide nanocomposite. The adhesion of bacteria on the coated-slides was verified after 24 h using confocal laser-scanning microscopy. Results indicated that the quercetin/multi-walled carbon nanotube/titanium dioxide nanocomposite had more negativity and higher recovery by glass surfaces than its counterpart. Moreover, coating surfaces with the quercetin-modified nanocomposite lowered both hydrophilicity and surface-attached bacteria compared to surfaces coated with the multi-walled carbon nanotubes/titanium dioxide nanocomposite.


Introduction
Designing surfaces for controlling biofilm development has become an important scope for both detrimental and beneficial biofilm technologies. Biofilms are surface-associated microbial communities encased in a self-produced extracellular polymeric substance (EPS) [1]. They can develop everywhere on almost all natural materials [2] like rock [3], sand [4], soil [5], skin, teeth, plants, etc. Also, artificial surfaces [6,7] such as plastics [8], glasses [9] and metals [10] are suitable substrata for biofilm formation. In water and wastewater treatment facilities, they are causative agents for corrosion [11] and contamination [12]. In addition, they reduce efficiency of heat exchangers [13]. On ship hulls, marine fouling results from the aggregation of microbial biofilms and larger marine organisms, can upsurge

Results
Morphological and spectroscopic characterization of the prepared MWCNTs/TiO 2 are illustrated in Figure 1. In Figure 1a, the XRD diffractogram showed sharp intense peaks at 25 [33] of calculated crystal size 36.17 nm. According to Wen et al. [34], the number of walls of MWCNTs was estimated to be 11. Interestingly, TiO 2 peak at 25.4 • was overlapping the main peak of MWCNTs at 26 was observed [34]. The oxide form of titanium was formed as a result of supplying titanium precursor; TTIP, by the alcoholic solvent, i-PrOH. Besides, anatase formation was developed by a gradual structural rearrangement of the titanium-oxygen lattice of the amorphous reactants, and thermal treatment. In addition to the slow reaction rate, an organic species was formed and acted as a capping agents controlling the crystal growth and influenced particle morphology as well as assembly behavior [35].

Results
Morphological and spectroscopic characterization of the prepared MWCNTs/TiO2 are illustrated in Figure 1. In Figure 1a, the XRD diffractogram showed sharp intense peaks at 25 and 70° for diffraction from {101}, {103}, {004}, {200}, {005}, {105}, {211}, {204}, {116} and {220} planes; respectively, indicating a high crystalline pure tetragonal anatase phase for TiO2 NPs (JCPDS 01-071-1167). Based on Scherrer's equation, the crystal size of TiO2 was 9.85 nm. Additional two peaks at 26.0° and 43.6° were attributed to {002} and {100} facets of hexagonal graphite structure of MWCNTs [33] of calculated crystal size 36.17 nm. According to Wen et al. [34], the number of walls of MWCNTs was estimated to be 11. Interestingly, TiO2 peak at 25.4° was overlapping the main peak of MWCNTs at 26 was observed [34]. The oxide form of titanium was formed as a result of supplying titanium precursor; TTIP, by the alcoholic solvent, i-PrOH. Besides, anatase formation was developed by a gradual structural rearrangement of the titanium-oxygen lattice of the amorphous reactants, and thermal treatment. In addition to the slow reaction rate, an organic species was formed and acted as a capping agents controlling the crystal growth and influenced particle morphology as well as assembly behavior [35].  After calcination, these organic stabilizers were decomposed [9] as confirmatively displayed by Raman spectrum (Figure 1b). The structural ordering of the nanocomposite was additionally analyzed by Raman Spectroscopy. Five peaks appeared at 143.8 (Eg), 198.0 (Eg), 395.7 (B 1g ), 516.6 (A 1g + B 1g ) and 637.6 (Eg) cm −1 which corresponds to the symmetric active modes of the anatase phase of the prepared TiO 2 NPs. Moreover, two extra distinct bands at 1347 cm −1 and 1575 cm −1 were known as D band and G band corresponding to the presence of defect sites and the integrity of hexagonal carbon; respectively. The ratio of D band to G band (I D :I G ≈ 1) indicated the presence of defective walls of CNTs. Furthermore, a weak shoulder appearing at 1600 cm −1 (G+ band) was also associated with the defects in the MWCNTs [34]. Figure 1c shows a TEM image illustrated the multi-walled carbon nanotubes (MWCNTs) with diameters ranging from 20 to 25 nm with a random aggregation of TiO 2 particles on CNTs. The defects in the CNT walls can be clearly seen which make the contact between TiO 2 and CNTs easier. These defects appeared after functionalization of MWCNTs by TiO 2 NPs as shown in the inset of Figure 1c. This TEM image confirmed the purity of the prepared bare MWCNTs as predicted by the Raman spectrum (see Figure 1b). The controlled growth of the crystal size during the drop-wised reaction in addition to the capping effect of the organic species led to uniform particle morphology [35] as presented by the TEM image (Figure 1c), which shows an irregular spherical shape NPs within a size range of 35-60 nm. Notably, the observed aggregated NPs were attributed to the attractive van der Walls forces [36]. Therefore, a large exposed surface area was predicted for MWCNTs/TiO 2 due to their nanosize. The mean external surface area of MWCNTs/TiO 2 was estimated to be 137 m 2 g −1 . The pore size was in a range of 2-50 nm and the cumulative pore volume was 0.45 cc g −1 (Figure 1d). Convincingly, the present MWCNTs/TiO 2 was described as a mesoporous material [37].
Optically, Q showed absorbance in the UV-visible region with three identified peaks in its spectrum at λ 204 , λ 258 and λ 374 nm (Figure 2a). According to the Beer-Lambert's law, the rate of Q relative recovery percentage by MWCNTs/TiO 2 over 36 h was represented by Figure 2b. The relative recovery of Q by MWCNTs/TiO 2 gradually increased to reach its maximum value i.e., 32.45 ± 0.93% of the dissolved Q after 24 h of incubation time. Definitely, the potentiality of MWCNTs/TiO 2 to adsorb Q was attributed to both the large surface area and its special surface characteristics [38]. The abundant active sites of CNTs like unsaturated suspending bonds, pentagons carbon loops, and pentagon-heptagon defect pairs can react with some hydroxyl groups of Q (see inset Figure 2a) resulting in adsorptions. Besides, Q energetically favored accumulation and assembly on the MWCNT interface due to being insoluble in water, with a strong hydrophobic property and high interfacial activity [38].
After calcination, these organic stabilizers were decomposed [9] as confirmatively displayed by Raman spectrum (Figure 1b). The structural ordering of the nanocomposite was additionally analyzed by Raman Spectroscopy. Five peaks appeared at 143.8 (Eg), 198.0 (Eg), 395.7 (B1g), 516.6 (A1g + B1g) and 637.6 (Eg) cm −1 which corresponds to the symmetric active modes of the anatase phase of the prepared TiO2 NPs. Moreover, two extra distinct bands at 1347 cm −1 and 1575 cm −1 were known as D band and G band corresponding to the presence of defect sites and the integrity of hexagonal carbon; respectively. The ratio of D band to G band (ID:IG ≈ 1) indicated the presence of defective walls of CNTs. Furthermore, a weak shoulder appearing at 1600 cm −1 (G+ band) was also associated with the defects in the MWCNTs [34]. Figure 1c shows a TEM image illustrated the multi-walled carbon nanotubes (MWCNTs) with diameters ranging from 20 to 25 nm with a random aggregation of TiO2 particles on CNTs. The defects in the CNT walls can be clearly seen which make the contact between TiO2 and CNTs easier. These defects appeared after functionalization of MWCNTs by TiO2NPs as shown in the inset of Figure 1c. This TEM image confirmed the purity of the prepared bare MWCNTs as predicted by the Raman spectrum (see Figure 1b). The controlled growth of the crystal size during the drop-wised reaction in addition to the capping effect of the organic species led to uniform particle morphology [35] as presented by the TEM image (Figure 1c), which shows an irregular spherical shape NPs within a size range of 35-60 nm. Notably, the observed aggregated NPs were attributed to the attractive van der Walls forces [36]. Therefore, a large exposed surface area was predicted for MWCNTs/TiO2 due to their nanosize. The mean external surface area of MWCNTs/TiO2 was estimated to be 137 m 2 g −1 . The pore size was in a range of 2-50 nm and the cumulative pore volume was 0.45 cc g −1 (Figure 1d). Convincingly, the present MWCNTs/TiO2 was described as a mesoporous material [37].
Optically, Q showed absorbance in the UV-visible region with three identified peaks in its spectrum at λ204, λ258 and λ374 nm (Figure 2a). According to the Beer-Lambert's law, the rate of Q relative recovery percentage by MWCNTs/TiO2 over 36 h was represented by Figure 2b. The relative recovery of Q by MWCNTs/TiO2 gradually increased to reach its maximum value i.e., 32.45 ± 0.93% of the dissolved Q after 24 h of incubation time. Definitely, the potentiality of MWCNTs/TiO2 to adsorb Q was attributed to both the large surface area and its special surface characteristics [38]. The abundant active sites of CNTs like unsaturated suspending bonds, pentagons carbon loops, and pentagon-heptagon defect pairs can react with some hydroxyl groups of Q (see inset Figure 2a) resulting in adsorptions. Besides, Q energetically favored accumulation and assembly on the MWCNT interface due to being insoluble in water, with a strong hydrophobic property and high interfacial activity [38].  After introducing Q, there was an increase in the size of nanocomposites from 155.33 ± 32.49 to 200 ± 69.3 nm; Q/MWCNTs/TiO 2 was quantified ( Figure 3a). The negative charge of the MWCNTs/TiO 2 ; −8.5 ± 2.95 mV (Figure 3b) was generated from adsorption of water's hydroxyl groups on the surface of the nanocomposite [39]. However, in the presence of Q, the negativity of the nanocomposite upturned into −45.0 ± 20.4 mV (Figure 3b). Likewise other biogenic polyphenols [40], the improved negativity of Q-based nanocomposite was attributed to five hydroxyl groups of Q [9] (see the chemical composition of Q inset Figure 2a). Furthermore, the immobilization of Q on MWCNTs/TiO 2 was confirmed by SEM analysis (Figure 3c,d). The higher intensity of the carbon peak revealed by EDX (inset Figure 3d) was an indicator for the content of Q in the Q-owned nanocomposite (inset Figure 2a). After introducing Q, there was an increase in the size of nanocomposites from 155.33 ± 32.49 to 200 ± 69.3 nm; Q/MWCNTs/TiO2 was quantified ( Figure 3a). The negative charge of the MWCNTs/TiO2; −8.5 ± 2.95 mV (Figure 3b) was generated from adsorption of water's hydroxyl groups on the surface of the nanocomposite [39]. However, in the presence of Q, the negativity of the nanocomposite upturned into −45.0 ± 20.4 mV (Figure 3b). Likewise other biogenic polyphenols [40], the improved negativity of Q-based nanocomposite was attributed to five hydroxyl groups of Q [9] (see the chemical composition of Q inset Figure 2a). Furthermore, the immobilization of Q on MWCNTs/TiO2 was confirmed by SEM analysis (Figure 3c,d). The higher intensity of the carbon peak revealed by EDX (inset Figure 3d) was an indicator for the content of Q in the Q-owned nanocomposite (inset Figure 2a). Gravimetrically, the relative recovery rates of Q, MWCNTs/TiO2, and Q/MWCNTs/TiO2 by the glass slides over 4 h are represented by Figure 4. The surface coverage of Q on the glass slides was 27.26 ± 0.22 mg mm −2 ; i.e., the adsorption capacity of the glass to Q was 8.45 ± 0.72% (Table 1). After introducing Q, there was an increase in the size of nanocomposites from 155.33 ± 32.49 to 200 ± 69.3 nm; Q/MWCNTs/TiO2 was quantified (Figure 3a). The negative charge of the MWCNTs/TiO2; −8.5 ± 2.95 mV (Figure 3b) was generated from adsorption of water's hydroxyl groups on the surface of the nanocomposite [39]. However, in the presence of Q, the negativity of the nanocomposite upturned into −45.0 ± 20.4 mV (Figure 3b). Likewise other biogenic polyphenols [40], the improved negativity of Q-based nanocomposite was attributed to five hydroxyl groups of Q [9] (see the chemical composition of Q inset Figure 2a). Furthermore, the immobilization of Q on MWCNTs/TiO2 was confirmed by SEM analysis (Figure 3c,d). The higher intensity of the carbon peak revealed by EDX (inset Figure 3d) was an indicator for the content of Q in the Q-owned nanocomposite (inset Figure 2a). Gravimetrically, the relative recovery rates of Q, MWCNTs/TiO2, and Q/MWCNTs/TiO2 by the glass slides over 4 h are represented by Figure 4. The surface coverage of Q on the glass slides was 27.26 ± 0.22 mg mm −2 ; i.e., the adsorption capacity of the glass to Q was 8.45 ± 0.72% (Table 1).
Gravimetrically, the relative recovery rates of Q, MWCNTs/TiO 2 , and Q/MWCNTs/TiO 2 by the glass slides over 4 h are represented by Figure 4. The surface coverage of Q on the glass slides was 27.26 ± 0.22 mg mm −2 ; i.e., the adsorption capacity of the glass to Q was 8.45 ± 0.72% (Table 1).  The glass slides adsorbed and recovered 68.75 ± 0.01% of the total suspended amount of MWCNTs/TiO2 adsorbate, producing a surface coverage 33.03 ± 0.07 × 10 −2 mg mm −2 (Table 1). However, the improved efficiency in Q/MWCNTs/TiO2 recovery by glass into 80.63 ± 0.38% to produce surface coverage of 35.63 ± 1.13 × 10 −2 mg mm −2 (Table 1) was a result of the multiple opportunities of forming H-bonds required for adsorption [29]. Moreover, the water contact angle of untreated and Q-coated glass surfaces was changed from 60° and 64° (a hydrophilic feature) to 24° and 47° (an improved hydrophilic behavior) after coating by MWCNTs/TiO2 and Q/MWCNTs/TiO2 (Figure 4a-d), respectively. The wettability behavior of the uncoated slides was attributed to the tendency of borosilicate glass to adsorb water owing to their Si-OH group terminated polar surfaces [41]. However, losing three-fifths of the contact angle value after coverage of the surface by MWCNTs/TiO2 was related to the interaction of water with CNTs. Briefly, the steep decrease in contact angle resulted from water condensation inside the CVD tubes based on the superior curvature of the inner interface of nanotubes and on their surfaces eventually in the space between the tube and the surface holder [42]. The reported hydrophobicity of Q molecules [43] can contribute to the decrease in the surface hydrophilicity (see Figure 5b).   The glass slides adsorbed and recovered 68.75 ± 0.01% of the total suspended amount of MWCNTs/TiO 2 adsorbate, producing a surface coverage 33.03 ± 0.07 × 10 −2 mg mm −2 (Table 1). However, the improved efficiency in Q/MWCNTs/TiO 2 recovery by glass into 80.63 ± 0.38% to produce surface coverage of 35.63 ± 1.13 × 10 −2 mg mm −2 (Table 1) was a result of the multiple opportunities of forming H-bonds required for adsorption [29]. Moreover, the water contact angle of untreated and Q-coated glass surfaces was changed from 60 • and 64 • (a hydrophilic feature) to 24 • and 47 • (an improved hydrophilic behavior) after coating by MWCNTs/TiO 2 and Q/MWCNTs/TiO 2 (Figure 4a-d), respectively. The wettability behavior of the uncoated slides was attributed to the tendency of borosilicate glass to adsorb water owing to their Si-OH group terminated polar surfaces [41]. However, losing three-fifths of the contact angle value after coverage of the surface by MWCNTs/TiO 2 was related to the interaction of water with CNTs. Briefly, the steep decrease in contact angle resulted from water condensation inside the CVD tubes based on the superior curvature of the inner interface of nanotubes and on their surfaces eventually in the space between the tube and the surface holder [42]. The reported hydrophobicity of Q molecules [43] can contribute to the decrease in the surface hydrophilicity (see Figure 5b). Nevertheless, because of the reported negative charges of the uncoated glass slide [41], the bacterial cells preferred to attach to the surface rather than the surrounding liquid medium (Figure 6a) with an estimated biovolume of 155 × 10 −2 ± 81.0 µm 3 µm −2 after 24 h of the incubation. Indeed, B. subtilis as a Gram-positive model bacteria had a high degree of hydrophobicity [44] and a great potency for adhesion. Therefore, the favorable attachment of bacillus cells to the glass surface was attributed to forming hydrogen bonding [45]. Successfully, B. subtilis can develop on the surface coated by Q with a biovolume of 133 × 10 −2 ± 0.91 µm 3 µm −2 ( Figure 6b); i.e., biovolume recovery of 85.81 ± 0.58%. However, the surface coated by TiO2/MWCNTs exhibited a great reduction in the biovolume of attached cells; 4.7 × 10 −2 ± 0.06 µm 3 µm −2 (Figure 6c) to produce levels of development that reached 3.03 ± 0.04% after 24 h. Initially, this anti-adhesion was attributed to the electrostatic repulsion [9] between the negatively charged TiO2/MWCNTs (see Figure 3b) and the negatively charged bacterial cells leading to weaken the cellular contact with the negatively charged substratum [9,46]. Moreover, the converse correlation between the bacterial cell attachment of micro-sized cells to nano-scaled surface (see Figure 3a) [46,47] can be contributing to such protection as the opportunity of cells to lay out between the narrow distance between nanoparticles was reduced [46]. Furthermore due to the porosity of TiO2/MWCNTs (see Figure 1d), the bacterial attachment to surfaces was minimized by small-diameter nanoscale pores [24]. Besides, the high hydrophilicity of the TiO2/MWCNTs coated surface (see Figure 5b) can efficiently diminish or inhibit bacterial adhesion as a result of the barrier hydration layer formed on the surface. This was resulted from the surface ability to take up enormous quantities of free water through both ion solvation and hydrogen bonding interaction [48]. Curiously, after 24 h of incubation, the surface coated by Q/TiO2/MWCNTs allowed the biofilm development of laboratory strain B. subtilis 168 with a slightly reduced bio-volume of 53.5 × 10 −2 ± 0.71 µm 3 µm −2 (Figure 5d) of which the recovery level was 34.52 ± 1.01%. Likewise, the repulsion between negative charges (see Figure 3b) enabled Q/TiO2/MWCNTs to save the substratum from bacterial physical attachment [9,46]. In addition, the relative decrease in attached biovolume of bacteria to the coated surface by Q/TiO2/MWCNTs was attributed to the improved hydrophilicity of Nevertheless, because of the reported negative charges of the uncoated glass slide [41], the bacterial cells preferred to attach to the surface rather than the surrounding liquid medium (Figure 6a) with an estimated biovolume of 155 × 10 −2 ± 81.0 µm 3 µm −2 after 24 h of the incubation. Indeed, B. subtilis as a Gram-positive model bacteria had a high degree of hydrophobicity [44] and a great potency for adhesion. Therefore, the favorable attachment of bacillus cells to the glass surface was attributed to forming hydrogen bonding [45]. Successfully, B. subtilis can develop on the surface coated by Q with a biovolume of 133 × 10 −2 ± 0.91 µm 3 µm −2 ( Figure 6b); i.e., biovolume recovery of 85.81 ± 0.58%. However, the surface coated by TiO 2 /MWCNTs exhibited a great reduction in the biovolume of attached cells; 4.7 × 10 −2 ± 0.06 µm 3 µm −2 (Figure 6c) to produce levels of development that reached 3.03 ± 0.04% after 24 h. Initially, this anti-adhesion was attributed to the electrostatic repulsion [9] between the negatively charged TiO 2 /MWCNTs (see Figure 3b) and the negatively charged bacterial cells leading to weaken the cellular contact with the negatively charged substratum [9,46]. Moreover, the converse correlation between the bacterial cell attachment of micro-sized cells to nano-scaled surface (see Figure 3a) [46,47] can be contributing to such protection as the opportunity of cells to lay out between the narrow distance between nanoparticles was reduced [46]. Furthermore due to the porosity of TiO 2 /MWCNTs (see Figure 1d), the bacterial attachment to surfaces was minimized by small-diameter nanoscale pores [24]. Besides, the high hydrophilicity of the TiO 2 /MWCNTs coated surface (see Figure 5b) can efficiently diminish or inhibit bacterial adhesion as a result of the barrier hydration layer formed on the surface. This was resulted from the surface ability to take up enormous quantities of free water through both ion solvation and hydrogen bonding interaction [48]. Curiously, after 24 h of incubation, the surface coated by Q/TiO 2 /MWCNTs allowed the biofilm development of laboratory strain B. subtilis 168 with a slightly reduced bio-volume of 53.5 × 10 −2 ± 0.71 µm 3 µm −2 (Figure 5d) of which the recovery level was 34.52 ± 1.01%. Likewise, the repulsion between negative charges (see Figure 3b) enabled Q/TiO 2 /MWCNTs to save the substratum from bacterial physical attachment [9,46]. In addition, the relative decrease in attached biovolume of bacteria to the coated surface by Q/TiO 2 /MWCNTs was attributed to the improved hydrophilicity of its surface (see Figure 5c), and the inverse relationship between the bacterial attachment of micro-sized bacteria to nano-scaled coated surfaces (see Figure 3a) [47]. However a superior value of the charged Q/TiO 2 /MWCNTs in comparison to its counterpart was observed, with the former protection being less effective than the later. Actually, the duplication of water contact angle value of the covered surface by Q/TiO 2 /MWCNTs reduced its hydrophilicity (see Figure 5d) which had a negative effect on the protection level of the substratum. its surface (see Figure 5c), and the inverse relationship between the bacterial attachment of microsized bacteria to nano-scaled coated surfaces (see Figure 3a) [47]. However a superior value of the charged Q/TiO2/MWCNTs in comparison to its counterpart was observed, with the former protection being less effective than the later. Actually, the duplication of water contact angle value of the covered surface by Q/TiO2/MWCNTs reduced its hydrophilicity (see Figure 5d) which had a negative effect on the protection level of the substratum.

Discussion
Overall, there was not an approximate potential growth of the bacterial cells on the coated surface by TiO2/MWCNTs. So, such surface can be used in industries where microbes induce dangerous effects like in food, pharmaceuticals, etc. Compared to dark conditions, coating glass slides with a negatively charged TiO2 (either anatase or amorphous) showed a repulsive activity against physical attachment of B. subtilis to the surface. Since most bacterial genera are negatively charged, the electrostatic repulsion weakened the cellular contact with the negatively charged substratum [9]. These nanocrystals can minimize the contact area between the bacterium and the surface leading to a reduction of bacterial adhesion [46]. However, quercetin had an impact on zeta potential and adsorption capacity of crystalline metal oxides; other experiments showed that the presence of Q has a meaningful impact on the ability of B. subtilis to develop a biofilm and consequently the application of the prepared materials. Interestingly, in comparison to the biomass

Discussion
Overall, there was not an approximate potential growth of the bacterial cells on the coated surface by TiO 2 /MWCNTs. So, such surface can be used in industries where microbes induce dangerous effects like in food, pharmaceuticals, etc. Compared to dark conditions, coating glass slides with a negatively charged TiO 2 (either anatase or amorphous) showed a repulsive activity against physical attachment of B. subtilis to the surface. Since most bacterial genera are negatively charged, the electrostatic repulsion weakened the cellular contact with the negatively charged substratum [9]. These nanocrystals can minimize the contact area between the bacterium and the surface leading to a reduction of bacterial adhesion [46]. However, quercetin had an impact on zeta potential and adsorption capacity of crystalline metal oxides; other experiments showed that the presence of Q has a meaningful impact on the ability of B. subtilis to develop a biofilm and consequently the application of the prepared materials. Interestingly, in comparison to the biomass growth on the uncoated glass, around two-thirds of the B. subtilis biovolume can develop on the coated surface by Q/TiO 2 /MWCNTs. Certainly, quercetin 2,3-dioxygenase was reported to be secreted by B. subtilis. This enzyme was described to be involved in the resistance mechanism of this bacteria. In addition, Q with its low water solubility, can provide opportunities for bacterial development. Such conductive bio-inspired nanocomposites in the presence of surface attached bacteria has a promising applicability in bio-electrochemical systems. Particularly, B. subtilis can generate electricity [49] and grow anaerobically [50]. Hence, examining the efficiency of such bio-electrodes to generate bio-energy will be an exciting prospective step.

Preparation of Nanomaterials
A composite of TiO 2 nanoparticles and multi-walled carbon nanotubes (MWCNTs/TiO 2 ) was prepared via sol-gel method [51]. Briefly, a 0.03 g of purified MWCNTs (EPRI) was added to a mixture of titanium (IV) isopropoxide (TTIP, Aldrich, St. Louis, MO, USA) and acidified isopropanol (i-PrOH, Fisher, Hampton, NH, USA) within a ratio 4:5 and pH range~2. The mixture was sonicated for 40 min followed by adding a diluted i-PrOH under vigorous stirring for 30 min at 25 • C. Stirring for the resulting colloidal solution continued for 1 h until sol was developed, followed by aging overnight to form the corresponding gel. The sample was dried at 120 • C in oven and annealed at 450 • C to induce the phase transformation to crystalline anatase TiO 2 while retaining the structural integrity of the CNTs [52]. Remarkably, the multi-walled carbon nanotubes (MWCNTs), have been synthesized via the Catalytic Chemical Vapor Deposition (CCVD) process [33]. To avoid nanoparticles agglomeration, MWCNTs were purified before through washing by strong acid (37% HCL, Aldrich). Furthermore, Q was immobilized on MWCNTs/TiO 2 by dispersing a known weight of MWCNTs/TiO 2 in the Q [1 × 10 −3 M] solution under sonication for 5 min followed by shaking (100 rpm) at room temperature over a time range stating from 3 h to 36 h. After the incubation time, Q/MWCNTs/TiO 2 was collected by centrifugation and washed 3 times by water then left for drying at 100 • C.

Characterization of Nanomaterials
The phases of prepared materials were identified by powder X-ray Diffraction (XRD) analysis using Analytical X'PERT PROMPD X-ray diffractometer, CuKα radiation of wavelength λ = 0.15406 nm, rating of 40 KV, 40 mA, step size = 0.02 and scan step time of 0.4 s in the 2θ range 20-80. The crystalline phases were matched to Joint Committee on Powder Diffraction Standards (JCPDS). The crystal size can be estimated using Debye-Scherrer's equation: where; D symbolizes the average crystallite size (nm) of x; TiO 2 or MWCNTs, β is the width of the peak at half maximum intensity (FWHM) of the main peak of anatase phase in radians and λ is the X-ray wavelength (λ = 0.154056 nm). The number of graphitic wall (N) of MWCNTs is calculated by the following relation [53]: The characteristic spectra of MWCNTs/TiO 2 were recognized by the dispersive Raman Microscopy (Model Sentera, Bruker, Germany) of power l0 mW at laser wavelength of 532 nm (doubled Nd:YAG; Neodymium-doped Ytrium Aluminum Garnet, Laser). MWCNTs/TiO 2 was checked using Transmission Electron Microscopy (TEM; JEOL JEM-2000EX Tokyo, Japan) to detect their morphology.
The surface area of the MWCNTs/TiO 2 was measured from nitrogen adsorption-desorption isotherms at liquid nitrogen temperature (77 K) using a Quantachrome AS1Win version 2.01 instrument. The samples were out-gassed for 3 h at 150 • C. The Brunauer-Emmett-Teller (BET) method was used for surface area calculation, while pore size distribution (pore diameter and volume) were determined by the Barrett-Joyner-Halenda (BJH) method. The concentration of adsorbed Q by MWCNTs/TiO 2 (c) was characterized over time (36 h) optically using UV-Visible spectrophotometry applying Beer-Lambert's law at λ 258 nm for the absorbance (A), the molar absorptivity of Q (ε) is 562.341 cm 2 mol −1 [54] and a path length of the sample (b) is 1 cm according to the relation: The relative adsorption of Q by MWCNTs/TiO 2 was expressed as a percentage as follow: where A 0 is the absorbance of Q at 0 time. The hydrodynamic diameter and zeta potential of the prepared nanomaterials were identified by dynamic light scattering spectroscopy (DLS; Malvern Instruments Ltd., Worcestershire, UK). The functionalization of MWCNTs/TiO 2 by Q was tested by scanning electron microscopy (SEM, HITACHI, Tarrytown, NY, USA) combined by Energy Dispersive X-ray detector (EDX, Bruker, Madison, WI, USA).

Coating Glass Surface and Characterization
Under sonication for 10 min, the glass cover-slides (22 mm × 22 mm × 0.17 mm) were cleaned by triplicate washing with deionized water, ethanol, and acetone. After drying, the polished slides were dipped in an aqueous suspension of tested materials [1 × 10 −3 M] for 3 h. The coverslips were dried in an oven at 100 • C. The mass of coating NPs (m) was estimated gravimetrically [55] as: where; m and m" are the mass of the cleaned glass slide before and after immersing in the tested suspension; respectively. The adsorption capacity was quantified using the following equation: Mwt. is the molecular weight of the tested nanomaterials. The recovery rate of the tested materials by the glass slide were characterized over time (4 h). Surface coverage (Γ) expressed in mg mm −2 was quantified applying the following relation [55]: where, S is the total surface area of glass slide. Measuring the static contact angle of distilled water; JT Baker, HPLC grade, (θ) to the coated surfaces was performed applying the sessile drop technique using a Tantec line of contact angle meter apparatus (Germany).
Microscopy: After 24 h, the tested slides were collected from the petri dishes, placed on microscopy glass slides and then covered with coverslips (24 × 50 mm). A layer of nail polish was applied on the corner of the coverslips in order to fix this arrangement. The slides were then observed using Zeiss LSM780 Confocal Laser-Scanning Microscopy (CLSM) fitted with a 488-nm laser an EC Plan-Neofluar 63× oil immersion objective (Carl Zeiss Microscopy GmbH, Jena, Germany). About 6-10 independent locations were selected and recorded at random positions on the glass slides from independent duplicated samples. The image stacks were analyzed using Comstat script [58] and the biomass of the attached cells was estimated from the calculated intensities.
Percentage of biovolume recovery: The percentage of biovolume recovery of B. subtilis on a coated surface, was evaluated based on the ratio between the biovolume developed by B. subtilis on coated surface (C) by nano-agent (x; Q, MWCNTs/TiO 2 or Q/MWCNTs/TiO 2 ) and uncoated glass slide (U) by the previously used equation [9]: Biovolume recovery (%) = C x U × 100 (8)

Conclusions
In summary, glass surfaces were coated with titanium dioxide-decorated multi-walled carbon nanotubes and quercetin titanium dioxide-decorated multi-walled carbon nanotubes nanocomposites to study the behavior of Bacillus subtilis biofilm development. Our results indicated that coating surfaces with titanium dioxide-decorated multi-walled carbon nanotubes changed the hydrophilicity of uncoated surfaces into super hydrophilicity and, in turn, protected the surface from bacterial adhesion. However, coating surfaces with quercetin titanium dioxide-decorated multi-walled carbon nanotubes nanocomposites improved the hydrophilicity of the glass surface to some extent and increased the relative efficiency of bacterial adhesion. The production of such bio-inspired conductive mesoporous nanomaterials is important for electrode applications such as microbial fuel cells.