Optimization of Antibacterial, Structures, and Thermal Properties of Alginate-ZrO 2 Bionanocomposite by the Taguchi Method

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Introduction
Despite improving treatment methods in recent years, treating some diseases, such as cancer, AIDS, autoimmune disease, and antimicrobial resistance, is still a global challenge [1][2][3]. Te emergence of antibiotic resistance has become one of the signifcant challenges due to the waste of large numbers of humans and economic losses [4]. Bacteria can form bioflms, but they pose additional challenges for antibiotic treatment [5]. S. mutans is a facultative anaerobic bacterium routinely found in dental plaque and can form bioflms by various mechanisms [6]. Tis bacterium can lead to dental caries, other cariogenic factors, pyogenic infections, and extraoral infections such as bacteremia and endocarditis [7,8].
Tere are many alternative methods to deal with the problems of routine antibiotic therapies against bacteria bioflm forming, including S. mutans, each of which has its challenges [5,9]. One of the successful alternatives to routine antibiotics and counteracting the spread of antibiotic resistance is nanotechnology and the synthesis of nanoparticles (NPs) and nanocomposites with antimicrobial properties [10,11]. Many applications of nanostructures are due to their physical properties, i.e., optical properties, small size, surface properties, and specifc shape [12,13]. Metal oxide NPs show much better antimicrobial activity than their bulky mass. In addition, it has been demonstrated that synthesizing nanocomposites containing natural polymers and incorporating metal NPs with them can enhance the biological activity of NPs [10,11,14].
Metal oxide NPs have been studied as one of the antimicrobial factors agents due to their optimal biocompatibility, including ZnO, TiO 2 , MgO, CuO, and ZrO 2 NPs. Meanwhile, the antimicrobial properties of zirconia NPs have been less studied despite their mechanical properties and optimal biocompatibility [15,16]. Zirconia can be a good candidate for many biomedical activities due to its strength, toughness, stability, relatively reasonable price, and biomimetic [17]. However, previous studies have shown that the synthesis of ZrO 2 NPs poses challenges due to agglomeration and poor crystallinity. In addition to these undesirable physical properties, it also exhibits minor biomedical activity [18,19]. However, using ZrO 2 in polymeric nanocomposites has shown good antibacterial and antifungal properties and excellent physical properties such as good temperature resistance [17].
Alginate is a natural linear polysaccharide typically extracted from seaweed and can be produced by some bacterial species [20]. Tis biopolymer has found wide applications in medicine due to its properties, such as nontoxicity, biodegradability, biocompatibility, and most importantly, its unique chemical structure [21]. Alginate is constituted of variable amounts of β-D-mannuronic (M) and α-L-guluronic (G), which are organized in an irregular pattern by β-1,4-glycosidic bonds [21,22]. Te ratio of G and M blocks in the alginate structure and the resulting diferent conformations can afect this biopolymer's physical and biological properties [22]. Alginate can be used as a matrix in the synthesis of many nanocomposites. Te presence of hydroxyl groups in the alginate structure allows it to interact with metal oxide NPs, which can improve its biological performance.
Tis research aimed to determine the ideal conditions for the synthesis of alginate/zirconia bionanocomposite as a novel antibacterial agent against S. mutans bioflm because the synthesis and antimicrobial activity of this bionanocomposite have not been assessed.

Synthesis of Zirconia Nanoparticles.
Using the biosynthesis process, Halomonas elongata synthesized zirconia NPs. For this purpose, 100 ml of bacterial culture medium containing 0.2 g glucose, 3 g NaCl, 0.046 g NH 4 Cl, 0.028 g MgSO 4 , 0.011 g K 2 HPO 4 , and 0.0001 g of FeSO 4 was prepared and inoculated with Halomonas elongata. After incubating bacterial cells at 37°C for 48 h, they were centrifuged at 5000 for 15 min. Te precipitate and supernatant were then separated, and the resulting supernatant was used to synthesize nanoparticles. Ten, 0.5 ml of bacterial culture supernatant was added to 1 mg/ml of zirconium chloride. In a shaking incubator, the mixture was incubated for 72 h at 120 rpm, 32°C, and 1 mg/ml zirconium chloride concentration. Final solutions containing NPs were fltered with Whatman flter paper to remove contaminants and then sterilized with a microbial flter to ensure sterility. Te product was put in an oven at 100°C for 24 h to obtain zirconia NPs powder.

Synthesis and Preparation of Alginate Biopolymer.
Te biosynthesis method produced an alginate biopolymer from the Azotobacter Vinelandi bacterium. Tis type of bacterium, IBRC-M 10786, was obtained from the Iranian center for biological resources for this purpose. Te bacterium was then added to a culture medium and incubated for 72 h at 29°C with 150 rpm in a shaking incubator [23]. Centrifugation carried out at 5000 rpm for 10 min was 100 ml of the fnal culture medium containing 10 ml of 0.1 M ethylenediaminetetraacetic acid (EDTA) and 10 ml of 1 M sodium chloride [19]. Te bacterial cells in the supernatant were precipitated, separated, transferred to a container containing isopropanol, and stirred with a magnetic stirrer for 20 min. To prepare an alginate biopolymer, a precipitate was collected, separated, and then dried in an oven at 40°C for 72 h using flter paper.

Synthesis of Alginate/Zirconia Bionanocomposite.
Nine experiments were planned using Qulitek-4 software and the Taguchi method to assess antibacterial activity and establish the best situations for synthesizing nanocomposites with strong antibacterial characteristics [14]. Using the Taguchi method, three levels of 60, 70, and 80 mg/ml of alginate biopolymer and three levels of 4, 6, and 8 mg/ml of zirconia NPs were used to synthesize alginate/zirconia bionanocomposites at stirring times of 40, 80, and 120 min. For synthesizing the nine studied nanocomposites, zirconia NPs and alginate biopolymer were separately dissolved in solutions with concentrations determined using the Taguchi method. Each was sonicated for 15 min to create a homogeneous solution after being stirred for 60 min. Drop by drop, the zirconia NPs solution was added to the alginate biopolymer solution in the container with the help of a magnetic stirrer. At 25°C, a magnetic stirrer was used to stir the fnal solutions for 40, 80, and 120 min before the sonication process, for 15 min, to produce nanocomposites with varying biopolymer to nanoparticle ratios. Te nanocomposites' powder was made by putting these solutions in an oven at 60°C.

Antibacterial Activity.
Tis bacterium S. mutans (ATCC 35668) was procured from the Iranian Research Organization for Science and Technology (IROST) to analyze the antibacterial efectiveness of synthesized alginate/zirconia bionanocomposites. One day was spent cultivating S. mutans on brain heart infusion (BHI) agar to produce a single colony. Te next step was to prepare a bacterial suspension at a concentration of 0.5 McFarland. For 72 h, the bacterial suspension was incubated at 37°C with a 96-well culture plate to form a bioflm. Refreshed BHI replaced the daily culture medium containing 2% sucrose and 1% mannose. After creating a bioflm, the surface was washed three times with phosphate-bufered saline (PBS) to eliminate planktonic S. mutans.
An incubation period of 24 hours was used to synthesize nanocomposite materials in each well. Bioflm viability was determined by isolating cells from well walls and incubating them at 37°C for 24 h. After three washes, the remaining cells adhering to the excellent wall were suspended in 1 ml of PBS bufer. Te resultant suspension was then homogenized for two min using a vortex. Te bacterial suspension was prepared by diluting ten times for the colony forming unit (CFU) test, then cultured on BHI agar plates and incubated at 37°C for 24 h. Te number of colonies was then counted, and their mean was calculated. All experiments had three replications [24,25].

Results and Discussion
3.1. Antibacterial Activity. Te alginate/zirconia bionanocomposites exhibiting the greatest antimicrobial properties were synthesized using the Taguchi method, which consists of nine experiments. Te viability of S. mutans was studied concerning nanocomposites synthesized under various conditions (Table 1). Experiment 6 revealed that the synthesized nanocomposite exhibited the highest antibacterial activity against S. mutans at 70 mg/ml of alginate biopolymer, 8 mg/ml of zirconia NPs, and 40 min of stirring. In its presence, the bacterial viability reached the lowest value, 0.87 CFU/ml.
Biopolymer alginate/zirconia NPs and stirring time all impact the viability of S. mutans, according to Table 2. Te outcomes showed that the stirring time at level 2, the biopolymer alginate factor at level 2, and the zirconia NPs factor at level 3 had the most signifcant efects on the viability of S. mutans. Te interaction of factors that afect the viability of S. mutans is depicted in Table 3. Te interaction between alginate biopolymer in the second level and stirring time in the frst level had the most signifcant efect on the viability rate of S. mutans at 64.50%. Te interaction between zirconia NPs at the third level and stirring time at the frst level signifcantly afected the viability of S. mutans by 18.67%. Alginate biopolymer had the least interaction intensity index in the second level, while zirconia NPs had the highest at 12.03%. Table 4 shows the variance analysis of the parameters afecting the viability of S. mutans. Te zirconia NPs had the highest impact on the viability rate of S. mutans (88.19%), followed by alginate biopolymer (7.05%) and stirring time (2.08%). Te ideal conditions for synthesizing alginate/zirconia bionanocomposites with the greatest antimicrobial activity were identifed following comprehensive information evaluation and analysis of the efects and interactions of each factor (Table 5). Accordingly, zirconia NPs had the highest level, and stirring time exhibited a minor level of infuence on the viability rate of S. mutans. At the same time, alginate biopolymer acted as a mediator between these two variables. Te second level was optimal for alginate biopolymer and stirring time factors, while the third level was optimal for zirconia nanoparticles. S. mutans viability was estimated to be reduced to 0.54 CFU/ml, which is the closest value to the results of experiment 6 and almost better, according to the results. Previous studies reported that zirconia NPs do not show good antimicrobial activity against Grampositive bacteria, especially S. mutans [17,26]. Tis could be due to the surface charges of these nanoparticles, which do not allow proper interaction with the bacterial cell walls of S. mutans [15]. However, synthesizing polymer-based nanocomposites with zirconia NPs can enhance the antibacterial properties of these NPs against Gram-positive bacteria, which accords with the fndings of the current research [27]. Te antibacterial activity of NPs depends on their size at the nanoscale, shape, and high surface energy level [28]. Although the antibacterial mechanisms of zirconia NPs are not fully understood, this property appears to be due to the ability to destroy cell  membranes as well as increased reactive oxygen species (ROS), which interferes with mesosome function, prevents DNA replication and cell division, and results in bacterial death [29]. Figure 1 depicts the FTIR spectra of alginate biopolymer (diagram (a)), zirconia NPs (diagram (b)), and synthesized nanocomposites (diagram (c)) in the 400-4000 cm −1 range of wavelength. Te hydroxyl groups' tensile fuctuations were observed in the spectrum of alginate with a wide peak at 3415 cm −1 . Te bands observed between 2920 and 1615 cm −1 were caused by C-H bond tension in the pyranoid ring and fuctuations in the carboxyl group. Additionally, the C-O bond tension was found to be associated with the peaks at 1290, 1100, and 940 cm −1 . Te bands observed between 885 and 770 cm −1 were of low intensity due to the C-C tensile bond and the internal rotation of the C-O bond, respectively [22,30]. Te FTIR spectrum of zirconia NPs showed a peak related to OH bond tension at position 3417 cm −1 and a peak associated with OH bond bending at position 1651 cm −1 . Also confrming the existence of a Zr-O bond, the peak was observed at 503 cm −1 [31]. Te observed peaks in the fnal spectrum of the nanocomposite were a combination of the peaks observed in the spectrum components. Alginate biopolymer and zirconia NPs form the fnal nanocomposite, indicating strong interactions between these two materials [32].

XRD Analysis.
XRD analysis investigated the crystal structure of alginate biopolymer samples, zirconia NPs, and alginate/zirconia bionanocomposites ( Figure 2). Te alginate biopolymer (a) XRD spectrum showed an amorphous structure for this material. Te XRD spectrum shows the synthesis of ZrO 2 NPs (b). Te dispersed peaks in the diffraction pattern of zirconia NPs at angles 2θ (°) were observed respectively at 28.52 for the plane (−111), 31.72 for the plane (111), 34.46 for the plane (120), 50.54 for the plane (022), and 60.38 for the plane (131) and confrmed the formation of ZrO 2 with a monoclinic crystal structure [33]. Te average crystal size was calculated to be 27 nm using the Scherrer equation for zirconia NPs. XRD spectra prepared from alginate/zirconia bionanocomposites (c) showed that the fnal structure was afected by NPs and alginate biopolymer, and the decrease in the intensity of the synthesized nanocomposite spectrum compared to the zirconia NPs confrmed the nanocomposite formation. Figure 3, the morphology of the alginate biopolymer (Figure 3(a)), zirconia NPs (Figure 3(b)), and prepared nanocomposite (Figure 3(c)) by feld emission scanning electron microscopy were examined. Te alginate biopolymer's SEM image showed this polymer's    interconnected network. Sscanning electron microscopy of zirconia NPs showed agglomeration of some of these NPs and cluster shapes due to their high surface-to-volume ratio.

SEM Analysis. In
Te analysis of these images also revealed that the synthetic zirconium oxide NPs have an almost spherical shape. According to the SEM image, the approximate size range of Journal of Nanotechnology 5 the synthesized zirconia NPs ranges between 20 and 80 nm. Figure 3(d) depicts the histogram of the size distribution of these NPs and the maximum frequency corresponding to the size range from 55 to 60 nm. Nanocomposite networks were synthesized and coated with zirconia NPs as reinforcement for the alginate biopolymer, which could be seen in images of both the biopolymer and the synthesized nanocomposite.

EDX Analysis.
Alginate biopolymer, zirconia nanoparticles, and alginate/zirconia bionanocomposites all had diferent constituent elements, according to energydispersive X-ray (EDX) results ( Figure 4). Tese elements in the polymer alginate include oxygen, sodium, carbon, sulfur, phosphorus, and chlorine, with the highest peak intensity and the highest weight percentage, respectively ( Figure 4(a)). Te EDX spectrum of zirconia NPs (Figure 4(b)) showed the presence of a high percentage of zirconium and an oxygen element, which confrmed the main constituents. Te EDX graph prepared from alginate/ zirconia bionanocomposites (Figure 4(c)) showed the presence of its constituent elements, which verifed the synthesis of the nanocomposite.
3.6. TEM Analysis. Figure 5 displays the TEM micrograph prepared from the alginate polymer ( Figure 5(a)), zirconia NPs ( Figure 5(b)), and synthesized nanocomposites ( Figure 5(c)). Te morphology and particle size distribution of zirconia were also carefully examined by transmission electron microscopy. Te shape of zirconia NPs was spherical. Also, the histogram of the size distribution of NPs showed that the maximum frequency was related to the size range from 50 to 55 nm ( Figure 5(d)). TEM micrograph analysis confrmed the formation of alginate/zirconia bionanocomposites. Te results demonstrated that the metal oxide NPs were dispersed in a biopolymer alginate matrix in a favorable manner and in various sizes.

UV-Vis
Analysis. UV-vis spectroscopy investigated the optical properties of alginate/zirconia bionanocomposites and their constituents between 200 and 800 nm ( Figure 6). Figure 6(a) shows an absorption band at 275 nm in the alginate biopolymer spectrum. Figure 6(b) also displays the UV-vis absorption spectrum for synthesizing zirconia NPs  Journal of Nanotechnology in the 200-800 nm scan range. In the spectrum of zirconia NPs, two peaks were detected between 214 and 236 nm. Te absorption spectra of the fnal synthesized nanocomposite ( Figure 6(c)) demonstrate that the spectrum of the fnal nanocomposite is a combination of the properties of its constituents and that the fnal nanocomposite has been formed [30]. Figure 7 presents the results of the thermal properties of the synthesized nanocomposite and alginate biopolymer. Te thermogravimetric analysis curve of alginate biopolymer revealed four temperature ranges at which a certain percentage of mass was lost. Temperatures between 25 and 210°C resulted in an 8 percent weight loss, while temperatures between 312 and 475°C resulted in a 5.18 percent weight loss, and temperatures between 475°C and 800°C yielded an 11 percent weight loss (Figure 7(a)). Te thermogravimetric diagram showed that the fnal synthesized nanocomposite has higher thermal stability than alginate biopolymer due to zirconia oxide NPs with high thermal stability in the synthesized nanocomposite (Figure 7(b)). Adding NPs to the polymer matrix altered the thermal stability of the nanocomposite, as manifested by a comparison of the thermogravimetric analysis curves for alginate polymer and nanocomposite samples.

Conclusions
Despite the variety of conventional antimicrobial agents, nanoparticles have a particular advantage because nanoparticles have a high surface-to-volume ratio. Terefore, it shows improved properties at their minimum concentration, and using nanoparticles as composite components can improve their stability and efectiveness. In this study, an alginate/zirconia bionanocomposite was tested for its antimicrobial property using the Taguchi method. Te results showed that the in situ-produced nanocomposite was effective against S. mutans bioflm. Based on the antibacterial activity fndings, it was estimated that under optimal conditions, nanocomposites could be synthesized (alginate 70 mg/ml, zirconia of 8 mg/ml, and stirring time of 80 min), and they signifcantly inhibited the formation of bacterial bioflm. In addition, various characterization methods confrmed that the investigated nanocomposite and its components had optimal structural properties. Due to its desirable antibacterial activity, this nanocomposite can be applied to various compounds in various felds. As an effective antibacterial agent, it can be a suitable alternative to the usual antibacterial agents with harmful efects.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.