Unveiling the role of β-Ag2MoO4 microcrystals to the improvement of antibacterial activity

Crystal morphology with different surfaces is important for improving the antibacterial activity of materials. In this experimental and theoretical study, the antibacterial activity of β-Ag2MoO4 microcrystals against the Gram-positive bacteria, namely, methicillin-resistant Staphylococcus aureus (MRSA), and the Gram-negative bacteria, namely, Escherichia coli (E.coli), was investigated. In this study, β-Ag2MoO4 crystals with different morphologies were synthetized by a simple co-precipitation method using three different solvents. The antimicrobial efficacy of the obtained microcrystals against both bacteria increased according to the solvent used in the following order: water <ammonia<ethanol. Supported by experimental evidence, a correlation between morphology, surface energy, and antibacterial performance was established. By using the theoretical Wulff construction, which was obtained by means of density functional calculations, the morphologies with large exposition of the (001) surface exhibited superior antibacterial activity. This study provides a low cost route for synthesizing β-Ag2MoO4 crystals and a guideline for enhancing the biological effect of biocides on pathogenic bacteria by the morphological modulation.

The morphological modulations can be achieved by the different solvents (water, ammonia, and ethanol) used in the co-precipitation synthesis method, as we described previously [28].
As a continuation of our work in this field of research, in this paper, we report the antibacterial activity of β-Ag 2 MoO 4 microcrystals against gram-positive bacteria, namely, methicillin-resistant Staphylococcus aureus (MRSA), and gram-negative bacteria, namely, Escherichia coli (E. coli). These bacteria are important because they are opportunistic pathogens that are often inherently resistant to antibiotics or capable of rapidly building resistance to many common antimicrobial agents [32]. More significantly, the biological effects of β-Ag 2 MoO 4 with different morphologies on the bacteria are systematically discussed. Additionally, to gain a deeper understanding of the atomic and electronic structure, and to establish a correlation among the morphology, surface energy, optical properties, and antibacterial activities, we conducted first principle calculations on the basis of density functional theory (DFT) to complement our experimental findings. This study intends to provide a more comprehensive insight into the development of novel biocide with a unique morphology and future potential for use in biological applications.

Results and Discussion
The β-Ag 2 MoO 4 microcrystals were synthesized by using different solvents (water, ammonia, and ethanol) and the co-precipitation method. The experimental method is described in the Supplementary Material (SM) section. The samples were structurally characterized by x-ray (XRD) diffraction to evaluate the order/disorder at long range. Figure 1 shows the XRD for the samples of silver molybdate. It was observed that all compounds presented a structure type assigned to a cubic spinel with the Fd3m space group, which is in agreement with the Inorganic Crystal Structure Database (ICSD) card 238013 [20]. The lattice parameters for the β-Ag 2 MoO 4 phase are a = b = c = 9.3170 Å and a = b = c 90 °. Moreover, Figure 1 shows clearly that there was no other additional peak; that is, there was no undesirable secondary phase. Figure   1 shows the cubic spinel structure type that was composed by distorted octahedral clusters [AgO 6 ] with Oh symmetry for the Ag sites, and distorted tetrahedral clusters [MoO 4 ] with Td symmetry for the Mo sites. The crystallinity degree of a structure (order/disorder), that is, the organization at long range is directly dependent of the manner of the ions organize themselves into the structure, being that it is totally dependent on the chemical environment in which the material is formed. Therefore, the change of solvent, or the addition of any species in the synthesis of the material may corroborate a change in the order/disorder. A factor related to structural order/disorder effects at long-range can be found by the analysis of the full width at half maximum Raman spectroscopy is a technique complementary to XRD for estimating structural order/disorder at short-range. Figure 2 shows the experimental spectra obtained for all samples. The β-Ag 2 MoO 4 belongs to the point-group symmetry ℎ 7 with the centrosymmetric inversion, which indicates five active Raman modes (A 1g , E g , and T 2g ) obtained from the decomposition of point Γ (Γ = A 1g + E g + 3T 2g + T 1g ) [20]. ] tetrahedral clusters [11,34]. Other vibrations are not observed because they appear below 400 cm -1 , outside the limit of the equipment used.
Theoretical investigations based on first principle calculations by ab-initio and quantum-chemical simulations have been increasingly used to complement the experimental findings, provide valuable information of the electronic, structural, and energetic properties, and simulate and predict the morphology of the materials [20,33,35]. Thus, by using this combined experimental and theoretical approach, previous studies determined that the most stable morphology for the β-Ag 2 MoO 4 can occur when the surface energies are 1.90,1.28, and 3.46 J/m 2 for the (001), (011), and (111) faces, respectively [28,33].
Considering that Ag 2 MoO 4 acquires different morphologies according to the synthetic method employed [28,30], in this study, the morphologies of the β-Ag    [30]. In this study, in addition to observing better anti-E. coli activity for the sample synthesized in ethanol, we also tested, for the first time, the efficiency against MRSA, which is a highly pathogenic bacterium.
It is worth noting that, for the three -Ag 2 MoO 4 microcrystals obtained in water, ammonia, and ethanol, the concentrations required to kill E. coli were always significantly lower than those required to kill MRSA. These findings can be attributed, at least in part, to the different cell wall structures. The Gram positive bacteria, namely, MRSA are composed of a cytoplasmic membrane and a thick, overlying peptidoglycan network (10-40 nm) composed of repeating units of a disaccharide-multipeptide building block that are polymerized and cross-linked to create a continuous network that envelops the cell [37]. This peptidoglycan network has several layers and contains mainly carboxyl, amide and hydroxyl functional groups [38], and teichoic acids (TAs).
Two distinct types of TAs have been identified: wall teichoic acids (WTAs) that are linked to and embedded into the peptidoglycan, and lipoteichoic acids (LTAs) extending into and anchored to the cell membrane (cytoplasmatic membrane) [39]. On the contrary, TAs were not found in gram-negative bacterial cells, such as E. coli [38,39]. Additionally, although gram-negative bacteria contain an outer membrane, they only have a single peptidoglycan layer that is thin (3-6 nm) and located in the periplasmatic space between the outer membrane and the inner (cytoplasmatic) membrane [37]. Given that the cell wall is crucial to the mechanical and chemical   Moreover, according to the Wulff crystal representation of optimized β-Ag 2 MoO 4 , which is shown in Figure 4, these faces have low surface energy (E suf = 1.28 J/m 2 ), as determined by the ab initio calculations. Therefore, they are more easily polarized, and able to generate OH*, O 2 ′, and O 2 H*, which are responsible for cell death.

Conclusion
We

Synthesis and Characterizations
The samples were synthesized as described by Fabbro et al [28]. The samples were characterized using x-ray diffraction (XRD) with a D/Max-2500PC diffractometer (Rigaku, Japan) involving CuK α radiation (λ =1.54056 Å) in the 10-70° 2θ range at a scan rate of 0.01º min −1 . X-ray photoelectron spectroscopy (XPS) analyses were obtained by Omicron-Scienta X-ray photoelectron spectrometer with Al K radiation.
Micro-Raman spectroscopy was carried out by using an iHR550 spectrometer (Horiba Jobin-Yvon, Japan) with a charge-coupled device (CCD) detector and an argon-ion laser

Computational Methods
The Vienna ab initio Simulation Package (VASP) [42][43][44] was used to perform the first-principle calculations. The Kohn-Sham equations were solved by using the generalized gradient approximation (GGA) in the Perdew-Burke-Ernzerhof (PBE) functional to determine the electron exchange and correlation contributions to the total energy [45,46]. The electron-ion interaction was described within a plane wave basis set by the projector augmented wave (PAW) method [47]. The (001), (011), and (111) surfaces of β-Ag 2 MoO 4 were characterized by an unreconstructed slab model using a calculated equilibrium geometry (a = 9.454 Å, and u(O) = 0.2351) and a (3×3×1) Monkhorst-Pack special k-point grid. A vacuum spacer of 15 Å was introduced in the zdirection such that the surfaces would not interact with each other [33].
E surf is defined as the total energy per repeating cell of the slab (E slab ) minus the total energy of the perfect crystal per molecular unit (E bulk ) multiplied by the number of the molecular units of the surface (n), and divided by the surface area per repeating cell of the two slab sides. To confirm the convergence of the total energy with respect to the slab thickness of different surface models, E surf was calculated for several low-index planes. The equilibrium shape of a crystal can be calculated by the classic Wulff construction [48], which minimizes the total surface free energy at a fixed volume, and provides a simple correlation between the surface energy (E surf ) of the (hkl) plane and its distance (rhkl) in the normal direction from the crystallite center. Microbiological experiments were performed on three different occasions, in triplicate.

Surface compositions (XPS)
XPS survey spectra (S1) were similar among the three samples tested, and demonstrated the presence of the elements Ag, Mo, O and C. In the three samples, the silver spectrum was similar, with the 3d 5/2 peak around 368 eV and the 3d 3/2 peak around 374 eV. The energy difference between the peaks is about 6 eV. The data found are in accordance with the literature [50][51][52]. S1: XPS survey spectra of the -Ag 2 MoO 4 microcrystals synthesized in water, ethanol and ammonia.
A summary of the data obtained by XPS analysis showed no significant differences between the three synthesized samples. The binding energy of all the samples were similar for the analyzed elements.