Growth and Characterization of Myristic Acid Crystals Doped with Co and Cu and Microbiological Assays for Potential Antimicrobial Applications

Abstract

In this study, pure myristic acid (MA) polycrystals and those doped with Co and Cu were synthesized and characterized to evaluate their structural features, thermal properties, and antimicrobial effects against the bacterium Xanthomonas citri. Scanning electron microscopy revealed that doping with Co and Cu altered the crystal surfaces. Specifically, pure MA polycrystals exhibited rougher and more porous surfaces, whereas Co and Cu doped MA polycrystals displayed more compact and less porous morphologies. Energy-dispersive X-ray spectroscopy confirmed the presence of Co and Cu in the samples. X-ray diffraction indicated that all samples crystallized in the same monoclinic structure; however, Co and Cu doping led to a slight decrease in unit cell volume and average crystallite size. Raman spectroscopy revealed changes in the vibrational bands of the crystalline lattice. Thermal analyses demonstrated that the addition of Co and Cu ions influenced the thermal stability of pure MA. In microbiological assays, all samples exhibited antimicrobial activity against X. citri. In particular, Co-doped MA polycrystals showed bactericidal properties at all tested concentrations, while pure MA polycrystals exhibited bacteriostatic action at lower concentrations (≤15.6 µg/mL) and bactericidal action at higher concentrations. Cu-doped MA polycrystals did not inhibit bacterial growth at lower concentrations (7.8 µg/mL) but were bactericidal at higher concentrations. These results demonstrated increased lethality against X. citri, particularly for Co-doped MA polycrystals, which exhibited the lowest LD₅₀ value (the toxicological dose required to inhibit 50% of the tested population). Overall, these findings indicate that metal-doped MA polycrystals may be effective for future antimicrobial applications.

1. Introduction

Myristic acid (AM, C14:0) is a long-chain saturated fatty acid with well-known applications in the pharmaceutical, cosmetic, and biomedical industries, alongside relevance in energy storage and functional coatings [1,2,3,4,5,6]. In particular, derivatives of MA exhibit antifungal and bactericidal activity, primarily through the inhibition of essential enzymes and disruption of bacterial membranes [7,8,9]. Recent studies, for instance, have demonstrated that incorporating MA-based coatings into metals can reduce surface bacterial growth by up to 90% [10]. Despite this evidence, however, the potential of MA crystals as a functional solid matrix for antimicrobial applications remains largely unexplored.

One promising approach to enhance the biological activity of organic crystals involves metal ion doping, which modifies their physicochemical characteristics and may improve antimicrobial performance. Transition metal ions are known to interact with bacterial cells through multiple mechanisms, including membrane permeability disruption, protein inactivation, and nucleic acid damage [11,12]. Among these metal ions, Co and Cu ions are of particular interest. Specifically, Cu ions are widely recognized for generating reactive oxygen species, which induce oxidative stress and irreversible cellular damage [13,14]. Meanwhile, Co ions act as catalytic centers in redox reactions, thereby enhancing bactericidal efficiency through enzymatic disruption [15,16]. Furthermore, Co and Cu based nanomaterials exhibit strong antimicrobial effects, with inhibition rates exceeding 90% at micromolar concentrations against both Gram-negative and Gram-positive bacteria [13,15,17]. Collectively, their high surface-to-volume ratio, magnetic properties, and biocompatibility support their selection as dopants for MA crystals in this study.

Xanthomonas citri is the causal agent of citrus canker, a devastating disease that can reduce citrus production by over 30% in severely affected orchards [18]. The growing resistance of X. citri to conventional antibiotics, such as streptomycin, which often fails to control infections in the field [19], underscores the urgent need for alternative antimicrobial strategies. Accordingly, X. citri was selected as the model microorganism for evaluating the antibacterial efficacy of the synthesized MA crystals. Establishing a link between the structural modification of MA crystals and the inhibition of X. citri provides a meaningful contribution at the intersection of materials science and agricultural biotechnology.

Herein, MA polycrystals were synthesized in three formulations and compared: undoped (PMA) and doped with 10% Co (CoMA) or 10% Cu (CuMA). These samples were characterized using high-resolution scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), Raman spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TG), and differential thermal analysis (DTA). Microbiological assays against X. citri, a phytopathogenic bacterium of agricultural relevance, were performed to evaluate the antimicrobial potential of the synthesized materials. An analysis of the obtained micrographs, corroborated by EDS results, revealed that metal incorporation notably altered the morphology of the doped crystals. Raman spectroscopy further indicated modifications in the crystal lattice, while XRD revealed a reduction in lattice parameters. In particular, the Co-doped sample exhibited a lower LD₅₀ (the toxicological dose required to inhibit 50% of the tested population) value than the undoped reference.

2. Experimental Procedures

2.1. Crystal Synthesis

CoMA crystals were synthesized using the slow evaporation method [20]. A solution of 0.45 g MA (C₁₄H₂₈O₂, 99% purity, supplied by Sigma-Aldrich, St. Louis, MO, USA) and 0.05 g cobalt sulfate heptahydrate (CoSO₄·7H₂O, supplied by Sigma-Aldrich, St. Louis, MO, USA, pure) was prepared in 25 mL of chloroform and 5 mL of deionized water (pH 5). The reagents fully dissolved after 45 min of stirring at 308 K using a magnetic stirrer. The resulting solution was filtered and maintained at 305 K to facilitate crystal nucleation. Polycrystalline CoMA formed after 10 days. Similarly, CuMA polycrystals were synthesized using copper(II) chloride dihydrate (CuCl₂·2H₂O, supplied by Sigma-Aldrich, St. Louis, MO, USA, pure). For comparison, pure MA (PMA) polycrystals were synthesized by dissolving 0.50 g MA in 25 mL of chloroform, stirring for 45 min at pH 5 and 308 K, and then allowing slow evaporation at 305 K for 10 days.

2.2. X-Ray Powder Diffraction (XRPD) Analysis

XRPD patterns of the polycrystalline samples were acquired using a PANalytical Empyrean diffractometer (Malvern, England) with Cu Kα radiation (λ = 1.5418 Å), operating at 45 kV and 40 mA. Diffractograms were recorded at room temperature over a 2θ range of 5–40°, with a step size of 0.02° and a counting time of 2 s per step. The patterns were analyzed using Rietveld refinement in GSAS-II software (version 286c6d) [21]. Initial lattice parameters and atomic positions for refinement [4] were sourced from the CIF file (ZZZOEG03 738618) available in the Cambridge Crystallographic Data Center.

2.3. Raman Spectroscopy

Raman spectra were recorded under ambient conditions using a HORIBA Jobin Yvon T6400 triple spectrometer (Palaiseau, France) equipped with a liquid-nitrogen-cooled charge-coupled device detector. A spectral resolution of 2 cm⁻¹ was achieved in triple-grating mode. An argon-ion laser (λ = 532 nm) served as the excitation source. The laser beam was focused onto the samples using an Olympus BX40 microscope fitted with a 20× objective (focal length = 20.5 mm, numerical aperture = 0.35).

2.4. Fourier-Transform Infrared (FT-IR) Spectroscopy

FT-IR spectra were acquired using a Bruker Vertex 70v spectrometer (Karlsruhe, Germany) across the spectral range of 40–3000 cm⁻¹. A platinum attenuated total reflectance module (A225/Q) was employed in conjunction with a wide-range RT-DLa TGS detector (6 mm aperture). Spectra were recorded at a resolution of 4 cm⁻¹ over 220 scans.

2.5. Morphological Characterization

The synthesized polycrystals were morphologically characterized using a high-resolution Tescan Mira field-emission gun SE microscope (Brno, Czech Republic) equipped with a four-lens electron optics column. Micrographs were captured at multiple sample regions using magnifications of 2000×, 5000×, and 10,000×. EDS was conducted using an Oxford X-Max-80 detector (80 mm² active area; 500,000 cps count rate) at an accelerating voltage of 5 keV and a working distance of 15 mm.

2.6. Thermogravimetric-Differential Thermal Analysis (TG-DTA)

Simultaneous TG–DTA analyses were performed using a Shimadzu DTG-60 analyzer (Kyoto, Japan) at a nitrogen flow rate of 100 mL/min. Measurements were conducted on 4 mg samples over a temperature range of 298–1000 K at a heating rate of 10 K/min. Crystal morphology was assessed using a JEOL JSM-7100F SE microscope with samples mounted on aluminum stubs.

2.7. Microbiological Assay (Minimal Inhibitory Concentration, MIC Test)

The MIC test [19,22] was performed to evaluate the antimicrobial activity of the synthesized materials against X. citri. A liquid culture medium containing 10 g sucrose, 8 g casein hydrolysate, 4 g yeast extract, 2 g K₂HPO₄, and 0.3 g MgSO₄·7H₂O (pH 6.9) per liter of distilled water was prepared. The medium was autoclaved at 393.15 K (20 psi) for 20 min. For the solid medium, 4.5 g agar was added per 100 mL before autoclaving, and the mixture was poured into 90 × 15 mm Petri dishes [18].

Bacterial activation was achieved by culturing X. citri on agar plates at 303.15 K for 24 h. A loopful of bacteria was subsequently inoculated into 3 mL of liquid medium and incubated at 303.15 K for another 24 h. The culture was standardized to approximately 1.0 × 10⁸ CFU/mL (colony-forming units).

For serial dilution, 1 mg of each sample—PMA, CoMA, and CuMA—was dissolved in 100 µL of dimethyl sulfoxide in 5 mL microtubes and vortexed. Subsequently, 900 µL of sterilized medium was added to each tube to prepare the stock solution. Streptomycin sulfate (1 mg/mL in distilled water) served as the control antibiotic and was diluted to 5 µL/mL in the medium.

The assay was conducted in 96-well tissue culture test plates, with three replicates per treatment. Each well initially received 100 µL of the medium. Subsequently, 100 µL of the stock solution was added to the wells in the first column (well A). Serial dilutions were performed by transferring 100 µL of the solution from well A to well B, mixing thoroughly, and continuing this process through well G. Finally, from well G, 100 µL solution was discarded. Well H served as the positive control and contained only the medium. Finally, 5 µL of the bacterial suspension was added to each well, and the plates were incubated at 303.15 K for 24 h.

Antimicrobial activity was visualized using 2% 2,3,5-triphenyltetrazolium chloride (TTC). Following the addition of 20 µL of TTC to each well, the plates were incubated for 2 h. Wells lacking bacterial growth remained colorless, whereas those with bacterial growth turned red [19,22].

To differentiate bacteriostatic from bactericidal effects, solutions from wells that did not turn red were reinoculated onto agar plates and incubated at 308.15 K for 24 h. The absence of bacterial growth indicated bactericidal activity, whereas the presence of growth indicated bacteriostatic effects.

Statistical analysis of variance (ANOVA) was conducted using R software version 4.3.3 (2024) and was followed by Duncan’s multiple range test [23,24] for post hoc comparisons. The significance threshold was set at p ≤ 0.05, and data were grouped according to similarity through cluster analysis. Duncan’s test, widely used in laboratory analyses, was performed to determine which treatment means differed significantly after a significant ANOVA result.

3. Results and Discussion

3.1. Crystal Voids

In materials science, doping is widely adopted to tailor a material’s physical and chemical properties through the introduction of foreign atoms or ions into its structure [25]. Analyzing voids within the unit cell of a material is essential for evaluating its doping capacity, as such voids can be utilized to accommodate dopants that modify the base compound’s properties [26,27]. As depicted in Figure 1, the presence of voids in the crystal structure of MA supports detailed investigations of its doping feasibility.

The voids within the MA unit cell, with an area of 706.86 Ų and a volume of 153.53 ų, correspond to an experimental porosity of approximately 10%, which is comparable to the porosity reported for a dipeptide by Soldatov et al. [28]. This observation indicates a non-fully dense structure containing regions capable of accommodating additional atoms or molecules. Such voids may result from inefficient molecular packing [26,27]. These regions serve as ideal sites for dopant incorporation, including transition or alkali metals, which can impart distinct functional properties.

The doping potential of MA, identified through the above void analysis of its unit cell using CrystalExplorer [27], offers a promising route for developing materials with enhanced properties. A detailed understanding of these voids enables targeted doping, allowing for the introduction of specific ions to optimize desired material characteristics. Consequently, doping not only broadens the application scope of MA but also underscores the importance of structural analysis in materials engineering.

3.2. SEM and EDS Analyses

High-resolution SEM was employed to characterize the PMA, CoMA, and CuMA samples, revealing distinct structural features across the three compositions. To capture morphological patterns, multiple micrographs were acquired from various regions of each sample at magnifications of 2000×, 5000×, and 10,000×.

Figure 2a–c presents SEM micrographs of the PMA sample at varying magnifications, revealing a rough surface with few pores and a moderate number of well-defined, circular grain aggregates, with an average diameter of approximately 600 nm. Meanwhile, Figure 2d–f presents SEM micrographs of the CoMA sample at varying magnifications. The surface of the CoMA sample appears considerably more compact than that of the PMA sample, with fewer pores and a lower density of grain aggregates.

Figure 2g–i presents those of CuMA. As depicted, the surface of the CuMA sample appears more compact than that of PMA, exhibiting fewer pores and a reduced number of grain aggregates. However, the CuMA sample displays more grain aggregates than the CoMA sample.

Figure 3 presents the EDS spectra of the PMA (a), CoMA (b), and CuMA (c) samples. The spectra confirm the presence of C and O, originating from MA molecules, while Au and Pd arise from sputter coating applied during SEM sample preparation. Additionally, Figure 3b presents the EDS spectrum of the CoMA sample, which reveals the presence of Co introduced during synthesis, along with trace levels of residual S from the CoSO₄·7H₂O reagent. Figure 3c displays the EDS spectrum of the CuMA sample, revealing the presence of Cu, incorporated during synthesis, and trace levels of residual Cl from the CuCl₂·2H₂O reagent.

3.3. XRPD Analysis

Figure 4 presents the experimental XRPD patterns of the polycrystalline (a) PMA, (b) CoMA, and (c) CuMA samples. The red line represents the computed pattern obtained through Rietveld refinement at room temperature. Notably, all samples crystallize in a monoclinic structure with the space group P2₁/c. Each unit cell comprises four molecules. Rietveld refinement yields the following lattice parameters: for PMA, a = 31.701(5) Å, b = 4.957(1) Å, c = 9.470(5) Å, α = γ = 90°, β = 95.347(0)°, and V = 1481.8(4) ų; for CoMA, a = 31.647(8) Å, b = 4.932(7) Å, c = 9.385(5) Å, α = γ = 90°, β = 94.284(0)°, and V = 1461.1(0) ų; and for CuMA, a = 31.570(7) Å, b = 4.939(2) Å, c = 9.456(1) Å, α = γ = 90°, β = 95.422(0)°, and V = 1467.9(6) ų. The lattice parameters for the PMA sample are consistent with those reported in the literature [4]. Unit cell volume reductions of approximately 1.4% and 1.0% are observed for CoMA and CuMA relative to PMA, respectively. The average crystallite size was estimated using the Scherrer equation [29,30], yielding values of 312.14 nm for PMA, 165.55 nm for CoMA, and 307.26 nm for CuMA—each notably smaller than the reference value of 675.14 nm reported in the literature (

Table 1. Lattice parameters of PMA, CoMA, and CuMA samples obtained from Rietveld refinement, compared with literature values [4].

	CIF [4]	PMA	CoMA	CuMA
a (Å)	31.628(2)	31.701(5)	31.647(8)	31.570(7)
b (Å)	4.966(9)	4.957(1)	4.932(7)	4.939(2)
c (Å)	9.492(2)	9.470(5)	9.385(5)	9.456(1)
α°	90.000(0)	90.000(0)	90.000(0)	90.000(0)
β°	95.105(7)	95.347(0)	94.284(0)	95.422(0)
γ°	90.000(0)	90.000(0)	90.000(0)	90.000(0)
Volume (Å)3	1485.25	1481.84	1461.10	1467.96
Crystallite (nm)	675.14	312.14	165.55	307.26
C. Structure	Monoclinic	Monoclinic	Monoclinic	Monoclinic
Space group	P21/c	P21/c	P21/c	P21/c

).

3.4. Raman and FT-IR Spectroscopy

Figure 5 presents the Raman spectra of the PMA, CoMA, and CuMA samples in the spectral range of 50–1800 cm⁻¹. The corresponding vibrational modes are listed in

Table 2. Raman and FT-IR vibrational modes. Adapted from [3,31].

Raman-Active Modes				IR-Active Modes
	(cm−1)		Assignments	(cm−1)		Assignments
	50		XX	600		XX
	161		τ(CCCC)(60)	672		sc(C14O2)(61)
	167		τ(CCCC)(65)	687		sc(C14O2)(61)
	181		δ(CCC)(78)	720		τ(C13C14O15H)(80)
	187		δ(CCC)(78)	728		τ(C13C14O15H)(80)
	200		XX	750		XX
	550		XX	1600		XX
	571		δ(CCC)(64)	1678		ν(C=O)(83)
	584		δ(CCC)(64)	1717		ν(C=O)(83)
	654		sc(C14O2)(61)	1800		XX
	671		τ(HO15C14C13)(80)
	742		ρ(CH2)(60)
	750		XX
	1350		XX
	1372		wag(C1H3)(84)
	1381		XX
	1411		sc(C13H2)(88)
	1437		sc(CH2)(69)
	1450		sc(CH2)(73)
	1462		sc(CH2)(55)
	1475		sc(CH2)(64)
	1507		sc(CH2)(71)
	1550		XX
	1600		XX
	1626		ν(C=O)(83)
	1649		ν(C=O)(83)
	1700		XX

ν = stretching; δ = bending; sc = scissoring; ρ = rocking; wag = wagging; τ = torsion.

[3,31]. Figure 6 highlights selected regions of the Raman spectra for detailed comparison. Specifically, Figure 6a displays the spectral region between 50 and 160 cm⁻¹. The vibrational modes in this region are associated with lattice vibrations caused by the movement of large portions of the crystal lattice [3]. Within this region, distinct spectral changes are observed for the CoMA sample relative to PMA, potentially resulting from the incorporation of Co²⁺ ions. The CuMA spectrum similarly reflects the incorporation of Cu²⁺ ions into the crystal structure. The bands at 161 and 167 cm⁻¹ are associated with carbon chain torsion (CCCC), whereas those at 181 and 187 cm⁻¹ correspond to chain bending (CCC). In the CoMA spectrum, these bands exhibit notable shifts, reflecting the structural influence of the incorporated Co²⁺ ions. These spectral shifts arise from the interstitial incorporation of Co²⁺ and Cu²⁺ ions, which exert stress on the unit cell and alter hydrogen bonding interactions.

Figure 6b presents the spectral region of 550–750 cm⁻¹. In this region, the bands at 571 and 584 cm⁻¹ are assigned to CCC bending, the band at approximately 654 cm⁻¹ corresponds to scissoring vibrations of the carboxyl group, the band at 671 cm⁻¹ is attributed to chain torsion, and the band at 742 cm⁻¹ is associated with CH₂ rocking deformation, as reported in the literature [3].

Figure 6c presents the spectral range of 1350–1550 cm⁻¹, where vibrations associated with CH₂ group motion are observed. The bands at 1372, 1381, 1411, 1437, 1450, 1462, 1475, and 1507 cm⁻¹ correspond to CH₂ scissoring vibrations [3]. A shift in the bands of the CoMA polycrystal is again evident, suggesting an indirect influence of the Co²⁺ ion on the CH₂ group.

Figure 6d presents the spectral range of 1600–1700 cm⁻¹. The bands observed at approximately 1626 and 1649 cm⁻¹ are attributed to carbonyl (C=O) stretching [3]. In this range, noticeable shifts in the vibrational bands of the CoMA sample indicate a pronounced change in stretching, driven by the influence of the metal ion.

Figure 7a presents the FT-IR spectra of the PMA, CoMA, and CuMA samples across the range of 400–1800 cm⁻¹. Figure 7b displays the spectral region between 600 and 750 cm⁻¹. Shifts in the bands at 672 and 687 cm⁻¹, attributed to carboxyl group scissoring vibrations, are noticed owing to the influence of Co²⁺ and Cu²⁺ ions. The bands at 720 and 728 cm⁻¹ are assigned to carbon chain torsional modes.

Figure 7c displays the 1600–1800 cm⁻¹ region, where the bands at 1678 and 1717 cm⁻¹, assigned as carbonyl (C=O) stretching, exhibit shifts relative to the PMA sample. These spectral shifts reflect the incorporation of Co²⁺ and Cu²⁺ ions into the crystal structure.

3.5. TG and DTA Analyses

Figure 8 presents the TG and DTA curves of the PMA, CoMA, and CuMA samples, while

Table 3. Thermal events observed in the TG and DTA analyses of the samples.

	TG				DTA
	Tonset (K)	Tmidset (K)	Tendset (K)	Weight Loss (mg)	T (K)
PMA	480.3	492.2	509.8	4.00	331
CoMA	477.4	491.3	509.8	3.99
CuMA	484.9	492.2	508.5	3.92

summarizes the corresponding thermal events observed during analysis. As depicted in Figure 8 (green line), the TG curve of PMA reveals a substantial mass loss between 480.3 K and 509.8 K (4 mg–100%), indicating that MA decomposes within this temperature range. The DTA curve displays an endothermic event at 331 K across all samples, which may correspond to a solid–liquid phase transition [32].

The incorporation of Co²⁺ and Cu²⁺ ions notably influences the onset (T_onset), midpoint (T_midset), and endpoint (T_endset) temperatures of the thermal events. As depicted in Figure 9 (blue line), the CuMA sample exhibits a decomposition profile similar to that of PMA (3.92 mg–98%), with a T_onset of 484.9 K, suggesting that Cu exerts a milder influence on MA’s thermal stability. Figure 9 (red line), in contrast, indicates that CoMA decomposes between 477.4 K and 509.8 K (3.99 mg–99%), with a slightly lower T_onset than that of PMA, implying that Co may facilitate the onset of decomposition [33].

The above comparative analysis reveals that the addition of Co alters the thermal stability and decomposition behavior of MA. In contrast, Cu exerts a more moderate effect, maintaining thermal stability at a level comparable with that of pure MA.

These findings are crucial for understanding the interactions between MA and different metal ions and provide insights into how such additives can be used to fine-tune the material’s thermal properties. This knowledge is essential for developing materials with tailored thermal stability, especially for applications wherein heat resistance or controlled decomposition are essential [34,35].

3.6. Biological Assay

Microbial viability was calculated based on spectrophotometric readings (Thermo Scientific Multiskan Go Microplate Spectrophotometer) and expressed as the percentage of MIC at various sample concentrations, according to the following equation 1:

$$\mathrm{\%}\mathrm{M}\mathrm{I}\mathrm{C}=\left[1- {\displaystyle \frac{A_c}{A_0}}\right]\times 100$$

(1)

where A_c is the mean absorbance at each sample concentration (after subtracting the absorbance of the substance alone, without bacteria), and A₀ is the mean absorbance of the microbial growth control (without the test substance). This value represents the percentage of microbial cells destroyed in the tested sample [36].

The MIC (%) results for antibacterial activity revealed that all samples, including PMA as well as those doped with the tested ions (CoMA and CuMA), demonstrated the ability to inhibit the growth of X. citri bacterial colonies. Most samples exhibited bactericidal activity, except—PMA at 15.6 µg/mL and 7.8 µg/mL, where the effect was bacteriostatic. For CuMA, the assay confirmed that no inhibitory effect was observed at the lowest tested concentration (7.8 µg/mL), while all other concentrations resulted in bacterial inhibition. All values of antimicrobial activity are given in

Table 4. Antimicrobial activity of PMA, CoMA, and CuMA samples against X. citri.

Samples	Inhibitory Activity
	Concentrations
	A	B	C	D	E	F	G
	500 µg/mL	250 µg/mL	125 µg/mL	62.5 µg/mL	31.3 µg/mL	15.6 µg/mL	7.8 µg/mL
PMA	*	*	*	*	*	**	**
CoMA	*	*	*	*	*	*	*
CuMA	*	*	*	*	*	*	N

(*) Bactericidal activity (**) Bacteriostatic activity (N) No Inhibitory activity.

.

Notably, the tested samples outperformed the antibiotic used in the control (streptomycin sulfate, Figure 10). This antibiotic exhibited no inhibitory action against the bacterium in the control, indicating that the organism was resistant to it.

One potential explanation for this resistance is a mutation in the rpsL gene, which encodes the ribosomal S12 protein. Mutations in this gene can prevent streptomycin from binding, thereby reducing or completely eliminating its efficacy and leading to the observed control failure. Furthermore, the administration of subinhibitory or excessive doses of antibiotics, as well as their indiscriminate use, can accelerate the development of antimicrobial resistance [37].

Statistical Analysis of the Microbiological Assay

Figure 9 displays the mean absorbance values obtained from the ELISA assay for treatments conducted using both the culture medium and bacterial cells. The x-axis includes the tested antibiotic (Ant.), culture medium (MB), PMA, CoMA, CuMA, and culture medium alone (CM). The corresponding mean absorbance values were 0.48, 0.40, 0.05, 0.06, 0.06, and 0.04, respectively.

According to the statistical analysis, the Ant. and MB samples were grouped under “a”, indicating no statistically significant difference between their mean absorbance values. Meanwhile, the PMA, CoMA, CuMA, and CM samples were grouped under “b,” also indicating no significant differences among their means. However, a significant difference was observed between groups “a” and “b”, corroborating the ELISA assay results that indicated microbial growth in the Ant. and MB samples and microbial inhibition in the remaining treatment groups.

An additional notable finding is that X. citri exhibited resistance to the antibiotic control included in the assay. Further, the findings of Duncan’s test confirmed the antimicrobial activity of PMA, CoMA, and CuMA.

Metals are known to exert antimicrobial effects by damaging bacterial membranes, inducing oxidative stress, and disrupting key metabolic pathways. Fatty acids alter membrane permeability, and their antimicrobial efficacy can be enhanced through metal doping. This synergistic effect primarily occurs at the membrane interface, where it regulates metal ion influx and leads to increased cellular damage. Moreover, the controlled release of metal ions may prolong the antimicrobial effect [38,39].

The surface morphology of the investigated materials (Figure 2) appeared to influence antimicrobial activity (Figure 9). In particular, PMA and CuMA featured surfaces with pronounced roughness, visible pores, and a substantial number of grain aggregates, while CoMA displayed regions with sharp, protruding structures [40]. These morphological characteristics promoted greater antimicrobial activity by inhibiting bacterial growth, resulting in a notable difference in antimicrobial activity compared to the Ant. and MB treatments. In general, surface roughness and porosity increase the contact area with bacteria, enhancing physical interactions, facilitating the adsorption of bacterial biomolecules, and enabling direct contact with cellular membranes [41].

Beyond this, polynomial regression [42] was applied to model the nonlinear relationship between %MIC and sample concentration, yielding a coefficient of determination (R²) greater than 0.80 (Figure 10). This result indicates that at least 80% of the variation in %MIC can be explained by the model, suggesting a good fit. From the regression curves, the LD₅₀ value (lethal dose 50%) [43], the toxicological dose required to inhibit 50% of the tested population, was also determined.

Given the parabolic distribution of the data points, a quadratic polynomial (y = ax² + bx + c) was identified as the most suitable model, with x representing the independent variable and y the dependent variable. The following equations (2, 3, and 4) were derived:

• Figure 10 (green line)—PMA sample:

y = −2.9 × 10⁻⁵ x² + 0.02x + 95.9 (R² = 0.81, LD₅₀ = 75.3 µg/mL).

(2)

• Figure 10 (blue line)—CuMA sample:

y = −3.2 × 10⁻⁵ x² + 0.02x + 95.9 (R² = 0.83, LD₅₀ = 86.3 µg/mL).

(3)

• Figure 10 (red line)—CoMA sample:

y = −5.4 × 10⁻⁵ x² + 0.04x + 94.5 (R² = 0.81, LD₅₀ = 73.0 µg/mL).

(4)

Although the quadratic polynomial effectively described the observed data, it may not reliably predict values beyond the tested range. Thus, more complex models should be investigated in future research.

The fitted model revealed a pronounced quadratic relationship between %MIC and sample concentration. Notably, an R² > 0.80 indicates that over 80% of the variability in inhibition is explained by the fitted curve. Further, the downwardly concave parabolic shape indicates increasing inhibition up to a concentration of 250 µg/mL.

4. Conclusions

In this study, pure and Co- and Cu-doped MA polycrystals were synthesized, and the effects of metal doping on their physicochemical properties and antimicrobial activity against X. citri were evaluated. Doping with Co and Cu affected the crystallization process, leading to changes in morphology, porosity, and grain size. XRD analysis revealed that doping reduced the lattice parameters and unit cell volume of the polycrystals. The most pronounced reduction occurred in the CoMA sample, which exhibited a unit cell volume of 1461.10 ų and a crystallite size of 165 nm. In comparison, the PMA sample had a unit cell volume of 1481.84 ų and a crystallite size of 312.14 nm. These findings imply enhanced bactericidal activity, as both particle morphology and size directly influence microbial inhibition. Raman and FT-IR spectroscopic analyses supported these structural changes, suggesting that metal ions were incorporated into the crystal lattice, where they influenced hydrogen bonding and lattice vibrations. The incorporation of Co altered the thermal stability of MA by lowering its initial decomposition temperature, while Cu produced a more moderate effect, preserving stability comparable to that of the undoped compound. All tested samples, namely PMA, CoMA, and CuMA, effectively inhibited the growth of X. citri, outperforming the control antibiotic, streptomycin sulfate, to which the bacterium was resistant. Notably, among these samples, CoMA exhibited the strongest effect, achieving bactericidal action at all tested concentrations. It also presented the lowest LD₅₀ value (73.0 µg/mL), suggesting that Co doping enhanced the bactericidal potency of the compound. Therefore, while Co and Cu doping did not alter the crystalline phase of MA, they did modify its structural characteristics and antimicrobial efficacy. In particular, CoMA emerged as the most promising formulation for future antimicrobial applications. Based on these findings, future research should investigate the mechanism of action of these compounds, broaden their microbiological scope, and assess their toxicity in mammalian cells. Additionally, efforts to optimize the synthesis by varying dopant concentrations and exploring alternative ions, such as Zn or Ag, may further enhance the material’s properties and antimicrobial activity.