Antibacterial performance of Co–Zn ferritenanoparticles under visible light irradiation

Research Article Received: 22 June 2024 Revised: 25 October 2024 Published online in Wiley Online Library: 20 November 2024 (wileyonlinelibrary.com) DOI 10.1002/jctb.7785 Antibacterial performance of Co–Zn ferrite nanoparticles under visible light irradiation Alicia G. Gubieda,a * Ana Abad-Díaz-de-Cerio,aAna García-Prieto,b M. Luisa Fdez-Gubieda,cLaura Cervera-Gabalda,d,e Eduardo Ordoqui-Huesa,fAlfonso Cornejofand Cristina Gómez-Polof Abstract BACKGROUND: To address water scarcity and promote sustainable resource management, more efficient and cost-effective water treatment solutions are necessary. Particularly, pathogens in drinking water are a topic of growing concern. One promising technology is the use of photocatalytic nanoparticles activated by visible light as antibacterial agents. This study focuses on the characterization and antibacterial properties of Co–Zn ferrite nanocatalysts, tested against Escherichia coli. RESULTS: The Co x Zn 1−x Fe 2 O 4 (x=0, 0.1, 0.4 and 0.6) ferrites were synthesized by the co-precipitation method. Structural, morphological and optical analyses confirmed that these nanoparticles have a cubic spinel structure, with sizes of around 10 nm, and band gap energies suitable for visible light activation (1.4–1.7 eV). The antibacterial efficacy of the nanoparticles against E. coli was tested and compared with their photocatalytic performance employing phenol as organic pollutant model (highest phenol degradation for x=0.6). Specifically, the antibacterial capacity of these nanoparticles was evaluated by comparing the ability of bacteria to grow after being incubated with the nanoparticles under visible light and in the dark. It was found that nanoparticles with lower cobalt content (x=0 and 0.1) significantly reduced bacterial culturability under visible light. Transmission Electron Microscopy analysis revealed that nanoparticles with cobalt content caused bacteria to secrete biofilm, potentially offering some protection against the nanoparticles. CONCLUSION: ZnFe 2 O 4 nanoparticles show the highest antibacterial effect amongst those tested. This is attributed to the combined action of Zn 2+ ion release and the photocatalytic effect under visible light. Furthermore, Zn might inhibit protective biofilm secretion, leading to higher antibacterial effects. © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). Keywords: Co–Zn ferrites; photocatalytic nanoparticles; antibacterial nanoparticles INTRODUCTION Infections related to contaminated water resources by waterborne pathogens are a major global concern for water quality. According to the 2030 Agenda for Sustainable Development's SDG 6, which aims to ensure access to water and sanitation for all, 2.2 billion people still lacked safely managed drinking water in 2022, including 703 million without a basic water service. 1 Diarrhea, mainly linked to limited access to improved water and sanitation, is the second leading cause of child morbidity and mortality worldwide, and responsible for more than 90% of deaths in children under 5 years of age in lowand middleincome countries. 2 Escherichia coli, besides being one of the major pathogens associated with waterborne diseases, is commonly used as an indicator of fecal contamination to verify water quality. 3,4 Furthermore, the extensive use of antibiotics in both veterinary and human medicine has promoted the occurrence of antibiotic-resistant bacteria, the primary contributor to antimicrobial resistance and one of the most worrying public health problems. In fact, antimicrobial resistance is expected to kill *Correspondence to: AG Gubieda, Departamento de Inmunología, Microbiología y Parasitología, Universidad del País Vasco (UPV/EHU), Leioa 48940, Spain, E-mail: [email protected] aDepartamento de Inmunología, Microbiología y Parasitología, Universidad del País Vasco (UPV/EHU), Leioa, Spain bDepartamento de Física Aplicada, Universidad del País Vasco (UPV/EHU), Bilbao, Spain cDepartamento de Electricidad y Electrónica, Universidad del País Vasco (UPV/EHU), Leioa, Spain dSpLine, Spanish CRG beamline at the European Synchrotron Radiation Facility, Grenoble, France eICMM-CSIC Cantoblanco, Madrid, Spain fDepartamento de Ciencias & Institute for Advanced Materials and Mathematics (INAMAT²), Universidad Pública de Navarra, Pamplona, Spain © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 428 10 million people by 2050 and cost the global economy $100 trillion. 5,6 Microbial pathogens are generally inactivated in wastewater treatments through conventional chemical oxidation agents like chlorine, chlorine dioxide, chloramines and ozone. 7 However, these often result in the generation of harmful disinfection byproducts. UV technologies offer an alternative disinfection technique, although the higher cost, energy consumption and lower effectiveness for UV-resistant pathogens limit their wide practical application. Among the current proposed technologies for the elimination of microbial pathogens, advanced oxidation processes stand out based on the generation of highly reactive oxygen species (ROS). 8 Particularly, photocatalytic antibacterial procedures stand out, where the generation of ROS is stimulated by light irradiation. 9 Heterogeneous photocatalysts such as TiO 2 and ZnO have been extensively studied for degrading a broad range of organic pollutants and pathogenic microorganisms. 10-13 13 However, most widely employed photocatalysts are characterized by a wide band gap (ca 3.2 eV, TiO 2 ), making them mainly active under UV light irradiation. For practical purposes, the activation of a semiconductor under visible (solar) light would facilitate the development of low-cost water disinfection systems, where the elimination of organic pollutants and microbial pathogens would be carried out synergistically. Accordingly, different visible-light-active photocatalytic semiconductors have been proposed for the simultaneous removal of organic and microbial pollutants, including semiconductor heterostructures 14-16 and doped TiO 2 (Ag, 17 Cr, 18 N 19 ), among others. Despite extensive research activity in recent decades, commercial applications of photocatalysis in wastewater treatment are currently scarce due to inherent process challenges (e.g. low photoconversion efficiency) and technological limitations (e.g. energy consumption). The separation of the photocatalytic agent from the medium, avoiding secondary contamination, and its subsequent reuse is one of the most important issues when optimizing a procedure. Thus, magnetic materials have emerged as potential alternatives for the easy and complete separation of catalysts using external magnetic field gradients. Most strategies are based on nanostructured magnetic materials, such as core–shell nanoparticles, where a magnetic core (i.e. Fe 3 O 4 )is usually coated with a photocatalytic active semiconductor. 20-22 The superparamagnetic regime (negligible values of coercivity and remanence) characteristic of magnetic nanoparticles is advisable in these systems to avoid particle aggregation and to achieve optimal dispersion in an aqueous medium. Spinel ferrite photocatalysts have demonstrated optimal performance for the degradation of organic pollutants in water under visible irradiation. 21,23 The combination of their narrow band gap (within the visible light range) and their ferrimagnetic nature, which allows reuse in several cycles through magnetic separation, makes these photocatalysts promising candidates for wastewater treatments. In particular, the photocatalytic response of Co–Zn spinel ferrites has been widely analyzed in the literature. 24-30 Furthermore, they have attracted significant interest due to their high magnetic moments (high saturation magnetization values), achieved by the incorporation of Co in the ZnFe 2 O 4 spinel cell structure. 31 Bulk ZnFe 2 O 4 (antiferromagnetic with Néel temperature T N =10 K) is a normal spinel, where Fe 3+ cations antiferromagnetically coupled occupy octahedral Bsites, and Zn 2+ cations (nonmagnetic) are preferentially located at the tetrahedral Apositions. However, mixed states (i.e. Fe 3+ cations in both Band Asites) give rise to the appearance of ferrimagnetism in this spinel and the desired superparamagnetism in the nanoscale regime. Since CoFe 2 O 4 is an inverse spinel, where Co 2+ cations tend to occupy Bsites, the replacement of Co by Zn leads to a displacement of A-Fe 3+ ions to the octahedral Bsites and to an initial increase of the net magnetic moment as Zn concentration increases followed by a decay for higher Zn concentrations that is ascribed to the null magnetic moment of Zn 2+ ions. 29,31 Thus, optimum magnetic response is found for mixed Co–Zn ferrite nanoparticles: maximum values of the magnetic moment of the spinel cell (maximum saturation magnetization) and superparamagnetic behavior. As for their photocatalytic response, most studies focus on the decomposition of dyes, reaching degradation rates close to 100%, 24-27 while the degradation of phenolic compounds is more limited and their photocatalytic efficiency significantly reduced. 29 Nevertheless, it is important to highlight that all studies show optimal photocatalytic behavior for those mixed compositions linked to maximum values of magnetic moments of the Co–Zn spinel cell. In fact, spin-polarization effects on the improvement of visible photocatalysis is a topic of current interest, 32 mostly linked to the inhibition of charge recombination. 28 However, despite numerous studies on their photocatalytic behavior, the activation of the microbial response by visible irradiation in Co– Zn ferrite nanoparticles (photocatalytic antibacterial effect)is scarcely analyzed in the literature and most studies are focused on ZnFe 2 O 4 , using diffusion-based methods. 33-35 Accordingly, the aim of the work reported here was to analyze the photocatalytic antibacterial activity of Co x Zn 1−x Fe 2 O 4 nanoparticles (x=0, 0.1, 0.4 and 0.6) employing E. coli. The selected compositions have been shown to present visible photocatalytic activity for the decomposition of phenol and toluene. 29 The culturability of E. coli was studied after incubating the bacteria with the nanoparticles under both visible light and in darkness, at different time points and concentrations. Results show an absence of antibacterial effect for Co-rich nanoparticles, despite their enhanced photocatalytic activity in phenol degradation. This is attributed to bacteria secreting a protective biofilm matrix, which is inhibited by Zn in the absence of Co. EXPERIMENTAL PROCEDURE Synthesis of photocatalysts Co x Zn 1−x Fe 2 O 4 nanoparticles (x=0, 0.1, 0.4 and 0.6) were synthesized by the co-precipitation method, 21 where Fe(NO 3 ) 3 ·9H 2 O (Lab Kem, 98.0–101.0%, CAS: 7782-61-8), Zn(NO 3 ) 2 ·6H 2 O (Sigma Aldrich, ≥99.0%, CAS: 10196-18-6) and Co(NO 2 ) 3 ·6H 2 O (Merck, ≥99.0%, CAS: 10026-22-9) were mixed stoichiometrically in an aqueous solution, adding NaOH solution (1 mol L −1 ) dropwise under constant stirring until the pH was 13. The precipitate, collected by centrifugation, was washed, dried at 50 °C overnight and calcined at 400 °C for 6 h. Characterization of photocatalysts The structure of the nanoparticles was analyzed through powder X-ray diffraction (XRD) (Bruker D8 Advance) with monochromated Cu K⊍1 radiation (⊗=1.54056 Å) and scanning transmission electron microscopy (STEM) with a high-angle annular dark field detector, STEM-HAADF (Tecnai field emission gun operated at 300 kV). Diffuse reflectance spectroscopy (DRS) was conducted with a UV/Vis/NIR Jasco V-670 to evaluate the band gap energy calculated using the Kubelka–Munk function. A SQUID magnetometer (Quantum Design MPMS XL7) was employed to Antibacterial performance of Co–Zn ferrite nanoparticles www.soci.org J Chem Technol Biotechnol 2025; 100: 428–437 © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). wileyonlinelibrary.com/jctb 429 10974660, 2025, 2, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jctb.7785 by Universidad Del Pais Vasco, Wiley Online Library on [17/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License characterize the high magnetic field magnetization (applied magnetic field of 6 T) at 50 and 300 K, and the hysteresis loops of the samples at room temperature. The photocatalytic activity of two selected samples (x=0and 0.6) was checked employing phenol as organic contaminant model in water. Phenol was selected as a model pollutant because it is a non-volatile contaminant commonly found in industrial wastewater. Its concentration in wastewater can range from <10 mg L −1 to several thousands of milligrams per liter. 36 The initial concentration was chosen based on reported toxicity levels of phenolic compounds in the literature, which range from 9 to 25 mg L −1 . 37 A suspension of nanoparticles (pH =6) was prepared by mixing 24 cm 3 of water, 1 cm 3 of phenol solution (500 mg dm −3 ) and 20 mg of photocatalyst and stirred in the dark which corresponds to an initial phenol concentration of 20 mg mL −1 . After 60 min in the dark, the suspension was photoirradiated with a 300 W xenon lamp (ScienceTech) employing a photoreactor (V=25 cm 3 ) equipped with a quartz window. The optical path included a cutoff filter that transmitted light of wavelength greater than 400 nm. Aliquots collected at different times (60 and 120 min) of reaction were analyzed employing a high-performance liquid chromatography (HPLC) system (Agilent 1100) equipped with refractive index detector and an UV detector (269 nm). The measurements were performed using a C18 column (Phenomenex Gemini 5 μm, 110A; 250 ×4.6 mm, PN 00G-4435-E0) working at 45 °C under isocratic flow conditions of 0.5 cm 3 min −1 . During HPLC measurements, a mobile phase composed of 70% acetonitrile (v/v), 29.5% water and 0.5% orthophosphoric acid (85% w/w solution) was used at a flow rate of 0.5 cm 3 min −1 . Characterization of leaching of metal ions (Fe 2+/3+ ,Zn 2+ and Co +3 ) in aqueous media was performed in samples diluted in Milli-Q water (pH 7). The photocatalysts were collected with centrifugation, to isolate the photocatalysts from the ions leached into the supernatant. Both the precipitated photocatalysts and the supernatant were digested in 300 μL of nitric acid at 80 °C for 24 h. The digested solutions were then diluted 60 times for analysis of Co, Zn and Fe concentration using inductively coupled plasma atomic emission spectrometry (ICP-AES; Agilent 5110). Antibacterial effect The antibacterial effect of the nanoparticles was evaluated on E. coli CECT 100. The strain was grown at 37 °C and shaking (250 rpm) in Luria–Bertani (LB) broth for 3 h to reach exponential growth phase, and harvested by centrifugation at 15 000 ×g using a tabletop centrifuge. The cells were washed three times in Milli-Q water and diluted to reach a density of 10 5 bacteria per milliliter. The bacterial suspensions were incubated for 5–8h in Milli-Q water (non-supplemented and supplemented with 1– 200 μgmL −1 of nanoparticles), at 25 °C and shaking (90 rpm), both in darkness and under visible light irradiation. The illumination system used for visible light irradiation was composed of Philips fluorescent tubes (TL5 HO 54W/965 and TL5 HO 54W/840), with irradiance set to 125 W m −2 . For each experimental condition, samples were periodically collected to determine the number of culturable bacteria (expressed as CFU mL −1 ). The number of culturable bacteria was assessed using the microdroplet method, on LB agar plates incubated for 24 h at 37 °C. The interaction of the nanoparticles with the bacteria was assessed with TEM. With this aim, bacteria were incubated at 10 8 bacteria per milliliter with 200 μgmL −1 of each ferrite for 4 h. After incubation, bacteria were collected by pipetting, avoiding centrifugation of the sample to prevent aggregation of the nanoparticles and changes in interaction with the bacteria. The samples were then deposited onto glow discharged carboncoated copper grids. TEM images were acquired in collaboration with the Analytic and High Resolution Microscopy in Biomedicine Service (SGIker), of the University of the Basque Country (UPV/EHU). Images were acquired with a JEOL JEM-1400 Plus electron microscope at an accelerating voltage of 120 kV. RESULTS Physicochemical and photocatalytic characterizations As previously reported, 29,31 the synthesis procedure provides spinel nanoparticles with spinel cell structure and mean grain sizes of around 10 nm. As an example, Figs 1and 2show, respectively, the XRD patterns and STEM micrographs of some selected samples: ZnFe 2 O 4 (x=0; Figs 1(a) and 2(a)) and Co 0.6 Zn 0.4 Fe 2 O 4 (x=0.6; Figs 1(b) and 2(c)). While CoZn nanoparticles are characterized by a single cubic spinel structure (Fd 3mspace group) (Fig. 1(b)), the occurrence of ZnO in x=0 nanoparticles as secondary phase (P63mc space group) is clearly visible (Fig. 1(a)). Rietveld refinement of the XRD diffractograms provides the following crystallite size, d, values for the set of Co x Zn 1−x Fe 2 O 4 analyzed samples: 20 40 60 80 ).u.a(ytisnetnI (b) Co 0.6 Zn 0.4 Fe 2 O 4 20 40 60 80 ).u.a(ytisnetnI 2θ (degrees) 2θ (degrees) (a)   ZnFe 2 O 4 ZnO ZnO ZnO Figure 1. XRD patterns for the selected Co x Zn 1−x Fe 2 O 4 nanoparticles: (a) x=0; (b) x=0.6. Experimental () and Rietveld fitting (—, in red) curves. The difference between the intensities is shown at the bottom (in blue). The Bragg reflections of ZnO are shown in (a) (j, in black), together with those of the cubic spinel structure (j, in red). www.soci.org AG Gubieda et al. wileyonlinelibrary.com/jctb © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). J Chem Technol Biotechnol 2025; 100: 428–437 430 10974660, 2025, 2, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jctb.7785 by Universidad Del Pais Vasco, Wiley Online Library on [17/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License x=0.6, d=5.4 nm; x=0.4, d=7.3 nm; x=0.1, d=6.0 nm. For x=0, d=5.1 nm (spinel, 88 wt%) and d=29.3nm (ZnO, 12 wt%). Slightly larger values for the nanoparticle dimensions can be deduced from the histograms of the nanoparticle diameters evaluated through the TEM micrographs (8.9, 8.4, 9.0 and 9.2 nm for x=0, 0.1, 0.4 and 0.6, respectively). Moreover, the biphasic nature of the ZnFe 2 O 4 sample can be also deduced from the analysis of its histogram (two nanoparticle distributions; Fig. 2(b)) in comparison with that shown by the rest of the analyzed samples (see Fig. 2(d), as an example). Regarding the surface area (Brunauer–Emmett–Teller (BET)), as previously reported, 29 the nanoparticles display the typical hysteretic curve type V (without initial knee ascribed to the formation of adsorption monolayer), according to IUPAC classification. No clear trend with xwas detected in either BET surface area or pore diameter (66.5–92.5 m 2 g −1 , 6.7–7.8 nm, respectively). As for the optical characterization through DRS, the analyzed nanoparticles display band gap energies, E g , corresponding to the visible range. In fact, the Tauc plot, estimated through the Kubelka–Munk function FRðÞ=1−RðÞ 2=2R(Ris diffuse reflectance), FR ðÞ hν ðÞ 1=2∝hν−Eg  (hνis incident energy), enables the estimation of E g (linear extrapolation in Fig. 3(a); 1.4 eV <E g <1.7 eV for the analyzed samples). Thus, optical absorption in the visible range is confirmed, which supports the visible photocatalytic performance of the analyzed nanoparticles. Regarding the magnetic response (Fig. 3(b)), the samples are characterized at room temperature by the typical anhysteretic behavior of superparamagnetic nanoparticles (negligible remanence and coercivity) leading to reduced nanoparticle aggregation due to negligible interparticle magnetic interactions. Furthermore, as discussed in the introduction, saturation magnetization increases with the Co content (x) as a consequence of the particular cation distribution (Zn 2+ ,Co 2+ and Fe 2+/3+ ) between the octahedral and tetrahedral sites 31 (inset of Fig. 3(b), high field magnetization at 6 T versus x). The marked increase in the initial magnetic susceptibility and saturation magnetization for x=0.6 provides this sample with optimal characteristics for magnetic separation (concentration under external magnetic fields) while maintaining superparamagnetic characteristics. Furthermore, the optimal photocatalytic performance for x=0.6 is confirmed. Figure 3(c) shows the evolution of the phenol concentration ratio after 2 h of visible irradiation compared to the photocatalytic response of ZnFe 2 O 4 (x=0) nanoparticles, where in this latter case negligible phenol degradation is achieved under the same experimental conditions. Previous studies clearly indicate the dominant role of Zn 2+ cations in the antibacterial activity of inorganic Zn compounds (i.e. ZnO). 32 Accordingly, the concentration of metal cations leached from the nanoparticles was studied by means of ICP-AES, by centrifuging the nanoparticles and analyzing both the precipitated nanoparticles and the supernatant. Table 1presents the 4 8 12 16 0 5 10 15 Counts Diameter (nm) (b) ZnFe 2 O 4 4 8 12 16 0 10 20 30 40 Counts Diameter ( nm ) (d) Co0.4Zn0.6Fe2O4 (a) ZnFe2O4 (c) Co0.4Zn0.6Fe2O4 Figure 2. (a, c) STEM images of selected nanoparticles and (b, d) their nanoparticle size distributions. Antibacterial performance of Co–Zn ferrite nanoparticles www.soci.org J Chem Technol Biotechnol 2025; 100: 428–437 © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). wileyonlinelibrary.com/jctb 431 10974660, 2025, 2, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jctb.7785 by Universidad Del Pais Vasco, Wiley Online Library on [17/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License cation concentration in the supernatant and in the nanoparticles in comparison with nominal concentration. The obtained concentration ratios of Co/Fe and Zn/Fe present in the supernatant for the Co x Zn 1−x Fe 2 O 4 ferrites (x=0.1, 0.4 and 0.6) are equivalent to the nominal concentration of these metal species in the nanoparticles. Thus, the detected cations would come from the absence of a proper centrifugation of the nanoparticles and no significant leaching can be concluded. However, for x=0, Zn cations are detected in the supernatant in a higher ratio than that present in the nanoparticles, indicating that Zn leaching is taking place. The total mass of Zn detected in the supernatant (0.652 mg L −1 ) corresponds to 0.12% of the total mass of Zn in the sample (540 mg L −1 ), indicating that this amount of Zn was leached from the ZnFe 2 O 4 particles. Antibacterial effect of ferrites To determine the photocatalytic antibacterial response of the selected Co–Zn nanoparticles, the growth of E. coli, the culturability, was analyzed in a 5 h time period under the following conditions (Fig. 4): (i) under visible light irradiation as a control; (ii) in the presence of 200 μgmL −1 of Co x Zn 1−x Fe 2 O 4 nanoparticles (x=0, 0.1, 0.4 and 0.6) in the dark; and (iii) in the presence of the aforementioned nanoparticles under visible light. Culturability refers to the capacity of the bacteria to undergo cell division after exposure to the previously mentioned conditions, and is represented as colony forming units per milliliter (CFU mL −1 ). ZnFe 2 O 4 nanoparticles significantly reduced E. coli culturability, both in the dark and under visible light irradiation, with clear differences observed between both conditions: while in darkness Figure 3. (a) Tauc plot (indirect band gap) for the analyzed Co x Zn 1−x Fe 2 O 4 nanoparticles. (b) Room temperature hysteresis loops (M: magnetization; ⊘ 0 H: applied magnetic field). Inset: magnetization at ⊘ 0 H=6T,(•) at 300 K and () 50 K. (c) Phenol concentration ratio C/C 0 (C 0 : initial concentration, 20 mg L −1 ) after 60 min in the dark (left) and 60 and 120 min of irradiation under xenon lamp (right). Table 1. Nominal metal ionconcentration of Co and Zn relative to Fe in Co x Zn 1−x Fe 2 O 4 particles, and ICP-AES analysis of metal ion concentrations in nanoparticles and supernatant, relative to iron concentration xNominal [Co]/[Fe] Nominal [Zn]/[Fe] Nanoparticle [Co]/[Fe] Nanoparticle [Zn]/[Fe] Supernatant [Co]/[Fe] Supernatant [Zn]/[Fe] 0—0.580 0.000 (1) 0.584 (4) 0.00 (2) 0.8 (1) 0.1 0.053 0.522 0.055 (1) 0.508 (2) 0.000 (3) 0.53 (6) 0.4 0.211 0.348 0.215 (3) 0.322 (1) 0.216 (1) 0.331 (2) 0.6 0.316 0.232 0.320 (1) 0.223 (1) 0.320 (4) 0.226 (1) www.soci.org AG Gubieda et al. wileyonlinelibrary.com/jctb © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). J Chem Technol Biotechnol 2025; 100: 428–437 432 10974660, 2025, 2, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jctb.7785 by Universidad Del Pais Vasco, Wiley Online Library on [17/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License these nanoparticles caused complete lack of culturability by the 4 h mark, their effect was significantly stronger under visible light irradiation, and complete lack of culturability was achieved by the 2 h mark. These results suggest that the ability of E. coli to survive and divide is strongly affected by the presence of these ferrite nanoparticles, and that the effect is synergistically enhanced by the combination of visible light irradiation and the nanoparticles. However, the effect on culturability was less pronounced for x=0.1 nanoparticles, being almost negligible for higher Co contents (x=0.4 and 0.6) either in dark conditions or under visible light irradiation. As Fig. 4(b) shows, for x=0.1 in darkness, only a 10-fold decrease in culturability was observed during the 5 h period, but under visible light irradiation the presence of nanoparticles led to a 100-fold decrease in culturability (10 5 CFU mL −1 in the visible light control versus 10 3 CFU mL −1 in the presence of nanoparticles and light). These findings suggest that the bacterial inhibition effect of the nanoparticles decreases as the Co/Zn ratio (x) increases. Focusing on the nanoparticles with enhanced antibacterial response (x=0), the effect of the nanoparticle concentration on the antibacterial effect was analyzed under similar experimental conditions (with and without visible light, after 5 h of incubation; Fig. 5). As can be seen, no significant changes in culturability were found for concentrations lower than 12.5 μgmL −1 under dark conditions, although at higher concentrations, x=0 nanoparticles showed toxicity in the dark (Fig. 5(a)). However, under light conditions a 10-fold decrease in culturability was observed at nanoparticle concentrations as low as 12.5 μgmL −1 . This effect is comparable to that observed with x=0.1 nanoparticles using a 16-fold higher concentration (200 μgmL −1 ) (Fig. 4(b)). Furthermore, when increasing the concentration of x=0 nanoparticles above 12.5 μgmL −1 under light conditions, a synergic effect was evident and the culturability sharply decreased reaching negligible values for concentrations above 50 μgmL −1 . Finally, culturability was analyzed at the selected concentration of 20 μgmL −1 for x=0 as a function of the incubation time (Fig. 5 (b)). While this concentration of nanoparticles caused a small effect on culturability in darkness (10-fold decrease), culturability decreased by four orders of magnitude after 8 h of incubation under visible light irradiation. These results indicate that concentration could be adjusted depending on the time of treatment desired. Interaction of ferrites and E. coli To investigate if the changes in bacterial culturability were due to changes in interaction between the nanoparticles and E. coli, the morphology of the bacteria after incubation with the ferrites was analyzed using TEM. For this experiment, bacteria were incubated in distilled water containing 200 μgmL −1 of nanoparticles under visible light irradiation for a 4 h period. As shown in Fig. 6, Zn ferrite nanoparticles (x=0) accumulated on the surface of the bacteria, suggesting interaction between the nanoparticles and the bacterial wall, although the bacterial structure remained seemingly intact. In contrast, Co–Zn ferrite nanoparticles induced changes in bacterial morphology, with the bacteria presenting secretion-like changes in their exterior. In bacteria incubated with x=0.1 nanoparticles, these secretions were visible mostly in the bacterial wall, and in some cases along the bacterial pili, in the area closest to the bacterial wall. Incubation with the x=0.4 Light Light+Nanoparticles (200 µg/mL) Nanoparticles (200 µ g /mL) 012345 10 0 10 1 10 2 10 3 10 4 10 5 10 6 time (h) CFU/mL 012345 10 0 10 1 10 2 10 3 10 4 10 5 10 6 time (h) CFU/mL 012345 10 0 10 1 10 2 10 3 10 4 10 5 10 6 time (h) CFU/mL x = 0 012345 10 0 10 1 10 2 10 3 10 4 10 5 10 6 time (h) CFU/mL (a) (b) (c) (d) x = 0.1 x = 0.4 x = 0.6 Figure 4. (a–d) Culturability of bacteria incubated in water under visible light irradiation, in the presence of 200 μgmL −1 of Co x Zn 1−x Fe 2 O 4 ferrites in darkness, and in the presence of 200 μgmL −1 of doped ferrites under visible light irradiation. Graphs indicate colony forming units at six time points, from 0to5h. Antibacterial performance of Co–Zn ferrite nanoparticles www.soci.org J Chem Technol Biotechnol 2025; 100: 428–437 © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). wileyonlinelibrary.com/jctb 433 10974660, 2025, 2, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jctb.7785 by Universidad Del Pais Vasco, Wiley Online Library on [17/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License nanoparticles on the other hand resulted in secretions both in the bacterial wall and in some cases along the full length of the bacterial pili. Lastly, incubation with x=0.6 nanoparticles resulted in a crown-like phenotype, with secretions of a greater size accumulating along the bacterial wall. In all three cases, the nanoparticles seemed to interact with the observed secretion. These Light + Nanoparticles Nanoparticles 02468 100 101 102 103 104 105 106 time (h) CFU/mL Light Light+Nanoparticles Nanoparticles x = 0 (20 µg/mL) (a) (b) x = 0 10 100 10–1 100 101 102 103 104 105 106 Concentration ( g/mL) CFU/mL Figure 5. (a) Effect of different concentrations of ZnFe 2 O 4 (x=0) nanoparticles on E. coli culturability, after 5 h of incubation with and without visible light irradiation. (b) Culturability of bacteria incubated in the presence of the nanoparticles in darkness and under visible light irradiation at a concentration of 20 μgmL −1 . Graph indicates colony forming units at five time points, from 0 to 8 h. Figure 6. TEM images of E. coli, after 4 h of visible light irradiation and incubation with Co x Zn 1−x Fe 2 O 4 nanoparticles. www.soci.org AG Gubieda et al. wileyonlinelibrary.com/jctb © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). J Chem Technol Biotechnol 2025; 100: 428–437 434 10974660, 2025, 2, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jctb.7785 by Universidad Del Pais Vasco, Wiley Online Library on [17/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License findings, in combination with the bacterial culturability assay results, suggest that the secretion observed in the TEM images could have a protective effect against the nanoparticles and their photocatalytic effect. DISCUSSION Zn-based nanoparticles (ZnO, ZnFe 2 O 4 ) have been extensively studied as antibacterial agents. 33-35,38 Two main mechanisms have been proposed for their antibacterial activity: (i) ROS generation, reinforced by the photocatalytic effect under light irradiation, with ROS generated by the photocharges being able to produce damage to the cell wall and other organelles of the bacteria, restricting their growth and leading to bacterial cell death; and (ii) zinc ion (Zn 2+ ) release, where the proteins in the bacterial cell wall (negatively charged at biological pH value) capture the charged Zn 2+ ions, leading to the formation of insoluble metal proteinates, the inhibition of respiratory chain enzymes and eventually bacterial death. 33 Other mechanisms, like membrane disruption or protein leakage, have also been proposed for the antibacterial effect of Zn ferrite nanoparticles. 33 However, the actual origin of the antibacterial response of nanoparticles is not entirely clear in the literature, and the contribution of each mechanism in the overall response of bacteria in the presence of nanoparticles is currently a topic of debate. 39 In this study the results point towards the release of Zn 2+ being a key factor for the antibacterial properties of the ferrites, as release of Zn 2+ was negligible in the nanoparticles with no antibacterial response (x=0.4 and 0.6), while the nanoparticles with the highest antibacterial response (x=0) presented Zn 2+ leaching. Other researchers have also found that Zn ferrites have better antibacterial effect than Co ferrites 40 ; however, the nanoparticle synthesis and tests performed are different, making comparison difficult. Furthermore, the enhanced antibacterial response under visible light irradiation suggests there is a synergic effect between Zn 2+ release and ROS generation, despite the reduced photocatalytic performance of Zn-based ferrite (x=0) observed in phenol degradation. However, and surprisingly, the most active visible photocatalytic nanoparticles (higher Co concentrations) do not promote any antibacterial effect. A potential explanation for this unexpected result is the bacterial secretion observed in bacteria incubated with Co–Zn nanoparticles, which might have a protective effect. Bacteria are known to produce an extracellular polymeric substance (EPS) to protect themselves from external threats and antimicrobial agents. This EPS matrix could limit the penetration of nanoparticles, thus reducing the antibacterial effect 13,41 and preventing bonding of the nanoparticles to the bacterial surface, a key mechanism for antibacterial action that increases membrane permeability and facilitates ROS transfer. 29,42 In addition, the EPS matrix could reduce the photocatalytic efficiency of nanoparticles through several mechanisms. For example, the EPS matrix could shield the nanoparticles from light, reducing their photocatalytic activity, or contain organic compounds that act as scavengers of ROS, preventing these molecules from reaching the bacteria. 43 Additionally, the EPS could reduce the surface area of the nanoparticles by adsorption, thus hindering photocatalytic reactions. 41 Nanoparticles with higher Zn concentration may have a greater ability to inhibit EPS production in E. coli than nanoparticles with a higher concentration of Co. Interestingly, zinc oxide nanoparticles have recently been observed to inhibit biofilm formation both in E. coli 44 and in Pseudomonas aeruginosa, another Gram-negative bacteria. 45,46 This work highlights the need not only to ensure accurate photocatalytic characterization of the nanoparticles but also to conduct a comprehensive analysis of their interaction with bacteria, as this interaction can have a profound impact on the overall antibacterial performance. Finally, the antibacterial response of ZnFe 2 O 4 , boosted by visible light, should be specially outlined. Concentrations as low as 20 μgmL −1 show a marked effect on the cell culturability after 8 h under visible light irradiation, with a noticeable reduction in the antibacterial capacity when the nanoparticles interact with the bacteria in the dark. This concentration is very low compared to other reported results, which indicate the use of ferrite concentrations above 100 μgmL −1 to obtain significant antibacterial response. 35 The highest reported dose of ZnFe 2 O 4 to exhibit no toxicity when tested in human cells is of 125 μgmL −1 , 33 and concentrations as high as 500 μgmL −1 have been shown to pose little risk to aquatic organisms when incubated for up to 100 h. 47 Therefore, the efficiency of a 20 μgmL −1 dose in this article is of special significance. Furthermore, the antibacterial effect is observed within 8 h, which is a relatively short period compared to other reported antibacterial nanoparticles. 42,47-49 CONCLUSIONS This study describes the photocatalytic antibacterial effect of Co– Zn ferrite (Co x Zn 1−x Fe 2 O 4 ,x=0, 0.1, 0.4 and 0.6) nanoparticles. The results show that the Co/Zn ratio mainly determines the photocatalytic activity of the nanoparticles and their antibacterial activity under visible light and dark conditions. Structural and morphological characterization (XRD and TEM) confirms the characteristic spinel structure without significant changes in the main nanoparticle sizes (around 10 nm). Band gap energy estimation and magnetic characterization support the optimal characteristics of the nanoparticles to be employed as photocatalytic agents for wastewater treatments: optical absorption in the visible range, high magnetic moment and superparamagnetic response at room temperature. The enhanced photocatalytic performance (phenol degradation) for the ferrite with the highest Co content (Co 0.6 Zn 0.4 Fe 2 O 4 , x=0.6) is confirmed. However, a light-induced decrease in culturability was only detected for nanoparticles with low Co content (ZnFe 2 O 4 and Co 0.1 Zn 0.9 Fe 2 O 4 ,x=0 and x=0.1). The optimum antibacterial effect of ZnFe 2 O 4 even at low doses (20 μgmL −1 )is explained as a synergic effect of Zn 2+ cation leaching, generation of ROS enhanced by visible light irradiation and inhibition of the secretion of a protective biofilm matrix. Finally, this work highlights the need not only to ensure accurate photocatalytic characterization of the nanoparticles, but also to conduct a comprehensive analysis of their interaction with bacteria. This interaction can have a profound impact on the overall antibacterial performance. ACKNOWLEDGEMENTS The work has been performed under grants IT1479-22 funded by the Basque Government and PID2020-116321RB-C21 funded by MCIN/AEI/10.13039/501100011033. The authors acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza for the microscopy. The authors thank SGIker (UPV/EHU/ERDF, EU) service for their technical and human support, especially the Phytotron; Analytic and High Resolution Microscopy in Biomedicine; and IBERCRON Services. Antibacterial performance of Co–Zn ferrite nanoparticles www.soci.org J Chem Technol Biotechnol 2025; 100: 428–437 © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). wileyonlinelibrary.com/jctb 435 10974660, 2025, 2, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jctb.7785 by Universidad Del Pais Vasco, Wiley Online Library on [17/01/2025]. 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