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Antibacterial membranes have attracted researchers’ interest in recent years as a possible approach for dealing with biofouling on the membrane surface. This research aims to see if blending AZ63 Mg alloy into a polyethersulphone (PES) membrane can improve antifouling and separation properties. The composite membranes’ pure water flux continued to increase from pristine PES to PES/AZ63 2.00 wt%. The results showed that PES/AZ63 2.00 wt% membrane supplied the highest permeate flux of E. coli. The steady-state fluxes of AZ63 composite membranes were 113.24, 104.38 and 44.79 L/m2h for PES/AZ63 2.00 wt%, 1.00 wt%, and 0.50 wt%, respectively. The enhanced biological activity of AZ63 was studied based on antioxidant activity, DNA cleavage, antimicrobial, anti-biofilm, bacterial viability inhibition and photodynamic antimicrobial therapy studies. The maximum DPPH scavenging activity was determined as 81.25% with AZ63. AZ63 indicated good chemical nuclease activity and also showed moderate antimicrobial activity against studied strains. The highest biofilm inhibition of AZ63 was 83.25% and 71.63% towards P. aeruginosa and S. aureus, respectively. The cell viability inhibition activity of AZ63 was found as 96.34% against E. coli. The photodynamic antimicrobial therapy results displayed that AZ63 demonstrated 100% bacterial inhibition when using E. coli.

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  • AZ63 Mg alloy was blended to polyethersulphone (PES) membrane.

  • Biologic activity of AZ63 was studied based on antioxidant activity, DNA cleavage, antimicrobial, anti-biofilm, bacterial viability inhibition and photodynamic antimicrobial therapy studies.

antifouling properties, AZ63 magnesium alloy, polyethersulfone membrane
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Due to a changing climate, an increasing population and economy, and changing lifestyles, water scarcity is becoming an increasing concern (Debaere & Kapral 2021). Water crises have become accepted as one of the world's greatest future concerns. Therefore, water recycling and reuse have become one of the most essential ways of avoiding scarcity (Ahmad et al. 2022). Membrane technologies are increasingly being used in tertiary processes for water recycling and reuse. However, while the treatment of water or wastewater, biofilm formation by gram-negative and gram-positive bacteria cause serious membrane biofouling, which decreases water flux and membrane lifetime (Kim et al. 2022). Antibacterial membranes have attracted researchers' interest in recent years as a possible approach for dealing with this problem(Jiang et al. 2022). Antibacterial materials are very important in membranes, and many different materials have been used in literature such as titanium, graphene, silver, etc. (Anis et al. 2022; Peng et al. 2022; Vatanpour et al. 2022). Magnesium (Mg) is often regarded as the best green material of the twenty-first century (Xu et al. 2019). Its low density, in particular, makes them popular for addressing major environmental pollution and energy problems (Yao et al. 2022). Excellent mechanical strength and stiffness, outstanding damping ability, huge hydrogen storage capabilities, and high theoretical specific capacity for batteries are only a few of the other physical and chemical features of magnesium alloys (Yang et al. 2021). Mg alloys are being used in a growing number of automotive components, including frames, seats, and steering wheels (Bu et al. 2020). They're also interesting candidates for many other uses, such as computer components, aerospace components, and defense equipment (Gottfried 2020; Han et al. 2020; Song et al. 2020). Furthermore, among the bio metal materials, Mg alloy has gotten a lot of interest because of its exceptional biocompatibility and degradability (Tong et al. 2022). In recent studies, it has been also understood that Mg has a good antibacterial ability due to the alkalinity that increases with the degradation (Chen et al. 2018; Yan et al. 2018; Ma et al. 2020). Consequently, in this study, highly efficient antibacterial membranes were prepared using an AZ63 magnesium alloy, and its biological activity was studied based on antioxidant, DNA cleavage, antimicrobial, photodynamic antimicrobial therapy, anti-biofilm and bacterial viability inhibition studies.

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Materials

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Polyethersulfone (PES Ultrason E6020P, MW: 58,000 g/mol) was supplied from BASF Company (Germany). N-methyl-2-pyrrolidone (NMP) was supplied from Sigma-Aldrich. The two-stage Millipore Direct-Q3UV purification system was utilized to get the distilled water used in all studies. The AZ63 Mg alloy powder (particle size < 45 μm) used in this study was obtained from a local provider in Istanbul.

Preparation of AZ63 Mg alloy blended PES membranes

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Pristine and AZ63 alloy blended PES membranes were prepared using the phase inversion method. Full description of the membrane synthesis procedures may be found elsewhere (Dizge et al. 2017). Table 1 lists the casting solution's composition. Before experiments, all membranes were pressured under 5 bar with deionized water.

Table 1

The manufactured membranes' casting solution composition (wt%)

Membrane samplePESNMPAZ63 Mg alloy
Pristine PES 14 86.0 0.00 
PES/AZ63-0.5 14 85.5 0.50 
PES/AZ63-1.0 14 85.0 1.00 
PES/AZ63-2.0 14 84.0 2.00 
Membrane samplePESNMPAZ63 Mg alloy
Pristine PES 14 86.0 0.00 
PES/AZ63-0.5 14 85.5 0.50 
PES/AZ63-1.0 14 85.0 1.00 
PES/AZ63-2.0 14 84.0 2.00 
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Characterization of pristine and composite membranes

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The surface morphology of the manufactured pristine PES and AZ63 blended PES membranes was characterized by scanning electron microscopy (SEM, Gemini Zeiss Supra 55). After drying at room temperature, the membranes were coated with a gold layer. The SEM images were taken using a 20.0 kV acceleration voltage. The filtration performances of the manufactured membranes were evaluated using a dead-end flat sheet membrane module (Sterlitech, HP4750) with a filtration area of 14.6 cm2 and an operating pressure of 1 bar.

The permeation flux (J) was calculated by using the following Equation (1) after collecting the filtered water in the prescribed intervals.
(1)
where, J is permeate flux (L/m2h); V is the volume of permeate pure water (L), A the effective area of the membrane (m2), and Δt the filtration time (h).

DPPH scavenging activity

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The antioxidant activity of new synthesized AZ63 on the DPPH radical was performed with the test described by Aǧirtaş et al. (2015). First, AZ63 alloys were prepared in test tubes with distilled water at five different concentrations (range from 12.5–200 mg/L). Then DPPH solution was added on top of them and vortexed to mix them well. The sample tubes were then incubated in the dark for 30 min at room temperature. Trolox and ascorbic acid were utilized as a standards and the procedures mentioned were also applied to them. Methanol was used as blank. After incubation, the absorbance of the samples was determined with a spectrophotometer at 517 nm, and the antioxidant activity was estimated using Equation (2).
(2)
Abscontrol: absorbance value of Trolox and ascorbic acid, Abssample: the absorbance value of the AZ63 and DPPH after 30 min.

DNA cleavage activity

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To examine the effect of AZ63 on DNA, E. coli pBR322 plasmid DNA was utilized. After homogeneously mixing three different concentrations of AZ63 Mg alloy, an amount equal to plasmid DNA was put on top and incubated for 60 min at 37 °C. The plasmid DNA used as a control was not treated with AZ63 Mg alloy. The negative control and mixes were placed onto the agarose gel at the end of the period. The electrophoresis parameters were 90 min, 80 volts, and 120 ampere. Finally, a transilluminator was used to visualize DNA molecules (Yıldız et al. 2017; Jawoor et al. 2018).

Antimicrobial activity

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The antibacterial activity of the synthesized AZ63 Mg alloy was studied using the microdilution technique. In this study, a total of 8 microorganisms were used including two yeast strains, three Gr−ve and three Gr + ve. Details about microorganism have been given elsewhere Farajzadeh et al. (2022). Two-fold serial dilutions of AZ63 were performed. Then, strains prepared in the range of 0.5 McFarland Scale were added to the wells in the same amount in a specific order. Finally, the plates were incubated at 37 °C for one day. The plates were examined at the end of the incubation time, and the miniumum inhibitory concentration (MIC) was determined as the lowest concentration that inhibited microbial growth.

Biofilm inhibition activity

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The ability of synthesized AZ63 Mg alloy to embarrass biofilm creation of S. aureus and P. aeruginosa was explored based upon crystal violet (CV) staining. The experiment was done on 24 well plates. Before the experiment, stock cultures were cultivated overnight. The microorganisms were inoculated with 2.9 × 108 CFU/mL to the wells. Bacterial strains were cultured in well plates containing various concentrations of AZ63 for 72 h at 37 °C. After 72 h, the wells were carefully emptied and cleaned two times with deionized water to eliminate the free-floating bacteria without damaging the biofilms. Then the plates were kept in an oven to dry the wells. Then, to stain biofilm formations, CV was added to the wells and treated for 45 min. The CV was removed after that, and the plates were carefully washed. The washing process was repeated twice. Ethanol was added to recover the absorbed CV, and it was left in a shaker incubator for 15 min. Biofilm inhibition was evaluated using a spectrophotometer, and the absorbance of biofilm inhibition was recorded at 595 nm. Wells with media containing only S. aureus and P. aeruginosa were used as positive controls. Equation (3) was used to calculate biofilm inhibition (Karaoğlu et al. 2021).
(3)

Bacterial cell viability activity and antibacterial photodynamic activity

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ATCC 10,536 E. coli strain was utilized to evaluate the bacterial cell viability inhibition ability and antibacterial photodynamic activity of AZ63 Mg alloy. The preparation steps of the E. coli used in the study before being treated with AZ63 Mg alloy were given in detail in our previous article (Farajzadeh et al. 2022). Prepared E. coli was treated with the synthesized AZ63 at various concentrations (125, 250, and 500 mg/L) for 90 min at 37 °C. Later, the mixtures were diluted in different proportions, inoculated in NB agar, and left to incubate at 37 °C for a day. In addition, in the antimicrobial photodynamic therapy study, the method in the microbial cell viability study was applied after the compounds were exposed to LED light for 20 min. When the incubation time was finished, colonies formed were counted and compared with the control plate, and finally inhibition of cellular viability was calculated using the equation below (4).
(4)
A(control): Numbers of colonies formed at control plate, A(sample): Numbers of colonies formed at sample plate

Utilization of PES membrane blended with AZ63 for E. coli removal and its antifouling performance

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ATCC 10536 E. coli strain was used to evaluated this parameter. Details about E. coli grown procedure have been given elsewhere (Saleh et al. 2021). After filtration experiments, the formed E. coli colonies were incubated then counted. The growth inhibition (%) was calculated using Equation (5)
(5)
A(control): Numbers of colonies formed at control plate, A(sample): Numbers of colonies formed at sample plate
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Characterization of AZ63 Mg alloy particles

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Figure 1(a) shows the shape of AZ63 Mg alloy particles which have homogeneous rod-shaped and flat form as seen in SEM images. SEM/energy-dispersive X-ray spectroscopy (EDX) spectra of AZ63 Mg alloy particles have also given in Figure 1(b). The elemental composition of Mg alloy was found 89.9% Mg, 8.93% Al, and 1.17% Zn which is similar to the literature (Li et al. 2016).
Figure 1
(a) SEM image and (b) SEM/EDX spectra of AZ63 powder.
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(a) SEM image and (b) SEM/EDX spectra of AZ63 powder.

Figure 1
(a) SEM image and (b) SEM/EDX spectra of AZ63 powder.
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(a) SEM image and (b) SEM/EDX spectra of AZ63 powder.

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Characterization of AZ63 Mg alloy particles blended PES membranes

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The surface SEM micrographs of the fabricated pristine and Mg alloy blended PES membranes are shown in Figure 2. A nearly smooth surface was visible on the pristine membrane (Figure 2(a)). The SEM micrographs (Figure 2(b)–2(d)) show that the AZ63 Mg alloy particles fall in a clear orientation and coated the whole surface of the PES membranes in a very smooth pattern.
Figure 2
AZ63 blended PES membranes (a) pristine PES membrane, (b) PES/0.5%AZ63, (c) PES/1.0%AZ63, (d) PES/2.0%AZ63.
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AZ63 blended PES membranes (a) pristine PES membrane, (b) PES/0.5%AZ63, (c) PES/1.0%AZ63, (d) PES/2.0%AZ63.

Figure 2
AZ63 blended PES membranes (a) pristine PES membrane, (b) PES/0.5%AZ63, (c) PES/1.0%AZ63, (d) PES/2.0%AZ63.
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AZ63 blended PES membranes (a) pristine PES membrane, (b) PES/0.5%AZ63, (c) PES/1.0%AZ63, (d) PES/2.0%AZ63.

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Flux performance of AZ63 Mg alloy blended membranes

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The variation of deionized water permeability coefficient (Jp) versus transmembrane pressure (TMP) was evaluated for pristine PES and AZ63 blended PES membranes (Figure 3(a)). According to the Darcy law, the permeate flow (Jp) of deionized water increases with the increase in transmembrane pressure (TMP) (Chikhi et al. 2008). The composite membranes' pure water flux continued to increase from pristine PES to PES/AZ63 2.00 wt%. It is known that the particles added to the membrane structure may increase the pore size in the literature. Sert et al. (2021) studied boron nitride quantum dots to improve the antifouling properties and flux performance of PES membranes. They reported that with the increase of nanoparticles, porosity and mean pore radius were increased. E. coli flux of pristine and AZ63 PES membranes is presented in Figure 3(b). It is well known that fouling has a major impact on the overall performance of polymeric membranes (Bagheripour et al. 2019). The most major challenges brought on by the fouling phenomena include limiting permeation flux, increasing operating and maintenance expenses, and decreasing membrane lifetime (Shang et al. 2022). Mg alloys with the right combination of alloying elements have significant antibacterial capabilities (Zhang et al. 2021). The initial and steady-state fluxes of E. coli for PES/AZ63 2.00 wt% were 113.24 and 45.6 L/m2h, and for PES/AZ63 1.00 wt% were 104.38 and 40.52 L/m2h, respectively. PES/AZ63 0.50 wt% was fouled and a severe decrease was observed at fluxes, however, the flux supplied was obviously higher than the pristine membrane's. The initial and steady-state fluxes of E. coli for PES/AZ63 0.50 wt% were 44.79 and 21.52 L/m2h, and for pristine membrane were 28.76 and 16.92 L/m2h, respectively. The pristine membrane supplied the lowest permeate flux of E. coli. The increase of fluxes after AZ63 alloys can be attributed to the enhancement of the antibacterial properties of membranes. Our findings are similar to the literature. Dadari et al. (2022) have used nickel-bentonite nanoparticles to enhance the antibacterial performance and antifouling properties of the PES membranes. They reported that composite membranes have higher antifouling performance than pristine PES membrane.
Figure 3
Variation of (a) permeability coefficient (Jp) of deionized water versus transmembrane pressure and (b) E. coli flux of pristine and AZ63 PES membranes.
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Variation of (a) permeability coefficient (Jp) of deionized water versus transmembrane pressure and (b) E. coli flux of pristine and AZ63 PES membranes.

Figure 3
Variation of (a) permeability coefficient (Jp) of deionized water versus transmembrane pressure and (b) E. coli flux of pristine and AZ63 PES membranes.
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Variation of (a) permeability coefficient (Jp) of deionized water versus transmembrane pressure and (b) E. coli flux of pristine and AZ63 PES membranes.

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DPPH scavenging activity

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Several antioxidant ways have been presented to measure and research the antioxidant feature and capability of diverse samples. DPPH free radical scavenging method is one of the most frequently utilized methods and presents the first approach for appraising antioxidant activity. Moreover, the DPPH radical scavenging way a simple, economic, easy, rapid, and effective method. DPPH radical has deep purple color; when the DPPH free radical is mixed with an antioxidant features molecule, which can give a hydrogen atom, it gives rise to the decreased form with the loss of this deep purple color. Antioxidant features substances play a crucial role in both in the human body as well as food systems to diminish oxidative and detrimental influences of reactive oxygen species (ROS) (Gulcin 2020). Magnesium and its alloys, owing to biodegradability, satisfactory mechanical properties and bioactive effects, are regarded to have great application prospects in various fields such as orthopedic applications (Lin et al. 2019). Therefore, the biological properties of AZ63, which has a limited number of studies, are worth more research. In this study, the antioxidant properties of AZ63 were investigated and the DPPH method was used for this. DPPH activity results of AZ63 are given in Figure 4. According to our results, it was determined that DPPH activity was concentration-dependent. DPPH activity was determined as 53.02%, 61.44%, 65.71%, and 72.59% at 12.5, 25, 50, and 100 mg/L concentrations, respectively. Ascorbic acid and Trolox were used as standards in the presented study. DPPH activity results of ascorbic acid, Trolox, and AZ63 at 200 mg/L concentration were 100%, 100%, and 81.25%, respectively. This result indicates that AZ63 can be used as an antioxidant agent in a variety of fields since it has good antioxidant activity.
Figure 4
DPPH radical scavenging activity.
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DPPH radical scavenging activity.

Figure 4
DPPH radical scavenging activity.
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DPPH radical scavenging activity.

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DNA cleavage ability

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The DNA cleavage activity of AZ63 Mg alloy was also investigated in the presented study. E. coli pBR322 plasmid DNA was used as the target for this. Plasmid DNA is Form I under normal conditions. If a single strand break occurs in the DNA chain, it becomes Form II, and if a double-strand break occurs, it becomes Form III. Moreover, in agarose gel, Form III appears somewhere between Form I and Form II. Figure 5 is showed the DNA cleavage activity results of plasmid DNA treated with two different concentrations of AZ63. As seen in Figure 5, since the plasmid DNA was broken into too small fragments in lines 2 and 3, no bands were seen in the gel. So, we can say that AZ63 was highly effective on the DNA molecule. DNA is an important target macromolecule for both antimicrobial agents and anticancer studies. In addition to these, the results of the DNA cleavage of this study may also be attributed to the antimicrobial activity of the AZ63. Moreover, it is known that magnesium alloys hold great promise because they are biocompatible (Brooks et al. 2018). Considering this information, the effect of AZ63 on DNA is promising for its use as an anticancer agent. AZ63 demonstrated an effective DNA cleavage activity according to our study, so it may be demonstrated to be effective by in-vivo studies, and then used as a DNA nuclease agent in various fields.
Figure 5
DNA cleavage activity of AZ63. Lane 1, pBR 322 DNA; Lane 2, pBR 322 DNA + 100 mg/L AZ63; Lane 3, pBR 322 DNA + 200 mg/L of and AZ63.
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DNA cleavage activity of AZ63. Lane 1, pBR 322 DNA; Lane 2, pBR 322 DNA + 100 mg/L AZ63; Lane 3, pBR 322 DNA + 200 mg/L of and AZ63.

Figure 5
DNA cleavage activity of AZ63. Lane 1, pBR 322 DNA; Lane 2, pBR 322 DNA + 100 mg/L AZ63; Lane 3, pBR 322 DNA + 200 mg/L of and AZ63.
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DNA cleavage activity of AZ63. Lane 1, pBR 322 DNA; Lane 2, pBR 322 DNA + 100 mg/L AZ63; Lane 3, pBR 322 DNA + 200 mg/L of and AZ63.

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Antimicrobial activity

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In the presented study, the antimicrobial activity of AZ63 on various microorganisms was investigated using the microdilution method. MIC values of the results are given in Table 2. The studied strains were found to have varying susceptibility to AZ63. MIC values of Gram-positive bacteria were 256 mg/L, 256 mg/L, and 512 mg/L for E. hirae, E. fecalis, and S. aureus, respectively. MIC values of fungal strains were 512 mg/L and 512 mg/L for C. parapisilosis and C. tropicalis respectively. MIC values were determined as 1,024 mg/L against Gram-negative bacteria E. coli, P. aeruginosa, and L. pneumophila. When we compared the susceptibility of the studied strains to AZ63, the order was as follows from most susceptible to least susceptible; Gr +ve> fungi >Gr –ve. Most researchers are reported that bacteria-killing of magnesium and its alloys are attributed to high pH levels (alkalinity) caused by their degradation (Lin et al. 2019). Antimicrobial activities of magnesium alloys have been reported in various studies. (Lin et al. 2019) reported that they studied the antibacterial activity of ZK60. They noticed that microbial growth reduction was observed when using ZK60. In another study, Lock et al. (2014) indicated that magnesium alloys reduced E. coli viability and decreased microbial growth over a 3-day incubation period in a synthetic urine solution when compared with now used trading polyurethane stent. Sun et al. (2020) reported that the contact-killing antibacterial ability against adherent E. coli and S. aureus of Mg alloys were evaluated via the bacteria counting process, in which the adherent bacteria were detached from the sample and recultured on agar plates. They found that the antibacterial activity of the Mg alloys was 63.50% against E. coli and 48.13% against S. aureus. Mandal et al. (2021) informed that Fe-Mn-Cu alloy showed significant bactericidal activity when compared to the base alloy against E. coli. Based on our results, AZ63 showed varying degrees of antimicrobial activity against all strains tested, which is consistent with published results. It can be concluded that AZ63 can find use as a surface material in various fields in the prevention of infections caused by microorganisms.

Table 2

The test microorganisms' minimum inhibitory concentration (MIC)

MicroorganismsAZ63
E. coli 1,024 
P. aeruginosa 1,024 
L. pneumophila subsp. pneumophila 1,024 
E. hirae 256 
E. fecalis 256 
S. aureus 512 
C. parapisilosis 512 
C. tropicalis 512 
MicroorganismsAZ63
E. coli 1,024 
P. aeruginosa 1,024 
L. pneumophila subsp. pneumophila 1,024 
E. hirae 256 
E. fecalis 256 
S. aureus 512 
C. parapisilosis 512 
C. tropicalis 512 

*mg/L.

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Biofilm inhibition activity

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Most of the microorganisms grow as biofilms in their native environments. These formations are comprised of bacterial communities being in a matrix formed of polysaccharides, lipids, extracellular DNA, and proteins (Wozniak & Grinholc 2018). Biofilm generation represents a preserved mode of growth that renders bacterial cells more resistant against antimicrobial agents and to killing by host immune effector systems and so facilitates the pathogens to survive in enemy environments and also to distribute and colonize newly niches. Device-connected infections, persistent infections in the lack of a foreign body, and even medical tool breakdown are all included in the biofilm infectious category (Del Pozo 2018). Considering the above information, the prevention of biofilm formation is important both in terms of human health and in terms of preventing economic losses. In this study, the biofilm inhibition activity of AZ63 was also evaluated. Two bacterial strains including Gr +ve (S. aureus) and Gr −ve (P. aeruginosa) were used. When the effect of AZ63 on biofilm formation of S. aureus and P. aeruginosa was examined, it was seen that P. aeruginosa was more sensitive than S. aureus. Possibly, this difference may be due to the difference in cell wall structure. The biofilm inhibition activities of AZ63 against S. aureus and P. aeruginosa were 43.05%, 52.13%, 71.63%, and 39.30%, 75.57%, 83.25 at 125 mg/L, 250 mg/L, and 500 mg/L concentrations, respectively (Figure 6). AZ63 biofilm inhibition activity can be attributed to, a convenient local alkaline medium that effectively inhibits the development of Gr +ve and Gr −ve bacteria by inhibiting ATP synthesis and stimulating oxidative stress (Ling et al. 2022). Many reports have indicated that magnesium alloys have perfect antibacterial features. For example, Brooks et al. (2018) informed that they studied biofilm inhibition activity of pure titanium surface and AZ91. They reported that a dense biofilm formation could be macroscopically seen on the pure titanium surface; but when they used AZ91 they did not observe visibly appreciable evidence of biofilm generation. Biodegradable magnesium alloy has an often-exfoliated surface, on which biofilm can scarcely build (Tie et al. 2020). For example, Cheng et al. (2016) implanted Zr and N into AZ91 Mg alloys and then compared the number of viable bacteria on Zr-N implanted AZ91, pure Ti, and un-implanted AZ91. As a result, they reported that the number of viable bacteria on pure Ti was considerably higher than that found on un-implanted and Zr-N implanted AZ91 Mg alloys, indicating that both Zr-N-implanted and un-implanted AZ91 Mg alloys inhibited bacteria adhesion. As a result, AZ63 can be implemented as an antibiofilm agent in the biotechnology and healthcare industries to exterminate the problems raised by biofilm formation.
Figure 6
Biofilm inhibition of P. aeruginosa and S. aureus.
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Biofilm inhibition of P. aeruginosa and S. aureus.

Figure 6
Biofilm inhibition of P. aeruginosa and S. aureus.
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Biofilm inhibition of P. aeruginosa and S. aureus.

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Bacterial cell viability activity and antibacterial photodynamic activity

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Magnesium alloys offer a potential alternating to recently dominant metallic biomaterials for medical implementations and they are promising objects because of their native biodegradability, favorable elastic modulus, inherent antibacterial properties, and good biocompatibility (Lock et al. 2014; Feng et al. 2016). E. coli were used in this investigation to examine the effect of AZ63 on cellular viability and anti-microbial photodynamic activity. The results of the study are presented in Figure 7. Accordingly, inhibition of E. coli by AZ63 was determined as 90.17% and 96.34% at 100 and 200 mg/L concentrations, respectively. It is reported that the in vitro antibacterial activity results were related to the AZ63 material (Brooks et al. 2018; Ling et al. 2022). In a study conducted by Lock et al. (2014), they noticed that magnesium alloys reduced the microbial viability and colony forming units of E. coli in a 3-day incubation period in a synthetic urine solution. In the presented study, it was determined that AZ63 caused a decrease in the cellular viability of E. coli, and our results are consistent with the aforementioned study. Antimicrobial photodynamic therapy is a promising alternative to the treatment of subjected or infected tissues. Antimicrobial photodynamic therapy includes the delivery of a photosensitizer to goal bacteria, followed by light irradiation for the localized formation of other ROS. As a result, rapid and irreversible oxidative damage occurs (Morales-de-Echegaray et al. 2020). In this study, an antimicrobial photodynamic therapy study was also performed by exposing the compounds prepared as mentioned above to LED light for 20 min. AZ63 were utilized as photosensitizers for antimicrobial photodynamic activity tests using LED irradiation. The related results are presented in Figure 8. After 15 min LED irradiation, AZ63 showed 100% photodynamic antimicrobial therapy at 100 mg/L and 200 mg/L concentrations. Lin et al. (2019) studied antimicrobial behavior irradiated PIII treated ZK60-UV. They reported that PIII treated ZK60 samples irradiated by UV light can effectively kill 99.31% of bacteria growth of S. aureus. And also, they indicated that antimicrobial activity of PIII treated ZK60-UV sample mainly ascribed to a surge of ROS generation other than pH change. In the present study, a 100% inhibition effect was observed on E. coli and these findings are similar to the aforementioned study. LED radiation may also have caused the production of ROS and thus the death of bacteria. As a result, both microbial cell viability inhibition and antimicrobial photodynamic therapy results are remarkable and we can say that AZ63 can be applied in various fields to prevent E. coli infections.
Figure 7
Bacterial cell viability inhibition.
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Bacterial cell viability inhibition.

Figure 7
Bacterial cell viability inhibition.
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Bacterial cell viability inhibition.

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Figure 8
Photodynamic antimicrobial activity.
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Photodynamic antimicrobial activity.

Figure 8
Photodynamic antimicrobial activity.
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Photodynamic antimicrobial activity.

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Utilization of PES membrane blended with AZ63 for E. coli removal

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Drinking and utility water is mainly obtained from natural or artificial lakes and dams. Although these water resources are given to drinking water networks after undergoing various processes, contamination in various ways is possible until consumption. For this reason, the demand for spring water and bottled drinking water has increased in recent years. Spring waters are generally safe from a microbial point of view, as they reach the earth's surface by infiltrating from the deep layers of the soil. However, if the source water is obtained from areas contaminated with fecal wastes, or inadequacy of hygiene in the warehouse and filling facilities, hygiene errors of the personnel, etc., bacterial contamination may occur with the effect of factors (Gonul & Karapinar 1991). For these reasons, alternative techniques are being developed every day for the removal of microbial pollution in water resources. In this investigation, the PES membrane was blended with different concentrations of AZ63, and bacterial suspension of E. coli was passed through the AZ63 blended PES membrane. The results are shown in Figure 9. According to these results, it was determined that the antimicrobial efficiency of the AZ63 blended PES membrane increased as the AZ63 concentration increased in the PES membrane. The antimicrobial activity of the PES membrane blended with 0.5, 1, and 2 mg/L AZ63 was found to be 99.99, 100, and 100%, respectively (Figure 9). Jesú et al. reported that Mg ions demonstrated an important antimicrobial effect on Staphylococcus epidermidis and E. coli. They stated that Mg ions' antibacterial effect is mostly due to an increase in osmotic pressure around bacterial cells. A similar mechanism of antimicrobial activity may be also observed in our study. The results suggest that AZ63-blended PES membranes can be used in water treatment systems for the removal of microorganisms.
Figure 9
Utilization of polyethersulfone(PES) membrane blended with synthesized AZ63 for E. coli removal. (a) Inlet E. coli suspension (b) permeate of PES membrane, (c) permeate of PES membrane blended with 0.5 wt% AZ63, (d) permeate of PES membrane blended with 1 wt% AZ63, and (e) permeate of PES membrane blended with 2 wt% AZ63.
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Utilization of polyethersulfone(PES) membrane blended with synthesized AZ63 for E. coli removal. (a) Inlet E. coli suspension (b) permeate of PES membrane, (c) permeate of PES membrane blended with 0.5 wt% AZ63, (d) permeate of PES membrane blended with 1 wt% AZ63, and (e) permeate of PES membrane blended with 2 wt% AZ63.

Figure 9
Utilization of polyethersulfone(PES) membrane blended with synthesized AZ63 for E. coli removal. (a) Inlet E. coli suspension (b) permeate of PES membrane, (c) permeate of PES membrane blended with 0.5 wt% AZ63, (d) permeate of PES membrane blended with 1 wt% AZ63, and (e) permeate of PES membrane blended with 2 wt% AZ63.
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Utilization of polyethersulfone(PES) membrane blended with synthesized AZ63 for E. coli removal. (a) Inlet E. coli suspension (b) permeate of PES membrane, (c) permeate of PES membrane blended with 0.5 wt% AZ63, (d) permeate of PES membrane blended with 1 wt% AZ63, and (e) permeate of PES membrane blended with 2 wt% AZ63.

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As a result, the biological activities of AZ63 were also investigated in the presented study and remarkable results were obtained. AZ63 showed 81.25% DPPH radical scavenging activity and exhibited excellent chemical nuclease activity. The investigated Mg alloy, AZ63, displayed good antibiofilm characteristics when incubated with P. aeruginosa and S. aureus. It was observed to have average antimicrobial activity on the strains studied. It was also observed that AZ63 displayed excellent cell viability effect on E. coli and antimicrobial photodynamic therapy activity of AZ63 was tested by using LED irradiation and 100% bacterial growth inhibition was observed. Moreover, the antimicrobial efficiency of the PES membrane blended with AZ63 was investigated and it was found that 100% E. coli removal efficiencies were achieved with PES membrane blended with 1 and 2 wt% AZ63. When all the biological activity results of AZ63 are evaluated together, we can say that this biocompatible material can find application in various fields after further studies.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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No funding was received to assist with the preparation of this manuscript.

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All relevant data are included in the paper or its Supplementary Information.

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The authors declare there is no conflict.

Aǧirtaş
S. M.
,
Karataş
C.
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Özdemir
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