SYNTHESIS, CHARACTERIZATION, ANTIMICROBIAL AND ELECTROCHEMICAL STUDIES OF BIOSYNTHESIZED ZINC OXIDE NANOPARTICLES USING THE PROBIOTIC BACILLUS COAGULANS (ATCC 7050)

and positive potential of 29±2 mV. The biosynthesized ZnO NPs showed potent antimicrobial activity against Gram-positive and Gram-negative bacteria as well as pathogenic yeast with minimum inhibition concentration (MIC) values of 500 and 800 μg/ml against Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 and 600 μg/ml against Candida albicans ATCC 10231. On the other hand, a voltaic cell composed from an immersed reduced copper (positive electrode) and platinum electrodes (negative electrode) in ZnO NPs/bacterial metabolites was connected to a voltameter and used to study the electrochemical activity of ZnO NPs/ B. coagulans metabolites. Electrochemical characterization of ZnO NPs/ B. coagulans metabolites was done using current density–voltage characteristic, power density and electrochemical impedance spectroscopy (EIS) analyses. ZnO NPs/ B. coagulans metabolites produced high current with voltage value ≈ >0.34 volt. The present study reported the ability of B. coagulans to produce nitrate reductase enzyme with enzyme activity 2.18 U/ml. The reduction pathway of nitrate (NO 3) into nitrite (NO 2) during the biosynthesis of ZnO NPs might help and stimulate the current production.


INTRODUCTION
Antimicrobial agents have two different effects on the bacterial cells either bactericidal or bacteriostatic action. However, many bacterial strains acquired a resistance against different current traditional antimicrobial agents due to the misuse and overuse of multiple antimicrobial agents throughout several industries including packaging, paints, food, building, water treatment moreover their applications in the healthcare and biomedical fields (Griffith et al., 2022;Hazra et al., 2022). This phenomenon increased and became great problematic which made disease treatment more complex and increased death rate. Thus, the development of innovative antimicrobial agents to address the persistent evolution of pathogens had a great attention in last decades. Lately, nanotechnology has developed as a safe and effective technology to overcome this problem. Nanomaterials (1 to100 nm) are an exceptional class of materials that have various distinctive physicochemical and biological properties compared to their bulks with larger scales (El-Zahed et al., 2022a; Raval et al., 2022;Zhao et al., 2020). There are different chemical and physical methods used for the synthesis of nanomaterials including Sol-Gel method, thermal decomposition, chemical vapor deposition, laser ablation and microwave synthesis (Khan, 2020;Rajput, 2015). But the synthesized nanomaterials were found to have cytotoxicity effects on human cells, which could have negative effects in medicinal applications. Alternatively, biological methods that uses plants and different microbes during the biosynthesis process produced biocompatible, stable, and non-toxic nanomaterials (Raina et al., 2020). Nano-metal oxides such as ZnO, AgO and CuO are one of the most recent nanomaterials that revealed strong antimicrobial action against different pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and On the other hands, a very real energy crisis is currently being caused by the rising worldwide demand for energy, the issues of scarcity and the environmental impact of existing sources. Petroleum will become more expensive and scarcer, and by that time, the negative effects of extensive usage of all fossil fuels on the climate will be clear. The unregulated and excessive use of current energy resources over the past few decades has contributed to the depletion of traditional resource types. In addition, these fossil fuels caused numerous diseases for human, animals, and plants ( Lisha et al., 2023). So, scientists compete to find alternative safe and ecofriendly energy sources. Electrical energy is one of the most reliable, effective, and efficient generation of energy. Over the next few decades, renewable electricity is considered the most important challenge and will be enormously increasing the influence of renewable energy compared to existing levels. Different biological systems such as algae, fungi, and bacteria were used as a natural source of energy. The biomass from biological systems was applied in several purposes, including cooking, heating, industries, and energy (Lisha et al., 2023). Bacteria was reported as electrically carrying microorganisms throughout getting rid of the electrons that make when they produce energy (Lovley & Phillips, 1988;Malvankar et al., 2011). The unique physical and chemical properties of ZnO NPs, such as their high chemical stability, strong electrochemical coupling coefficient, broad spectrum of radiation absorption, paramagnetic nature, and high photostability, make them a versatile material (Parihar et al., 2018). Regarding the employment of hazardous bacteria, this is an alternate method of generating energy as a source of sustainable energy. As an illustration, some solar cells contain toxic materials (Mariotti et al., 2020). This energy is supplied from renewable resources and is not dependent on fossil fuels. Because of their distinct optical, electrical, and electrochemical properties, ZnO NPs have received much research in electrochemical applications (Faizan et al., 2021). ZnO NPs have been investigated as electrode materials for electrochemical sensing and energy storage devices such as batteries and supercapacitors. ZnO NPs can be utilised as an electrode material in electrochemical sensing to find diverse analytes, like gases, biomolecules, and heavy metals. ZnO nanoparticles are a good candidate for sensitive and selective electrochemical sensing applications due to their large surface area and distinctive electrical characteristics (Thareja & Kumar, 2021). Due to its great cycling stability and high specific capacity, ZnO NPs have been employed as an electrode material for lithium-ion batteries in energy storage systems. Because of their great Nowadays, nanotechnology has been used to overcome many global problems such as growing worldwide demand for energy and problems of microbial antibiotic resistance. The presented study used the probiotic Bacillus coagulans (ATCC 7050) as a nano-factory for the biosynthesis of zinc oxide nanoparticles (ZnO NPs). UV-visible spectroscopy, FTIR spectroscopy, XRD, TEM and Zeta analysis confirmed the formation of spherical ZnO NPs with a mean size of 10-19 nm and positive potential of 29±2 mV. The biosynthesized ZnO NPs showed potent antimicrobial activity against Gram-positive and Gram-negative bacteria as well as pathogenic yeast with minimum inhibition concentration (MIC) values of 500 and 800 μg/ml against Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 and 600 μg/ml against Candida albicans ATCC 10231. On the other hand, a voltaic cell composed from an immersed reduced copper (positive electrode) and platinum electrodes (negative electrode) in ZnO NPs/bacterial metabolites was connected to a voltameter and used to study the electrochemical activity of ZnO NPs/B. coagulans metabolites. Electrochemical characterization of ZnO NPs/B. coagulans metabolites was done using current density-voltage characteristic, power density and electrochemical impedance spectroscopy (EIS) analyses. ZnO NPs/B. coagulans metabolites produced high current with voltage value ≈ >0.34 volt. The present study reported the ability of B. coagulans to produce nitrate reductase enzyme with enzyme activity 2.18 U/ml. The reduction pathway of nitrate (NO3 -) into nitrite (NO2 -) during the biosynthesis of ZnO NPs might help and stimulate the current production.

ARTICLE INFO
electrical conductivity and high surface area, they have also been employed as a component in supercapacitors (Zhang et al., 2017). The present study aimed to use the probiotic Bacillus coagulans (ATCC 7050) as a new electrically carrying microorganisms to produce renewable and available alternative source of electrical energy as -to our knowledge-a first record in addition to extracellular biosynthesize of antimicrobial ZnO NPs and further subjected to characterization and evaluation of antimicrobial action against different pathogens.

Extracellular biosynthesis of ZnO NPs
Lyophilized culture of Bacillus coagulans (ATCC 7050) was obtained from the Microbiology Laboratory, Faculty of Science, Damietta University (Damietta, Egypt), subcultured for 24 hr at 37°C on de Man, Rogosa and Sharp (MRS, Oxoid) agar plates and reactivated three times before use. After incubation time, 3 single colonies were inoculated aerobically in a nutrient broth medium (Oxoid) and incubated at 37°C and 150 rpm for 24 hr. Bacterial supernatants were collected by centrifugation at 5000 rpm for 15 min under aseptic conditions. Then, cell-free supernatants were obtained by passing the supernatants through millipore filters. 20 ml of cell-free supernatant was mixed with 100 ml of zinc sulphate (ZnSO4, Sigma Aldrich, ACS reagent, 99%) solution (3 mM) and stirred for 24 hr at room temperature (25°C) and dark conditions. The biosynthesis of ZnO NPs was observed via the visible colour change and light-yellow precipitate formation. Mixtures were centrifugation centrifugated at 8000 rpm for 15 min to collect ZnO NPs precipitate and dried at 80 °C. The precipitate was dried completely and calcined at 400°C for 2 hr (Rajabairavi et al., 2017).

Minimum inhibition concentration (MIC) and minimum microbicidal concentration (MBC)
MIC and MBC for the biosynthesized ZnO NPs were studied using the broth dilution method (Clinical and Laboratory Standards, 2000). Aliquot 50 ml of Mueller-Hinton or Sabouraud dextrose broth media (Oxoid) supplemented with 100-1000 μg/ml ZnO NPs, penicillin or fluconazole were inoculated with 100 µl of overnight bacterial (10 8 CFU/ml) or yeast culture (10 6 CFU/ml), respectively, and incubated at 37°C and 150 rpm for 24 hr. MIC of the ZnO NPs (no apparent growth) was determined spectrophotometrically at 600 nm. To determine the MBC, aliquot 10 µl of each set was inoculated on MHA or SDA plate for bacteria or yeast culture, respectively, and incubated at 37°C for 24 hr. The MBC of the ZnO NPs (no apparent growth plates) was recorded.

Ultrastructure study
The antibacterial action of the ZnO NPs was investigated using P. aeruginosa as a model organism. Centrifugation was used to separate untreated and ZnO NPstreated bacteria with MIC values from 24 hr old cultures grown on nutrient broth media. After being fixed for three minutes in phosphate buffer solution and 5 min in potassium permanganate solution, the bacterial pellets were washed with 3% glutaraldehyde. The fixed samples were dehydrated for 15 min in a series of ethanol dilutions ranging from 10 to 90%, and then for 30 min in pure ethyl alcohol. The samples were filtrated with epoxy resin and acetone through a graded series till finally in pure resin. Ultrathin sections were collected on copper grids, then stained. The stained sections were observed with a TEM JEOL JEM-2100, Japan (El-Dein et al., 2021).

Electrode preparation
Voltaic cell composed of an emersed reduced copper electrode in cell-free supernatants of ZnO NPs/B. coagulans (ATCC 7050) as a positive electrode in addition to a platinum electrode as a negative electrode that connected to voltammeter (µA) was prepared and used for measuring the produced voltage ( Figure 1). Also, cell-free supernatants of ZnO NPs/B. coagulans was tested to light a led lamp in the voltaic cell connected to a LED lamp instead of the voltmeter.

Electrochemical analyses of cell-free supernatants of ZnO NPs/B. coagulans
Current density-voltage characteristic and power density cell-free supernatants of ZnO NPs/B. coagulans were measured by Metrohm Autloab (PGSTAT204) Nova2 software. Then, electrochemical impedance spectroscopy (EIS) analysis of the fabricated DSSC employing samples was carried out between 0.1 Hz and 100 kHz at an open-circuit AC potential of 10 mV using a frequency response analyzer and fitted to an equivalent circuit model using the Metrohm Autloab (PGSTAT204) Nova2 software (Optoelectronics Lab, Physics Department, Faculty of Science, Damietta University, Egypt).

Total protein estimation and NADH dependent enzymes assay
Total protein content of B. coagulans (ATCC 7050) was estimated by Bradford method (Bradford, 1976). Nitrate reductase (NR) activity was measured spectrophotometrically at 540 nm according to Harley (1993) depending on the reduction of nitrate into nitrite as a model for NADH dependent reductase enzyme. Nitrite standard curve was prepared and used to calculate the amount produced by enzyme activity. Production of one μmol nitrite/h/ml was defined as one unit of NR activity (U/ml).

Statistical analysis
SPSS software version 18 was used to analyse results using the ANOVA test. The threshold for significance was fixed at 0.05. The experiments were carried out three times. All values were reported as the mean and standard error (SE) (O'connor,  2000).

RESULTS AND DISCUSSION
People take B. coagulans for irritable bowel syndrome (IBS), diarrhea, gas, airway interpret, and many other conditions, but there is no good scientific evidence to support these uses (Liu et al., 2018). The bacteria that cause gangrene (Clostridium perfringens) and hospital-acquired infections (Enterococcus faecalis) and some disease-causing streptococcus bacteria were used to produce electricity (Mukhaifi & Abduljaleel, 2020). On the other hand, the presented study used B. coagulans, similarly to Lactobacillus sp. and other probiotics as "beneficial" bacteria, to biosynthesize ZnO NPs with potent antimicrobial activity and studied their electrochemical behaviour.

Biosynthesis and characterization of ZnO NPs
Many studies had documented the ability of bacteria to biosynthesize different NPs via several mechanisms (Pandit et al., 2022). One of the recommended pathway for NPs biosynthesis at dark conditions is the bioreduction of metal ions using NADH dependent enzymes into nanometals or nanometal oxides (Yusof et al., 2019). This biosynthesis method may occur either intracellularly or extracellularly (Mahdi et al., 2021). However, the extracellular methods are more favourable due to the intracellular methods requires extra-steps such as cell breakdown, extraction and purification of the produced NPs (Mahdi et al., 2021). B. coagulans (ATCC 7050) could biosynthesize ZnO NPs extracellularly within 24 hr at room temperature (25°C) and dark conditions. ZnO NPs formation was confirmed and characterized by different techniques. The colour change into pale yellow indicated to the biotransformation of zinc ions in the presence of B. coagulans cell-free supernatant to ZnO NPs which assigned to excitation of ZnO NPs surface plasmon resonance (SPR) (Rehman et al., 2019). The UV-visible spectrometer absorption peak was appeared at 318 nm, which is specific for ZnO NPs (Figure 2A). This absorption peak agree well with the ZnO NPs absorption band reported by (Mousa & Khairy, 2020). On the other hand,  (Lai et al., 2011). The hydroxyl groups stretching appeared as broad peaks at 3390.4, 3271.6 cm -1 . The secondary amines vibration band located at 2929.5 and 2860 cm -1 . Vinyl and cis-tri substituted absorption peaks raised at 1645.1 cm −1 . Amines stretching vibrations appeared at 1457.3, 1259.3 and 1394.3 cm -1 . The carbon-oxygen bond stretching located at 1072.3 cm −1 while carbon-hydrogen bond stretching presented at 881.5 and 781.1 cm -1 . XRD of ZnO NPs was analysed between 10° and 80° of 2θ range ( Figure 2C). ZnO crystalline peaks appeared at 32.3°, 35.2°, 36.6°, 57.2°, 64.3° and 67.2° corresponding to the lattice planes (100), (002), (101), (110), (103) and (112) that matched with results. Debye-Scherrer equation (D = kλ/βcosθ; where D; average crystalline particle size, λ; wavelength of x-ray (1.5406 Å), k; Scherer's constant (0.9), θ; diffraction angle, and β; XRD peak full width at half maximum) was applied to calculate ZnO NPs size. The average size was found to be 17±1.5 nm which matched with the TEM results. According to several studies, the effectiveness of antibacterial activity of nanoscaled particles depends on the size of the particles  ZnO NPs had a surface positive charge equal to 29±2 mV according to Zeta potential test ( Figure 2D). The TEM micrograph showed the good dispersion of the biosynthesized spherical-shaped ZnO NPs ( Figure 2E). Also, the spherical shaped ZnO NPs with 5.40 -6.79 nm range were demonstrated using B. foraminis as demonstrated by EL-Ghwas (2022). While B. subtilis ZBP4 biosynthesized ZnO NPs with irregular spherical shape and size range of 22-59 nm as reported by Hamk et al. (2022). The current results confirmed the high stability of the biosynthesized ZnO NPs due to the high repulsion forces between the particles due to its high surface positive charge values which prevent their aggregation and agglomeration. In addition, the FTIR results confirmed the presence of different proteins that will act as capping agents (El-Dein et al., 2021).
A typical issue that reduces the biological potential of nanoparticles is the low stability and their tendency to aggregate. Capping agents were used to decrease and stop nanoparticles agglomeration and aggregation (Duan et al., 2015). The existence of proteins as capping agents for ZnO NPs was confirmed by the FTIR spectrum and may also act as stabilising agents. Additionally, Siddique et al.
(2013) speculate that charged nanoparticles may strengthen the attraction between particles, reducing the likelihood of particle aggregation. The biosynthesized ZnO NPs had a positive potential reached to 29±2 mV which produce strong repulsion force between the particles preventing its aggregation. Also, TEM micrograph showed the homogeneity of the biosynthesized ZnO NPs moreover their monodispersity in size.

Antimicrobial activity of biosynthesized ZnO NPs
Antimicrobial activity results showed that ZnO NPs were effective against both Gram-positive and Gram-negative bacteria as well as yeast (Figure 3). It was found that ZnO NPs showed higher antibacterial potential against the Gram-positive than Gram-negative bacteria due to the difference in their cell wall composition ( found that ZnO NPs showed inhibition zones (7,8,9,9 and 10 mm) increases with increasing concentrations (62.5, 125, 250, 500 and 1000 μg/ml, respectively).

Figure 3
Antimicrobial activity of ZnO NPs comparing to other antimicrobial agents (1000, 3000 and 5000 μg/ml) against S. aureus ATCC 25923, P. aeruginosa ATCC 27853 and C. albicans ATCC 10231. Each column represents the mean of three replicates ± SE. Columns with common letters are not significantly different at P < 0.05. The MIC and MBC values of ZnO NPs in the current study were 800 and 900 µg/ml for P. aeruginosa, 500 and 700 µg/ml for S. aureus and 600 and 700 µg/ml for C. albicans, respectively showing a dose-related inhibitory effect (Figure 4). ZnO NPs revealed moderate to strong antibacterial activity compared to the standard antibacterial drug (penicillin

Ultrastructure of ZnO NPs-treated bacteria
The ZnO NPs impact on the ultrastructure of P. aeruginosa was assessed by TEM examination (Figure 5). Control bacterial cells had a smooth rod-shaped cell membrane and homogeneous cytoplasm. In contrast, ZnO NPs-treated bacterial cells showed irregular shapes and distinct morphological changes on the cell surface ( Figure 5B). The malformation of the treated cells resulted in total cell lysis, release of cytoplasmic material, formation of lipids, separation of the outer membrane from the plasma membrane, and total loss of cytoplasm. Although the precise ZnO NPs antimicrobial mechanism is still unknown, some reported hypotheses suggested that ZnO NPs might interact with cellular proteins in cell membranes, attacking respiration and cell division and ultimately killing cells. In addition, the bactericidal effects of ZnO NPs were reported in different studies including formation of reactive oxygen species, cell wall damage and injuries in membrane permeability (Sirelkhatim et al., 2015). The antimicrobial mechanism of ZnO NPs might be clarified through further studies.

Electrochemical studies of cell-free supernatants of ZnO NPs/B. coagulans
Regarding the use of bacteria in electrical current production, this refers to a process known as microbial electrochemical systems (MES). In an MES, microorganisms are used as biocatalysts to convert organic matter into electrical current. The use of harmful bacteria in an MES is common and can be beneficial because they can break down complex organic matter that other microorganisms cannot, thus increasing the efficiency of the system. However, it is important to note that the ZnO NPs/B. coagulans metabolites produced high power density. To have a better knowledge of the electrochemical process at electrodes/solution interface and inside the solution, EIS Analyses were performed on two identical platinum electrodes with solution in between. Figure 6 illustrates current density-voltage and power density of B. coagulans. The power density curve was designed to examine voltage where power density as a function of current density. Short circuit current density is -0.0012 A.cm -2 which negative sign refers to output electricity and open circuit voltage is 0.335 V. Maximum value of power density 0.113 mW. cm -2 . The produced electricity is without external influences or catalyst factor, and this is attributed to the movement of ions inside the cell (Marshall & May, 2009).

Figure 6
Current density-voltage characteristic and power density generated from ZnO NPs/B. coagulans metabolites. Figure 7 shows EIS Nyquist plot Pt dummy cells whereas series resistance is 12.19 Ω.cm 2 , Q = 1.244e-3 F.s^(a -1), n= 0.644 4, Rre = 493.3 Ω.cm 2 , and Cch= 950.15e-6 F from these data we calculated ion conductivity (σ= 0.164 Ω -1 .cm -1 ). On the other hand, Figure 8 illustrates EIS Nyquist plot Cu dummy cells whereas series resistance is 17.59 Ω.cm 2 , Q = 1.24e-3 F.s^(a -1), n= 0.751 6, Rre = 256.3Ω.cm 2 , and Cch= 847.93e-6 F from these data we calculated ion conductivity (σ= 0.113 Ω -1 .cm 1-). Figure 8 Shows output power density from ZnO NPs/B. coagulans metabolites. EIS analysis was carried out for two (dummy) Pt electrodes and identical electrodes of Cu with ZnO NPs/B. coagulans metabolites in between two electrodes. The primary goal of EIS analysis was to comprehend the behavior of ions at interface B. coagulans/electrode. There are critical parameters (chemical capacitor (Cch), the recombination resistance (Rre), constant phase element (Q), constant phase (n), and series resistance (Rs) that are calculated from EIS analysis to understand ions performance in cell at electrodes. These parameters are computed using a proper equivalent circuit to fit the experimental data (Rs +Q1/( Rre +W2)). B. coagulans had a specific pathway to reduced nitrate (NO3 -) into nitrite (NO2 -) using extracellular NR which might produce the current. The bacterial total protein concentration (1277.6 ± 0.03 µg/ml) of B. coagulans was estimated by plotting of standard curve for bovine serum albumin protein sample with different known concentrations against absorbance values, the unknown samples were determined directly from the equation derived from the curve. The activity of NR was measured and calculated in the B. coagulans crude metabolite, at the rate of 2.18 U/ml. In addition, B. coagulans may transport electrons through their chemical compounds that found in its secondary metabolites into the environment as tiny currents which could assisted by ubiquitous flavin molecules enhancing electrons flowing into the electrodes (Paquete, 2020). Although work on MES-capable bacteria is still in its early phases and the majority of applications are still in the development stage, there is great potential for making important advances in the field.

CONCLUSIONS
Zinc oxide nanoparticles (ZnO NPs) were biosynthesized using the cell-free bacterial supernatant of Bacillus coagulans (ATCC 7050). The provided biosynthesis approach was rapid, simple, cheap, and eco-friendly with high stability. To the best of our knowledge, this is the first report on the study of B. coagulans for biosynthesis of ZnO. The biosynthesized nanoparticles had higher biocidal activity against Gram-positive than Gram-negative bacteria moreover their strong antifungal action against Candida albicans. ZnO NPs/B. coagulans metabolites had high current value (voltage value ≈ >0.34 volt) which was enough to light a LED lamp as a new microbial electrochemical system (MES). The obtained results are considered as promising results for an applicable, cheap, renewable, and safe source of energy, especially in the field of electricity moreover their strong microbicidal action.