Bioremediation of zinc metals and microplastics by biosynthesizing zinc oxide nanoparticles from isolated bacteria of the Caspian Sea, Iran

Bioremediation of zinc metals and microplastics by biosynthesizing zinc oxide nanoparticles from isolated bacteria of the Caspian Sea, Iran | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bioremediation of zinc metals and microplastics by biosynthesizing zinc oxide nanoparticles from isolated bacteria of the Caspian Sea, Iran Motahare Haghighatjoo, Parichehr Hanachi, Ali Mohammadi, Omran Moradlou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2851887/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The high levels of environmental pollutants like heavy metals and microplastics have prompted researchers to invent new remediation strategies, which might be accompanied by drawbacks, including the risk of secondary pollution, being costly, and inefficient. So, the aim of this study is the reduction of these environmental pollutants by the application of nanoparticle-producing bacteria. For this purpose, from 22 screened bacterial isolates of the Caspian Sea, the isolate ( N1.5.2 ) was used to produce ZnO NPs, which were characterized by techniques like FTIR, XRD, Raman, SEM, EDX, UV-vis spectroscopy, Zeta potential, and DLS. Subsequently, different protocols of bacterial nanoparticle production (using supernatant, extract, biomass, and dilution) were compared by UV-vis spectroscopy. Then, the produced nanoparticles were applied to polystyrene microplastics to degrade them. This process was monitored by SEM and FTIR. The selected bacterial isolate ( N1.5.2 ) was identified and its phylogenetic tree of life was depicted. The results of the blast illustrated 100% similarity of N1.5.2 to Bacillus cereus . The produced ZnO NPs peaked at 360 nm in their UV-vis spectrum. They showed a crystalline structure in the zincite phase and dispersed in the range of 171–262 nm in an aqueous environment with a PDI of 0.444 and zeta potential of -5.60 meV. Moreover, the comparative study between different production protocols has shown that ZnO nanoparticles produced by bacterial extract have higher UV-vis peak values. Furthermore, the photocatalytic activity of ZnO NPs and microplastic degradation has been proved by SEM and FTIR. Bacterial Nanoparticle Production Bioremediation Microplastics Photocatalytic Activity Zinc Oxide Nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The proliferation of technology and industry has emerged as a significant factor contributing to the escalation of environmental pollution, which has resulted in irrevocable and detrimental consequences. For instance, heavy metals are highly toxic to human and other organisms. Their toxicity highly depend on their concentration and doses[ 1 ]. The accumulation of these pollutants in the environment is the result of some human activities, like mining, smelting, and foundries [ 2 ]. Take microplastics as another example. These tiny plastic particles are the main culprit for the health issues of human and other organisms [ 3 ]. Heavy metals are characterized by high densities, atomic weight or atomic numbers, and are highly toxic to humans and other organisms. Their toxicity levels depend on their concentration and doses [ 1 ]. Some of them, like zinc, are required for body functions. They are associated with several enzymes as a cofactor through processes like protein synthesis or DNA replication [ 4 ]. However, excess levels of them in the environment might be toxic [ 5 ]. For example, the normal concentration of zinc in the soil that is essential for crops is between 30 to 200 µg/g [ 6 ]. Excess levels of zinc as a result of human activities may alter soil microbial communities, consequently affecting the growth of crops. Moreover, the extra amount of this element in the human body may cause gastrointestinal distress with symptoms like cramps, diarrhea, nausea, and vomiting [ 5 ]. So, the high concentration of heavy metals is considered a type of environmental pollution. Since zinc and other heavy metal pollutants tend to aggregate in different environmental matrices, many researchers focus on developing different remediation methods. Most industrial remediation techniques are classified into three groups of physical, chemical, and biological methods [ 7 ]. Thermal desorption, vertification, encapsulation, vapor stripping soil washing, sorting, and Electrokinetic remediation are examples of physical methods. These methods are usually low-efficient. So, applying a replacement technique seems necessary. The chemical methods include different techniques like distillation, electrodialysis, chemical precipitation, and ion exchange. Chemical precipitation includes sulfide precipitation, hydroxide precipitation, chelating precipitation, Xanthate precipitation, and carbonate precipitation [ 8 ]. Additionally, biological methods, as the alternative strategies for the remediation of heavy metals, are commonly used for their high efficiency, fast-growing, and easy controlling [ 8 ]. Their primary procedure is Biosorption, which is a physiochemical technique in which pollutants like metal ions from an aqueous environment bind onto the presented functional groups on the organism’s surfaces. This method is efficient and selective [ 9 ]. Some groups of bacteria have the ability to transform pollutants like metallic ions into less harmful structures such as metallic nanoparticles [ 10 ]. Nanoparticle production by microorganisms is a detoxifying mechanism to reduce the toxic metals to nanoparticles, which is considered a defense mechanism in bacteria against heavy metals stresses. In this way, the concentration of heavy metals like zinc will be reduced and metallic nanoparticles will be produced at the same time [ 10 ]. The production of nanoparticles by microorganisms might be intracellularly or extracellularly [ 10 ]. Usually, this process consists of three steps: capturing, reducing, and capping. In the beginning, the positive ions are captured on the surface of the cell. Then they can be reduced to metal nanoparticles by active molecules like enzymes through a redox reaction. Finally, the secreted biomolecules by microbes could act as capping or stabilizing agents. Serratia ureilytica can synthesize ZnO nanoflowers which have antimicrobial properties against S.aureus 1 and E.coli 2 . Moreover, Lactobacillus plantarum and Aeromonas hydrophila are other examples of ZnO nanoparticle-producing bacteria [ 11 ]. The production of nanoparticles in this way would be efficient. By applying a proper protocol, the total expenses in nanoparticle production may be reduced to 1/10 in comparison to the chemical method. These nanoparticles show a high level of catalytic activity and they do not aggregate because of the capping agents. This method of nanoparticle production is efficient, safe, and environmentally friendly [ 10 ] Microplastics, as another environmental pollutant, are less than 5mm in their size and are composed of different chemical components. They can be found in different ecological conditions [ 12 ][ 3 ]. They are classified into primary and secondary microplastics. The first group is produced in order to be applied in different industries like the production of face wash, toothpaste, and cosmetics. However, the second one is undesirably created through the degradation of plastics in natural habitats. The annual production of plastics and microplastics is soaring. Thus, in 2018, the global production of plastics exceeded 360 million metric tons [ 13 ]. Since the biological interactions of these particles are size-dependent, smaller particles are more active and dangerous than larger ones. Their chemical additives, like coloring agents and plasticizers, are toxic to living organisms. Moreover, they can easily be transferred along the food chain and piling up in cells and different tissues. So, disruption of cellular metabolisms and their physiological activities can cause inflammation and metabolic disorders [ 14 ]. Therefore, these toxic side effects have made scientists focus on different remediation techniques, including physical, chemical, and biological methods. The physical forces, like adsorption and filtration, are the main triggering factor for the collection of microplastics from an environment [ 15 ]. Although these techniques are commonly used, their protocols become too complicated in the case of small particles. So, they are highly costly and require a large amount of budget allocation in terms of energy and facilities [ 16 ]. Moreover, chemical remediation contributes with coagulants, like Fe- or Al- salts to remove these types of pollutants. Furthermore, one of the novel strategies to reduce the number of plastics in nature is the application of photocatalists. Most metal oxide nanoparticles have photocatalytic properties which can be used to degrade some organic and polymeric pollutants. In other words, a new microplastic remediation technique is photodegradation. UV radiation alters the electron structure of photocatalysts like zinc oxide nanoparticles by forming a pair of electrons and holes. This active structure can trigger a redox reaction that leads to the degradation of microplastics into smaller molecules like carbon dioxide and water. The oxidation and reduction reactions should take place simultaneously. In this case, semiconductors are the best options, since they have a moderate band gap which provides the possibility of oxidation and reduction reactions. In this case, the most suitable semiconductor are those that have band gaps between 1.5–3.5 eV. Metal oxides have this feature [ 17 ]. In this study, we seek to find a solution to solve the challenge of microplastic pollutants and zinc compounds in the environment. The proposed scheme is to use the route of biological production of nanoparticles. In more detail, zinc compounds are absorbed by bacteria for the production of nanoparticles. Subsequently, the produced nanoparticles can be used as a photocatalyst to degrade microplastics. 2. Material and methods Zinc sulfate monohydrate (ZnSO4.H2O), Sodium Chloride (NaCl), Magnesium sulfate heptahydrate (MgSO4.7H2O), Magnesium chloride hexahydrate (MgCl2.6H2O), Calcium chloride dihydrate (CaCl2.2H2O), Potassium chloride (KCl), Ammonium sulfate ( \({\left(N{H}_{4}\right)}_{2}S{O}_{4}\) ), yeast extract, and agar were purchased from Sigma Aldrich. 2.1. Sampling and bacterial isolation Ten marine samples, comprising of both water and sediment, were meticulously collected at a depth of 50 cm from four distinct regions, namely Chamkhaleh, Amir Abad, Kiashahr, and Chaf, located in Gilan, Iran, during the period spanning from September 23, 2020, to September 26, 2020. To preserve the integrity of these samples, they were carefully transferred to sterile tubes and stored in close proximity to ice prior to being transported to the laboratory [ 18 ]. The marine samples were subjected to culture on three distinct mediums, namely SW agar, marine agar, and Sea Water salt medium. The SW agar, with constituents comprising of NaCl (9.1 g/l), MgSO 4 .7H 2 O (0.75 g/l), MgCl 2 .6H 2 O (0.17 g/l), CaCl 2 .2H 2 O (0.22 g/l), KCl (0.13 g/l), \({\left(N{H}_{4}\right)}_{2}S{O}_{4}\) (0.86 g/l), FeSO 4 .7H 2 O (0.009 g/l), peptone (10.0 g/l), yeast extract (2 g/l), and agar (15 g/l) ; had a pH of 8.2. On the other hand, the Sea Water salt medium was formulated using yeast extract (0.2 g/L), peptone (1.0 g/L), and agar (15 g/L), in combination with filtered Caspian Sea water, and had a pH of 8.2. Subsequent to an incubation period, 22 isolates were successfully obtained. [ 19 ]. 2.2. Studying the zinc tolerance of the bacterial isolates To investigate the bacterial isolates' tolerance to zinc, a suspension of 0.5 McFarland of each isolate was inoculated into 3 ml of nutrient broth with varying concentrations of ZnSO 4 .H 2 O (0.25 to 5 mg/mL). Following incubation, the growth and resistance of each culture were evaluated by separate culturing on nutrient agar[ 20 ]. The isolates demonstrating the highest level of resistance were identified as potential candidates for ZnO nanoparticle production. 2.3. Bacterial production of the zinc oxide nanoparticles The utilization of a bacterial extract for the production of nanoparticles involved several precise steps. Initially, a suspension containing 2.0g of cell biomass (dry weight) in 100 ml of deionized water was incubated on a rotating incubator (150 rpm) for 48 hours. Following this period, the suspension underwent centrifugation (7000 rpm, 15 minutes), and the resulting supernatant was filtered. Subsequently, 20ml of ZnSO 4 .H 2 O (0.10 M) was added to the supernatant, and the mixture was incubated at 30˚C on a shaker incubator (120 rpm) for 48 hours under dark conditions. The resulting solution was then utilized with positive and negative controls, utilizing the cell extract and the salt solution, respectively [ 21 ]. Ultimately, after undergoing centrifugation (10000 rpm, 20 minutes), washing with distilled water and ethanol, the ZnO nanoparticles (ZnO Nps) were obtained.[ 22 ]. 2.4. ZnO nanoparticles characterization The biosynthesized ZnO nanoparticles (ZnO Nps) underwent a comprehensive evaluation process utilizing various analytical techniques. The UV-vis spectrophotometer (UV-2150; Unico, US) was employed to examine the nanoparticles in the range of 200–600 nm with a resolution of 1nm. To further analyze the crystalline phases of the ZnO NPs, X-ray diffraction analysis (Rigaku) was conducted using Cu Kα radiation (λ = 1.541874 A˚) within the range of 2Ѳ of 0–80˚. Fourier-transform infrared (FTIR) spectroscopy (Bruker, Germany) was implemented to investigate the functional groups and bioactive molecules that played a significant role in the reduction, stabilization, or capping of the ZnO NPs, utilizing a wavenumber range of 400–4000 cm − 1 at a resolution of 4cm − 1 . In addition, dynamic light scattering (DLS) (Malvern Instruments, UK) was utilized to identify the polydispersity index (PDI), hydrodynamic diameter, and zeta potential of the biosynthesized nanoparticles[ 22 ][ 21 ][ 23 ]. Furthermore, the surface characteristics of the nanoparticles were analyzed by a field-emission scanning electron microscope (FESEM; S-4800, Hitachi, Japan). 2.5. Comparative study on different bacterial methods of ZnO NPs production In this study, the production of ZnO NPs from four different protocols involving bacterial culture, extract, biomass, and supernatant were compared via UV-vis spectroscopy. To obtain NPs from bacterial biomass and supernatant, the overnight culture of N1.5.2 was centrifuged at ×5000, and the biomass and supernatant were collected. The biomass underwent washing with phosphate-buffered saline and was then suspended in 50 mL of Zn 2+ solution (0.5M). Followed by incubation at 30˚C and 150 rpm agitation. After centrifugation, the ZnO NPs were obtained via ultrasonic disruption. For the supernatant route, 100 mL of filtered supernatant was added to 100 mL of ZnSO4.H2O solution (0.1M) and incubated on a shaker incubator for 24 hours. The formation of white precipitation indicated the production of ZnO NPs in the solution [ 22 ]. To synthesize nanoparticles directly from bacterial culture, 20 mL of ZnSO4.H2O (0.1M) was added to the overnight culture and incubated at 30˚C with 120 rpm agitation for 72 hours. The final product was collected after centrifugation [ 21 ]. Subsequently, the produced nanoparticles were collected, purified, and dried, after which UV-vis spectroscopy was utilized to evaluate and compare these different synthetic methods. 2.6. Bacterial identification 2.6.1. Staining of bacteria In order to investigate the morphological and physiological features of the chosen isolate, Gram and spore staining were performed using established protocols outlined in Laboratory Techniques In Microbiology And Biotechnology [ 24 ]. These techniques are widely recognized as key tools for characterizing microbial isolates and provide valuable insights into their cellular structure and metabolic properties. 2.6.2. DNA extraction, PCR, and electrophoresis For the amplification of the 16S-rRNA gene, genomic DNA was extracted using the boiling protocol, and the universal primers 8F (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1492R (5´-CACGGATCCTACGGGTACCTTGTTACGACTT-3´) were utilized. To prepare the PCR reaction, a mixture with a total volume of 25 µL was prepared, including 1.25 µL each of forward and reverse primers, 0.5 µL of dNTP (10 mM), 2.5 µL of PCR buffer, 0.75 µL of MgCl2 (50 mM), 1 µL of template DNA, 1.25 µL of DMSO, 0.5 µL of SmartAq DNA polymerase (Cinnagen, Iran), and 16 µL of dH2O[ 25 ]. The PCR was conducted under the following conditions: an initial denaturation step at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 45 seconds, annealing at 55°C for 1 minute, extension at 72°C for 1.5 minutes, and a final extension at 72°C for 10 minutes. To verify the results of the PCR analysis, gel electrophoresis was used to separate and visualize DNA fragments based on their size. 2.6.3. Sequencing and phylogenic tree The forward sequencing of the PCR product was carried out by Niagene Noor, Iran. Subsequently, the resulting sequence was subjected to analysis using BLAST, and the phylogenetic tree of life was constructed using MEGA11. To ensure proper documentation and accessibility, the obtained sequence (669 bp) was deposited in GeneBank under the accession number OM185305.1. 2.7. Photocatalytic studies The photocatalytic activity of biosynthesized nanoparticles on polystyrene microplastics (1cm×1cm) was investigated using a photoreactor equipped with a 6-watt UV-C Hg vapor lamp. The reaction was conducted for a period of 96 hours in a reaction vessel containing the biosynthesized ZnO nanoparticles and deionized water, with the vessel positioned at a distance of 10cm from the UV lamp. To prepare a suspension of ZnO NPs for microplastic degradation, a concentration range of 1–3 mg/L in distilled water was utilized. In order to enhance the likelihood of effective nanoparticle-microplastic collisions, a magnetic stirrer was employed to circulate the contents of the reaction vessel. The reaction was initiated upon placement of the vessel under the UV lamp. Every 48 hours, 2 µl of the suspension were extracted for further analysis. The surface morphology of polystyrene microplastics and the variation of their surface functional groups were studied using scanning electron microscopy (GEMINI Ultra 55, Carl Zeiss, AG, Germany) and Fourier transform infrared spectroscopy over the range of 400 to 4000 cm − 1 , respectively [ 26 ]. 3. Results and discussion The community of bacteria present in marine environments is characterized by its extensive diversity in terms of composition, genetic materials, and metabolisms. Of particular note is the fact that bacteria are responsible for 98% of primary production [ 27 ]. As such, researchers have focused on exploring the potential applications of marine bacteria in both industrial and clinical settings, with the goal of harnessing their secondary metabolite production capabilities [ 28 ]. One particularly promising source of bacterial diversity is the Caspian Sea, which is considered the world's largest inland body of water. This unique ecosystem contains a multitude of distinct bacterial communities [ 29 ], making it an attractive target for screening efforts aimed at identifying nanoparticle-producing bacteria. Through targeted sampling and culturing techniques, we have successfully isolated 22 distinct bacterial strains from this environment. These isolates represent a valuable resource for further study and exploration of potential biotechnological applications. 3.1. Studying the resistance of bacterial isolates against zinc salt Environmental stressors, such as the presence of toxic compounds or excessive concentrations of heavy metals, have been found to significantly impact bacterial communities. Chen et al. conducted research indicating that the presence of heavy metals, such as zinc compounds within bacterial habitats, triggers the expression of metal resistance genes (MRGs). This response may ultimately lead to the effective remediation of heavy metals from polluted areas[ 30 ]. Therefore, bacterial resistance to heavy metals like zinc is one of the main key factors for the bioremediation of a polluted area, and the production of metallic nanoparticles like ZnO by bacteria is not an exception to this rule [ 31 ]. In line with this research, 22 bacterial isolates were cultured in media containing varying concentrations of zinc, ranging from 0.25 to 5.0 mg/L. The goal of this approach was to identify the most resistant isolates capable of producing nanoparticles. Through this investigation, four highly resistant isolates were identified and subsequently utilized for nanoparticle production, namely: N0.5.2, N1.1, N1.5, and N1.5.2. 3.2. The production of zinc oxide nanoparticles Bacteria, as eco-friendly catalysts, play an important role in the bioremediation of natural environments through redox reactions. Their bioactive molecules alter the ionic state of heavy metals which may affect their solubility and bioavailability which might lead to the alteration of their movements in the natural environment. Microbial active molecules and enzymes can reduce toxic metal ions like zinc salts to less toxic compounds like zinc nanoparticles [ 32 ]. This process of nanoparticle production can take place inside or outside of the cell [ 22 ]. In this study, the bioactive molecules of bacterial extract converted the aqueous zinc ions to ZnO NPs. Based on Yousof et al. the first sign of ZnO NPs production is the appearance of white precipitation. Zinc sulfate monohydrate was added to the bacterial extract of four selected isolates ( N0.5.2 , N1.1 , N1.5 , and N1.5.2 ), just the Erlenmeyer containing the extract of N1.5.2 became turbid. Sambalova et al. produced Silver nanoparticles by using bacteria. They suggested carboxylate functional groups mediate this reaction [ 33 ]. In order to confirm the production of ZnO NPs, the obtained white precipitation was studied via UV-vis spectroscopy in the range of 300–600 nm. Spectral analysis revealed a prominent peak at 360nm in the spectrum of the nanoparticles produced by the N1.5.2 bacterial isolate, thus providing further confirmation of successful ZnO NP production (Fig. 1 (a)) and (Fig. 1 (b)). 3.3. ZnO NPs Characterization 3.3.1. X-Ray diffraction X-ray diffraction (XRD) analysis is a widely-used technique for confirming the phases of ZnO nanoparticles (NPs). The XRD pattern of ZnO NPs obtained in this study is depicted in Fig. 1 (c), which indicates that the observed diffraction peaks at 31.84˚, 34.44˚, 36.28˚, 47.60˚, 56.66˚, 62.90˚, 68.06˚, and 72.62˚ can be characterized as (100), (002), (101), (012), (110), (013), (112), and (001) reflections, respectively. By comparing these results with the data from Match 3 software (database reference: COD-Inorg 2021.12.14), it was confirmed that the ZnO NPs exhibited a hexagonal phase with a Zincite structure (Fig. 1 (c)). This finding provides further evidence to support the successful production of ZnO NPs using the bioactive molecules present in the bacterial extract from the N1.5.2 isolate. 3.3.2. Particle size distribution by DLS Dynamic Light Scattering (DLS) was employed to determine the size distribution profile and hydrodynamic diameter of Zinc Oxide Nanoparticles (ZnO NPs) in an aqueous solution, as reported by reference [ 34 ]. The study revealed that the resulting particles were dispersed in an aqueous medium with a size range of 171-262.6 nm. The Polydispersity Index (PDI) value of the biosynthesized nanoparticles was 0.444, with a Z-average of 232.5 nm, as presented in (Fig. 1 (d)). Notably, the DLS analysis showed that the biosynthesized nanoparticles had medium dispersion in the aqueous solution, which contributed to their PDI value. It is pertinent to mention that the measured size obtained via DLS was larger than what was estimated by SEM. Such a difference can be attributed to the tendency of water molecules in the aqueous solution to surround the nanoparticles, forming hydrodynamic shells. The PDI is a characteristic feature of the homogeneity of nanoparticles, as referenced in [ 35 ]. This finding is distinct from previous research conducted by Yedurkar et al., using Ixora Coccinea Leaf Extract to produce monodisperse ZnO NPs with a size range of 78 to 145 nm, and Yusof et al., who applied the bacterial extract of Lactobacillus Plantarum to synthesize ZnO NPs with a size of 292.6 ± 83.2 nm. These results underscore the notion that the characteristics of the produced nanoparticles are highly dependent on the source utilized, as outlined in [ 36 ][ 37 ]. 3.3.3. Zeta potential analysis The primary objective of the utilization of zeta potential is to characterize the surface charge potential of biosynthesized nanoparticles, which serves as an important factor indicative of their stability in an aqueous solution. Electrostatically stable nanoparticles typically possess a zeta potential value exceeding + 30 mV or falling below − 30 mV. In this regard, (Fig. 1 (e)) demonstrates the obtained zeta potential graph of biosynthesized nanoparticles. Notably, the zeta potential measurement of the produced nanoparticles was found to be -5.60 mV, as depicted in (Fig. 1 (e)). Such a relatively low value implies the low stability of Zinc Oxide Nanoparticles (ZnO NPs) in an aqueous solution. It is pertinent to mention that deionized water served as a dispersant for the analysis conducted. 3.3.4. Raman Spectroscopy The Raman spectrum of the produced nanoparticles is presented in (Fig. 1 (f)), showing distinct peaks observed at 341.7, 438, 638.3, 708.5, 807.2, and 999.5 cm − 1 . Among these observed peaks, those appearing at 341.7 and 438 cm − 1 are attributed to the ZnO NPs [ 38 ]. 3.3.5. FTIR spectroscopy The Fourier Transform Infrared (FTIR) test that provides information on the chemical bonding between Zinc (Zn) and Oxygen (O), as elaborated in reference [ 38 ]. A comparison of the FTIR spectra of the biosynthesized ZnO NPs with those obtained from bacterial extract clearly reveals specific absorption peaks in both cases, as shown in (Fig. 1 (g)). However, there are several modifications concerning the location, shape, and number of peaks in the nanoparticle samples relative to those observed in the extract. For instance, the peaks within the range of 1400 to 1600 cm − 1 correspond to the stretching mode of the acetate group (-COOH). Additionally, the emergence of peaks around 3400 cm-1 suggests the presence of hydroxyl groups (-OH), as outlined in [ 39 ]. Furthermore, according to the study cited in, [ 38 ] the peak located at 457 cm − 1 and 545 cm − 1 can be attributed to the ZnO NPs. 3.3.6. Field Emission Scanning Electron Microscope (FESEM) In order to investigate the surface morphology, topography, and size of the biosynthesized nanoparticles, this study is carried out. The analysis revealed that the particles exhibited an average size range of 130 nm, with hexagonal shapes visible in (Fig. 2 (a-d)). However, some particles appeared to be aggregated, forming larger structures, which could potentially be attributed to solvent evaporation [ 35 ]. These findings provide valuable insight into the physical characteristics of the synthesized nanoparticles, which have significant implications for their potential applications in various fields. 3.3.7. Energy Dispersive X-Ray (EDX) In order to determine the composition of the synthesized nanoparticles (Fig. 2 (e)), EDX analysis was employed as described in reference [ 40 ]. The results revealed the presence of both zinc and oxygen in the structure, indicating that ZnO nanoparticles had been produced, as depicted in (Fig. 2 (f)). Specifically, the sharp peak observed at 1 Kev suggests that zinc is the major element in the composition, representing 52.57% of the total content. Additionally, the resulting peak at 0.5 Kev corresponds to oxygen, which accounts for 36.62% of the overall composition, as shown in (Fig. (2f)). These findings provide valuable information regarding the chemical makeup of the synthesized nanoparticles, which has important implications for their potential applications in various fields. 3.4. Investigation of different bacterial ZnO NPs production methods There are two types of intracellular and extracellular methods for the production of nanoparticles by bacterial cells [ 41 ]. In order to produce nanoparticles extracellularly, the bacterial supernatant or the cultured bacteria can be used. The production of nanoparticles by these methods relies on extracellular enzymes and active biomolecules. However, intracellular methods like bacterial Biomass depend on intracellular biomolecules and enzymes. In this process, metal ions are absorbed and then reduced to metallic nanoparticles inside the bacterial cells [ 42 ]. So, the cell should be ruptured and purified to get access to the biosynthesized nanoparticles. Intracellular nanoparticles are usually smaller than extracellular ones. One of our goals for doing this study is to use the process of nanoparticle production to bioremediate zinc compounds in natural habitats, like in the study of Ando et al. In their study, the dead biomass of Rhodotorula mucilaginosa was used to remove copper pollutants from the wastewater and produce copper nanoparticles [ 43 ]. Moreover, our second goal was the production of nanoparticles, the same as the study of Yusof et al. using bacterial supernatant and biomass to produce ZnO NPs (Fig. 3 (a)). They compare these two methods of nanoparticle production and found the UV-vis peak value of ZnO nanoparticles produced by cell biomass was lower than the one produced by bacterial supernatant [ 36 ]. The same result was achieved in our study. As shown in (Fig. 3 (b)), the UV-vis peak value of nanoparticles produced with bacterial biomass is lower than the rest of the methods, including the bacterial supernatant. So, using N1.5.2 biomass for the production of nanoparticles does not seem to be a suitable method. The cultured bacteria were used in the study of Kundu et al. to directly reduce zinc salt ions and produce hexagonal nanoparticles with a size range of 100–120 nm, and UV-vis peak at 340nm [ 21 ]. In our research, we used the same protocol to produce ZnO nanoparticles. The UV-vis peak of produced nanoparticles was located at 371 nm. The study of Yusof et al proved that the nanoparticles produced by different methods have different morphologies and characteristics. For example, nanoparticles produced by cell-free supernatant had flower shapes, while those produced by cell biomass had irregular- shapes. This is because the active biomolecules and enzymes of supernatant are different from those present in biomass. They also proved that other features of nanoparticles can be affected by the method of production, like the wavelength of the UV-vis peak in the spectrum[ 36 ]. In our study, the shape and wavelength of peaks contributed to the method of nanoparticles produced by N1.5.2 . This can be attributed to different available biomolecules and enzymes that are involved in the process of nanoparticle production. N1.5.2 has the potential to produce ZnO NPs from all of these routes. All these methods can be used for nanoparticle production in the natural environment, each of which produces a nanoparticle with a certain shape and special properties[ 36 ]. 3.5. Bacterial identification The morphological and physiological characteristics of pure colonies were studied by Gram and spore staining. The selected isolate is gram-positive (Fig. 4 (a)), motile, bacilli, and white in Nutrient agar (Fig. 1 (b)). It has the ability to produce spores in an undesirable situation (Fig. 4 (b)). The result was deposited at GeneBank (Acc. No. OM185305.1). the Blast showed that this sequence had the highest level of similarity (100%) with Bacillus cereus strain L14 (Acc. No. LN890010.1). then the phylogenetic tree of life is depicted in (Fig. 4 (c)). 3.6. Microplastics degradation Zinc oxide nanoparticles (ZnO NPs) have been identified as a promising tool for various environmental applications, including bioremediation and photocatalysis. These nanoparticles are activated by exposure to ultraviolet (UV) radiation, resulting in the generation of highly reactive hydroxyl and superoxide radicals. This photochemical process has been found to be effective in the degradation of pollutants such as microplastics, through mechanisms such as branching, chain breaking, and oxidation. In light of their demonstrated efficacy, ZnO NPs are increasingly being explored as a feasible solution for the mitigation of environmental pollution. The potential of these nanoparticles extends beyond environmental remediation, as they have also been shown to possess antimicrobial properties, thus presenting opportunities for application in the fields of disinfection and sterilization.[ 44 ]. The findings presented in (Fig. 5 (a-c)) indicate that an increase in cracks and spots on the surface occurred over time, providing evidence of the effective functioning of the photocatalyst. These observations are consistent with prior research conducted by Razali et al., who employed SEM micrographs to demonstrate the photocatalytic activity of ZnO NPs via the amplification of both the number and size of cracks on the surface of microplastics [ 26 ]. However, it is notable that some cracks were present on the control sample surface; these may be attributed to defects introduced during the manufacturing process of the plastics. Such manufacturing flaws and weak bonds can act as initiation points for the oxidation process, ultimately leading to the degradation of microplastics and the initiation of the photocatalytic reaction [ 44 ]. 3.7. Studying chemical changes by FTIR Photocatalysts have the capacity to break down molecular bonds and modify functional groups, which can be readily detected through Fourier Transform Infrared Spectroscopy (FTIR). (Fig. 6 ) displays several modifications in chemical structure and functional groups before and after treatment. A comparison with the control revealed various modifications that peaked at 1500–2000 cm-1, which corresponded to the stretching of aromatic (C-H) and (C-C) vibrations. Additionally, absorption peaks were observed at 1600.8 cm − 1 , 1492.7 cm − 1 , and 1452.2 cm − 1 , indicating aromatic (C = C) stretching vibration. The presence of (C-H) was also confirmed by the absorption peaks at 756.0 and 698.2 cm − 1 [ 44 ]. It is worth noting that the study conducted by Bandyopadhyay and Basak in 2007 reported similar alterations at 3300–3500 cm − 1 and 1706 cm − 1 , corresponding to (O-H) and (C = O) vibrations [ 45 ]. These observations demonstrate that FTIR is a powerful technique for characterizing photocatalytic reactions and provides insight into the specific chemical changes brought about by photochemical processes. 4. Conclusion In conclusion, the production of nanoparticles through the isolation of bacteria from the Caspian Sea presents a promising opportunity for bioremediation of natural environments that are contaminated with heavy metal pollutants and microplastics. The confirmation of ZnO nanoparticle production was achieved through the appearance of a peak in the UV-vis spectrum. The resulting nanoparticles were found to exhibit a hexagonal shape and possess a crystalline structure in the zincite phase, with a size distribution ranging from 171–216 nm when dispersed in water. Additionally, the surface characteristics of the ZnO nanoparticles were analyzed using FTIR spectroscopy, which revealed the presence of carboxyl and hydroxyl groups. Bacterial extract of N1.5.2 was identified as a highly effective protocol for the production of nanoparticles, as evidenced by a greater UV-vis peak compared to other methods. These produced nanoparticles also demonstrated photocatalytic activity, enabling them to degrade microplastics over time. The degradation process was further investigated through SEM analysis, which revealed the formation of cracks and holes on the surface of the nanoparticles. Finally, changes in the surface functional groups of the nanoparticles were tracked using FTIR, highlighting the chemical transformations that occurred during the degradation process. Together, these findings underscore the potential of bacterial nanoparticle production as an effective and environmentally sustainable approach to bioremediation. Further research is warranted to explore the full range of applications for these nanoparticles in environmental remediation and beyond. Declarations Ethics declarations Ethical approval and consent to participate Not applicable Consent to publication Not applicable Competing interests The authors declare no competing interests Acknowledgment The authors gratefully acknowledge Biotechnology Department, Alzahra University laboratory for support this study. Funding Not Applicable Author information Authors and Affiliations Department of Biotechnology, Faculty of Biological Science, Alzahra University, Tehran, Iran Motahare Haghighatjoo, Parichehr Hanachi Department of Microbiology, Faculty of Biological Science, Alzahra University, Tehran, Iran. Ali Mohammadi Department of Analytical Chemistry, Faculty of Chemistry, Alzahra University, Tehran, Iran. Omran Moradlou Contributions Motahare Haghiaghatjoo was the first author and she performed the research, collected and analyzed the results. Parichehr Hanachi supervised and designed the research. Ali Mohammadi, and Omran Moradlou advised the research. All authors contributed to the revision of the manuscript. All the authors approved the final version of the manuscript. Corresponding Author Correspondence to Parichehr Hanachi Department of Biotechnology, Faculty of Biological Science, Alzahra University, Tehran, Iran [email protected] References P. B. Tchounwou, C. G. Yedjou, A. K. Patlolla, and D. J. Sutton, “Molecular, clinical and environmental toxicicology Volume 3: Environmental Toxicology,” Mol. Clin. Environ. Toxicol. , vol. 101, pp. 133–164, 2012, doi: 10.1007/978-3-7643-8340-4. J. Briffa, E. Sinagra, and R. Blundell, “Heavy metal pollution in the environment and their toxicological effects on humans,” Heliyon , vol. 6, no. 9, p. e04691, 2020, doi: 10.1016/j.heliyon.2020.e04691. S. Karbalaei, P. Hanachi, G. Rafiee, P. Seifori, and T. R. ‎Walker, “Toxicity of polystyrene microplastics on juvenile Oncorhynchus mykiss (rainbow trout) after individual and combined exposure with chlorpyrifos‎,” J. Hazard. Mater. , vol. 403, no. June 2020, 2021, doi: 10.1016/j.jhazmat.2020.123980. J. P. McClung and A. G. Scrimgeour, “Zinc: An essential trace element with potential benefits to soldiers,” Mil. Med. , vol. 170, no. 12, pp. 1048–1052, 2005, doi: 10.7205/MILMED.170.12.1048. S. Hussain et al. , “Zinc Essentiality, Toxicity, and Its Bacterial Bioremediation: A Comprehensive Insight,” Front. Microbiol. , vol. 13, no. May, 2022, doi: 10.3389/fmicb.2022.900740. H. Kaur and N. Garg, “Zinc toxicity in plants: a review,” Planta , vol. 253, no. 6, pp. 1–28, 2021, doi: 10.1007/s00425-021-03642-z. A. Selvi et al. , “Integrated remediation processes toward heavy metal removal/recovery from various environments-A review,” Front. Environ. Sci. , vol. 7, no. May, 2019, doi: 10.3389/fenvs.2019.00066. M. Kumar, A. Seth, A. K. Singh, M. S. Rajput, and M. Sikandar, “Remediation strategies for heavy metals contaminated ecosystem: A review,” Environ. Sustain. Indic. , vol. 12, no. December 2020, p. 100155, 2021, doi: 10.1016/j.indic.2021.100155. I. Michalak, K. Chojnacka, and A. Witek-Krowiak, “State of the art for the biosorption process - A review,” Appl. Biochem. Biotechnol. , vol. 170, no. 6, pp. 1389–1416, 2013, doi: 10.1007/s12010-013-0269-0. R. T. Kapoor, M. R. Salvadori, M. Rafatullah, M. R. Siddiqui, M. A. Khan, and S. A. Alshareef, “Exploration of Microbial Factories for Synthesis of Nanoparticles – A Sustainable Approach for Bioremediation of Environmental Contaminants,” Front. Microbiol. , vol. 12, no. June, 2021, doi: 10.3389/fmicb.2021.658294. S. Ghosh, R. Ahmad, M. Zeyaullah, and S. K. Khare, “Microbial Nano-Factories: Synthesis and Biomedical Applications,” Front. Chem. , vol. 9, no. April, 2021, doi: 10.3389/fchem.2021.626834. H. Yu, Y. Zhang, W. Tan, and Z. Zhang, “Microplastics as an Emerging Environmental Pollutant in Agricultural Soils: Effects on Ecosystems and Human Health,” Front. Environ. Sci. , vol. 10, no. March, pp. 1–18, 2022, doi: 10.3389/fenvs.2022.855292. H. Park and B. S. Park, “Review of microplastic distribution, toxicity, analysis methods, and removal technologies,” Water (Switzerland) , vol. 13, no. 19, 2021, doi: 10.3390/w13192736. A. Amobonye, P. Bhagwat, S. Raveendran, S. Singh, and S. Pillai, “Environmental Impacts of Microplastics and Nanoplastics: A Current Overview,” Front. Microbiol. , vol. 12, no. December, 2021, doi: 10.3389/fmicb.2021.768297. R. Ahmed, A. K. Hamid, S. A. Krebsbach, J. He, and D. Wang, “Critical review of microplastics removal from the environment,” Chemosphere , vol. 293, no. January, p. 133557, 2022, doi: 10.1016/j.chemosphere.2022.133557. M. T. Sturm, H. Horn, and K. Schuhen, “Removal of microplastics from waters through agglomeration-fixation using organosilanes—effects of polymer types, water composition and temperature,” Water (Switzerland) , vol. 13, no. 5, pp. 1–15, 2021, doi: 10.3390/w13050675. M. B. Tahir, M. Sohaib, M. Sagir, and M. Rafique, “Role of Nanotechnology in Photocatalysis,” Encycl. Smart Mater. , no. January, pp. 578–589, 2021, doi: 10.1016/B978-0-12-815732-9.00006-1. M. Faezi, S. Javanbakht, F. A. Mohseni, and F. Elham, “International Journal of Molecular and Clinical Microbiology Isolation and characterization of bacterial strains from shoreline waters of Caspian Sea with novel antibacterial activity,” vol. 8, no. 2, pp. 982–992, 2018. A. Makhdoumi, “Bacterial diversity in south coast of Caspian Sea: Culture-dependent and culture-independent survey,” Casp. J. Environ. Sci. , vol. 16, no. 3, pp. 259–269, 2018. R. Esmail, A. Afshar, M. Morteza, A. Abolfazl, and E. Akhondi, “Synthesis of silver nanoparticles with high efficiency and stability by culture supernatant of Bacillus ROM6 isolated from Zarshouran gold mine and evaluating its antibacterial effects,” BMC Microbiol. , vol. 22, no. 1, pp. 1–10, 2022, doi: 10.1186/s12866-022-02490-5. D. Kundu, C. Hazra, A. Chatterjee, A. Chaudhari, and S. Mishra, “Extracellular biosynthesis of zinc oxide nanoparticles using Rhodococcus pyridinivorans NT2: Multifunctional textile finishing, biosafety evaluation and in vitro drug delivery in colon carcinoma,” J. Photochem. Photobiol. B Biol. , vol. 140, pp. 194–204, 2014, doi: 10.1016/j.jphotobiol.2014.08.001. M. Yusof et al. , “Sustainable microbial cell nanofactory for zinc oxide nanoparticles production by zinc ‑ tolerant probiotic Lactobacillus plantarum strain TA4,” Microb. Cell Fact. , pp. 1–17, 2020, doi: 10.1186/s12934-020-1279-6. B. Balraj et al. , “Synthesis and characterization of Zinc Oxide nanoparticles using marine Streptomyces sp. with its investigations on anticancer and antibacterial activity,” Res. Chem. Intermed. , vol. 43, no. 4, pp. 2367–2376, 2017, doi: 10.1007/s11164-016-2766-6. T. Tiwari, Hoondal, Laboratory_Techniques_In_Microbiology__amp__Biotechnology.pdf . 2009. R. Rohban, M. A. Amoozegar, and A. Ventosa, “Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran,” J. Ind. Microbiol. Biotechnol. , vol. 36, no. 3, pp. 333–340, 2009, doi: 10.1007/s10295-008-0500-0. N. A. RAZALI, A. N. , WAN RAFIZAH WAN ABDULLAH, and M. ZIKIR, “EFFECT OF THERMO-PHOTOCATALYTIC PROCESS USING ZINC OXIDE ON DEGRADATION OF MACRO/MICRO-PLASTIC IN AQUEOUS ENVIRONMENT,” J. Sustain. Sci. Manag. , vol. 15, pp. 1–14, 2020. M. L. Sogin et al. , “Microbial diversity in the deep sea and the underexplored ‘rare biosphere,’” Proc. Natl. Acad. Sci. U. S. A. , vol. 103, no. 32, pp. 12115–12120, 2006, doi: 10.1073/pnas.0605127103. P. Velmurugan, C. K. Venil, A. Veera Ravi, and L. Dufossé, “Marine Bacteria Is the Cell Factory to Produce Bioactive Pigments: A Prospective Pigment Source in the Ocean,” Front. Sustain. Food Syst. , vol. 4, no. November, pp. 1–7, 2020, doi: 10.3389/fsufs.2020.589655. N. Mahmoudi et al. , “Microbial community composition and diversity in Caspian Sea sediments,” FEMS Microbiol. Ecol. , vol. 91, no. 1, pp. 1–11, 2015, doi: 10.1093/femsec/fiu013. J. Chen, J. Li, H. Zhang, W. Shi, and Y. Liu, “Bacterial heavy-metal and antibiotic resistance genes in a copper tailing dam area in northern China,” Front. Microbiol. , vol. 10, no. AUG, pp. 1–12, 2019, doi: 10.3389/fmicb.2019.01916. S. Iravani and R. S. Varma, “Bacteria in Heavy Metal Remediation and Nanoparticle Biosynthesis,” ACS Sustain. Chem. Eng. , vol. 8, no. 14, pp. 5395–5409, 2020, doi: 10.1021/acssuschemeng.0c00292. V. Pande, S. C. Pandey, D. Sati, P. Bhatt, and M. Samant, “Microbial Interventions in Bioremediation of Heavy Metal Contaminants in Agroecosystem,” Front. Microbiol. , vol. 13, no. May, pp. 1–16, 2022, doi: 10.3389/fmicb.2022.824084. O. Sambalova et al. , “Carboxylate Functional Groups Mediate Interaction with Silver Nanoparticles in Biofilm Matrix,” ACS Omega , vol. 3, no. 1, pp. 724–733, 2017, doi: 10.1021/acsomega.7b00982. P. Hanachi, S. Karbalaei, T. R. Walker, M. Cole, and S. V. Hosseini, “Abundance and properties of microplastics found in commercial fish meal and cultured common carp (Cyprinus carpio),” Environ. Sci. Pollut. Res. , vol. 26, no. 23, pp. 23777–23787, 2019, doi: 10.1007/s11356-019-05637-6. Z. Gharari, P. Hanachi, and T. R. Walker, “Green synthesized Ag-nanoparticles using Scutellaria multicaulis stem extract and their selective cytotoxicity against breast cancer,” Anal. Biochem. , p. 114786, 2022, doi: 10.1016/j.ab.2022.114786. H. Mohd Yusof, N. A. Abdul Rahman, R. Mohamad, U. H. Zaidan, and A. A. Samsudin, “Biosynthesis of zinc oxide nanoparticles by cell-biomass and supernatant of Lactobacillus plantarum TA4 and its antibacterial and biocompatibility properties,” Sci. Rep. , vol. 10, no. 1, pp. 1–13, 2020, doi: 10.1038/s41598-020-76402-w. S. Yedurkar, C. Maurya, and P. Mahanwar, “Biosynthesis of Zinc Oxide Nanoparticles Using Ixora Coccinea Leaf Extract—A Green Approach,” Open J. Synth. Theory Appl. , vol. 05, no. 01, pp. 1–14, 2016, doi: 10.4236/ojsta.2016.51001. K. Handore et al. , “Novel green route of synthesis of ZnO nanoparticles by using natural biodegradable polymer and its application as a catalyst for oxidation of aldehydes,” J. Macromol. Sci. Part A Pure Appl. Chem. , vol. 51, no. 12, pp. 941–947, 2014, doi: 10.1080/10601325.2014.967078. A. Sharma, B. P. Singh, S. Dhar, A. Gondorf, and M. Spasova, “Effect of surface groups on the luminescence property of ZnO nanoparticles synthesized by sol-gel route,” Surf. Sci. , vol. 606, no. 3–4, 2012, doi: 10.1016/j.susc.2011.09.006. L. F. A. Anand Raj and E. Jayalakshmy, “Biosynthesis and characterization of zinc oxide nanoparticles using root extract of Zingiber officinale,” Orient. J. Chem. , vol. 31, no. 1, pp. 51–56, 2015, doi: 10.13005/ojc/310105. N. Gürsoy, B. Yilmaz Öztürk, and İ. Dağ, “Synthesis of intracellular and extracellular gold nanoparticles with a green machine and its antifungal activity,” Turkish J. Biol. , vol. 45, no. 2, pp. 196–213, 2021, doi: 10.3906/biy-2010-64. S. Ghosh, R. Ahmad, K. Banerjee, and M. F. Alajmi, “Mechanistic Aspects of Microbe-Mediated Nanoparticle Synthesis,” vol. 12, no. May, pp. 1–12, 2021, doi: 10.3389/fmicb.2021.638068. A. Ando, A. Oller, and B. Corre, “Intracellular Biosynthesis and Removal of Copper Nanoparticles by Dead Biomass of Yeast Isolated from the Wastewater of a Mine in the Brazilian Amazonia,” vol. 9, no. 1, pp. 1–9, 2014, doi: 10.1371/journal.pone.0087968. T. S. Tofa, K. L. Kunjali, S. Paul, and J. Dutta, “Visible light photocatalytic degradation of microplastic residues with zinc oxide nanorods,” Environ. Chem. Lett. , vol. 17, no. 3, pp. 1341–1346, 2019, doi: 10.1007/s10311-019-00859-z. A. Bandyopadhyay and G. C. Basak, “Studies on photocatalytic degradation of polystyrene,” no. March 2007, 2016, doi: 10.1179/174328407X158640. Footnotes Staphylococcus aureus Escherichia coli Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2851887","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":208138467,"identity":"463a84f8-34e3-429c-badb-37c1d3e7505b","order_by":0,"name":"Motahare Haghighatjoo","email":"","orcid":"","institution":"Alzahra University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Motahare","middleName":"","lastName":"Haghighatjoo","suffix":""},{"id":208138469,"identity":"0a513c6c-431d-4eb7-8fb5-dc23b0098862","order_by":1,"name":"Parichehr Hanachi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYFACHjCZwMbewHiAgeEAVNSAGC08BxhI1MIgkYCsBQ8wZ+A9+Lmgxi6PT/Lxg4M/au7k8zcwP/zAUHAPpxbLBr5k6RnHkovZpNMMDkgce2Y54wCbsQSDQTFOLQYHeAykedgOJLZJJxgcMGA7bAB0mxlQPAGfFuPfPP+AWiSPfziQ8O+wgfwB9m+EtJhJ87YBtUjwGBw42HbYACSCV4tlM4+ZNW9fcmIbT07Bwca+wwaGh3mKJRLwaDFn7zG+zfPNLnF++/GND398O2wgd7x944cPf/A4jBlDCCSCWwOBWB4Fo2AUjIJRAAYAOZJS9gTW/UUAAAAASUVORK5CYII=","orcid":"","institution":"Alzahra University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Parichehr","middleName":"","lastName":"Hanachi","suffix":""},{"id":208138471,"identity":"1b44233c-436c-465b-abb8-d27dfdc57542","order_by":2,"name":"Ali Mohammadi","email":"","orcid":"","institution":"Alzahra University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Mohammadi","suffix":""},{"id":208138473,"identity":"9accde8c-06fd-4910-b274-cc5bd0a45f87","order_by":3,"name":"Omran Moradlou","email":"","orcid":"","institution":"Alzahra University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Omran","middleName":"","lastName":"Moradlou","suffix":""}],"badges":[],"createdAt":"2023-04-23 18:14:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2851887/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2851887/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":38407108,"identity":"74575666-ec7a-4e49-aa92-ad2c04d9acac","added_by":"auto","created_at":"2023-06-12 14:52:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1267648,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV-vis spectrum of produced nanoparticles by four isolates. A sharp peak was observed at 360nm related to the spectrum of biosynthesized nanoparticles of N1.5.2 (these spectra are depicted by Excel); (b) N1.5.2 on Nutrient agar; (c) XRD of ZnO NPs (it is depicted by Excel); (d) Size distribution analysis; (e) Zeta potential spectrum analysis; (f) Raman spectrum of ZnO nanoparticles (it is depicted by Origin); (g) FTIR spectrum, the spectra related to biosynthesized nanoparticles is depicted by a black solid line, and the spectra of bacterial extract indicated by a dotted line (these are depicted by Spectragryph).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2851887/v1/189eb91fbe55224b6b9c51b0.png"},{"id":38407109,"identity":"db15f477-ff37-4b5a-ad61-6420b5f6404b","added_by":"auto","created_at":"2023-06-12 14:52:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2031267,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fe-SEM of ZnO nanoparticles ×5000; (b) Fe-SEM of ZnO nanoparticles ×30000; (c) FE-SEM of ZnO nanoparticles ×60000; (d) FE-SEM of ZnO nanoparticles ×100000 (e)EDX spectrum of biosynthesized zinc oxide nanoparticles. (f) The powder of biosynthesized nanoparticles\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2851887/v1/dc1088c296f8302824d52ad8.png"},{"id":38408509,"identity":"83711f83-3613-4d15-83a1-fbf282acb909","added_by":"auto","created_at":"2023-06-12 15:00:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":650868,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent methods of ZnO nanoparticle production. (a) Bacterial Supernatant; the right vessel includes the produced nanoparticles from N.1.5.2 supernatant and ZnSO4.H2O, 0.1M. The positive and negative controls were supernatant and ZnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO solution. Bacterial Extract; in this method, the cellular extract of \u003cem\u003eN1.5.2\u003c/em\u003e was used to produce ZnO NPs from zinc salt solution (ZnSO4.H2O, 0.1M). The appearance of white precipitation or turbidity is the first sign of ZnO NPs production. Bacterial Extract and zinc salt solution (ZnSO4.H2O, 0.1M) were used as positive control and negative control, respectively. Bacterial Biomass; the biomass of \u003cem\u003eN1.5.2\u003c/em\u003e was used to produce ZnO NPs. The bacterial biomass was used as a positive control and the salt solution was used as a negative control. The cultured Bacteria; the cultured N1.5.2 was applied to produce ZnO from a zinc salt solution. The cultured N1.5.2 and salt solution were used as positive and negative controls. (b) The UV-vis spectra of produced nanoparticles (they were depicted by Excel). The bacterial extract showed a higher peak than the rest.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2851887/v1/9d6dfcbfe362ea71334f78a4.png"},{"id":38408511,"identity":"1f719c08-112d-4dfe-af5c-34738ab21fa6","added_by":"auto","created_at":"2023-06-12 15:00:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":823504,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of N.1.5.2 isolate. (a) The result of Gram stain under the light microscope (×100 X); Gram-positive bacilli are clearly obvious. (b) The result of spore stain under the light microscope (×100 X); green spores after staining indicate the capability of bacteria to produce this structure. (c) The phylogenetic tree depicted in Mega 3.1 by the Neighbor-joining method.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2851887/v1/692ab93467495a28d0a434dc.png"},{"id":38407117,"identity":"53167236-e1b7-479e-89b5-a05fc0f83f6e","added_by":"auto","created_at":"2023-06-12 14:52:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2954726,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM micrograph of polystyrene microplastics after treatment of 48h and 96h by using 1-3 g/l ZnO.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2851887/v1/7a1a4163446302f15b35808e.png"},{"id":38407112,"identity":"d014aae2-528e-42d7-a9bf-83197166ab51","added_by":"auto","created_at":"2023-06-12 14:52:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":532695,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of polystyrene microplastics after treatment of 48h and 96h by using 1-3 g/l ZnO. (they are depicted by Spectragryph)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2851887/v1/8b302c7df3ae86d3ab569717.png"},{"id":57006053,"identity":"4e804b47-d85b-4ae9-88e8-a821ce1ed0cb","added_by":"auto","created_at":"2024-05-23 10:04:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13409312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2851887/v1/440c47b1-6197-4851-9926-c13e2a045b37.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioremediation of zinc metals and microplastics by biosynthesizing zinc oxide nanoparticles from isolated bacteria of the Caspian Sea, Iran","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe proliferation of technology and industry has emerged as a significant factor contributing to the escalation of environmental pollution, which has resulted in irrevocable and detrimental consequences. For instance, heavy metals are highly toxic to human and other organisms. Their toxicity highly depend on their concentration and doses[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The accumulation of these pollutants in the environment is the result of some human activities, like mining, smelting, and foundries [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Take microplastics as another example. These tiny plastic particles are the main culprit for the health issues of human and other organisms [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHeavy metals are characterized by high densities, atomic weight or atomic numbers, and are highly toxic to humans and other organisms. Their toxicity levels depend on their concentration and doses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Some of them, like zinc, are required for body functions. They are associated with several enzymes as a cofactor through processes like protein synthesis or DNA replication [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, excess levels of them in the environment might be toxic [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For example, the normal concentration of zinc in the soil that is essential for crops is between 30 to 200 \u0026micro;g/g [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Excess levels of zinc as a result of human activities may alter soil microbial communities, consequently affecting the growth of crops. Moreover, the extra amount of this element in the human body may cause gastrointestinal distress with symptoms like cramps, diarrhea, nausea, and vomiting [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. So, the high concentration of heavy metals is considered a type of environmental pollution. Since zinc and other heavy metal pollutants tend to aggregate in different environmental matrices, many researchers focus on developing different remediation methods. Most industrial remediation techniques are classified into three groups of physical, chemical, and biological methods [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thermal desorption, vertification, encapsulation, vapor stripping soil washing, sorting, and Electrokinetic remediation are examples of physical methods. These methods are usually low-efficient. So, applying a replacement technique seems necessary. The chemical methods include different techniques like distillation, electrodialysis, chemical precipitation, and ion exchange. Chemical precipitation includes sulfide precipitation, hydroxide precipitation, chelating precipitation, Xanthate precipitation, and carbonate precipitation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, biological methods, as the alternative strategies for the remediation of heavy metals, are commonly used for their high efficiency, fast-growing, and easy controlling [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Their primary procedure is Biosorption, which is a physiochemical technique in which pollutants like metal ions from an aqueous environment bind onto the presented functional groups on the organism\u0026rsquo;s surfaces. This method is efficient and selective [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Some groups of bacteria have the ability to transform pollutants like metallic ions into less harmful structures such as metallic nanoparticles [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Nanoparticle production by microorganisms is a detoxifying mechanism to reduce the toxic metals to nanoparticles, which is considered a defense mechanism in bacteria against heavy metals stresses. In this way, the concentration of heavy metals like zinc will be reduced and metallic nanoparticles will be produced at the same time [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe production of nanoparticles by microorganisms might be intracellularly or extracellularly [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Usually, this process consists of three steps: capturing, reducing, and capping. In the beginning, the positive ions are captured on the surface of the cell. Then they can be reduced to metal nanoparticles by active molecules like enzymes through a redox reaction. Finally, the secreted biomolecules by microbes could act as capping or stabilizing agents. \u003cem\u003eSerratia ureilytica\u003c/em\u003e can synthesize ZnO nanoflowers which have antimicrobial properties against \u003cem\u003eS.aureus\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e and \u003cem\u003eE.coli\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e. Moreover, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and \u003cem\u003eAeromonas hydrophila\u003c/em\u003e are other examples of ZnO nanoparticle-producing bacteria [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The production of nanoparticles in this way would be efficient. By applying a proper protocol, the total expenses in nanoparticle production may be reduced to 1/10 in comparison to the chemical method. These nanoparticles show a high level of catalytic activity and they do not aggregate because of the capping agents. This method of nanoparticle production is efficient, safe, and environmentally friendly [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eMicroplastics, as another environmental pollutant, are less than 5mm in their size and are composed of different chemical components. They can be found in different ecological conditions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. They are classified into primary and secondary microplastics. The first group is produced in order to be applied in different industries like the production of face wash, toothpaste, and cosmetics. However, the second one is undesirably created through the degradation of plastics in natural habitats. The annual production of plastics and microplastics is soaring. Thus, in 2018, the global production of plastics exceeded 360\u0026nbsp;million metric tons [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Since the biological interactions of these particles are size-dependent, smaller particles are more active and dangerous than larger ones. Their chemical additives, like coloring agents and plasticizers, are toxic to living organisms. Moreover, they can easily be transferred along the food chain and piling up in cells and different tissues. So, disruption of cellular metabolisms and their physiological activities can cause inflammation and metabolic disorders [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, these toxic side effects have made scientists focus on different remediation techniques, including physical, chemical, and biological methods. The physical forces, like adsorption and filtration, are the main triggering factor for the collection of microplastics from an environment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although these techniques are commonly used, their protocols become too complicated in the case of small particles. So, they are highly costly and require a large amount of budget allocation in terms of energy and facilities [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, chemical remediation contributes with coagulants, like Fe- or Al- salts to remove these types of pollutants. Furthermore, one of the novel strategies to reduce the number of plastics in nature is the application of photocatalists. Most metal oxide nanoparticles have photocatalytic properties which can be used to degrade some organic and polymeric pollutants. In other words, a new microplastic remediation technique is photodegradation. UV radiation alters the electron structure of photocatalysts like zinc oxide nanoparticles by forming a pair of electrons and holes. This active structure can trigger a redox reaction that leads to the degradation of microplastics into smaller molecules like carbon dioxide and water. The oxidation and reduction reactions should take place simultaneously. In this case, semiconductors are the best options, since they have a moderate band gap which provides the possibility of oxidation and reduction reactions. In this case, the most suitable semiconductor are those that have band gaps between 1.5\u0026ndash;3.5 eV. Metal oxides have this feature [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we seek to find a solution to solve the challenge of microplastic pollutants and zinc compounds in the environment. The proposed scheme is to use the route of biological production of nanoparticles. In more detail, zinc compounds are absorbed by bacteria for the production of nanoparticles. Subsequently, the produced nanoparticles can be used as a photocatalyst to degrade microplastics.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cp\u003eZinc sulfate monohydrate (ZnSO4.H2O), Sodium Chloride (NaCl), Magnesium sulfate heptahydrate (MgSO4.7H2O), Magnesium chloride hexahydrate (MgCl2.6H2O), Calcium chloride dihydrate (CaCl2.2H2O), Potassium chloride (KCl), Ammonium sulfate (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\left(N{H}_{4}\\right)}_{2}S{O}_{4}\\)\u003c/span\u003e\u003c/span\u003e ), yeast extract, and agar were purchased from Sigma Aldrich.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sampling and bacterial isolation\u003c/h2\u003e \u003cp\u003eTen marine samples, comprising of both water and sediment, were meticulously collected at a depth of 50 cm from four distinct regions, namely Chamkhaleh, Amir Abad, Kiashahr, and Chaf, located in Gilan, Iran, during the period spanning from September 23, 2020, to September 26, 2020. To preserve the integrity of these samples, they were carefully transferred to sterile tubes and stored in close proximity to ice prior to being transported to the laboratory [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe marine samples were subjected to culture on three distinct mediums, namely SW agar, marine agar, and Sea Water salt medium. The SW agar, with constituents comprising of NaCl (9.1 g/l), MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO (0.75 g/l), MgCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO (0.17 g/l), CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO (0.22 g/l), KCl (0.13 g/l), \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\left(N{H}_{4}\\right)}_{2}S{O}_{4}\\)\u003c/span\u003e\u003c/span\u003e (0.86 g/l), FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO (0.009 g/l), peptone (10.0 g/l), yeast extract (2 g/l), and agar (15 g/l) ; had a pH of 8.2. On the other hand, the Sea Water salt medium was formulated using yeast extract (0.2 g/L), peptone (1.0 g/L), and agar (15 g/L), in combination with filtered Caspian Sea water, and had a pH of 8.2. Subsequent to an incubation period, 22 isolates were successfully obtained. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Studying the zinc tolerance of the bacterial isolates\u003c/h2\u003e \u003cp\u003eTo investigate the bacterial isolates' tolerance to zinc, a suspension of 0.5 McFarland of each isolate was inoculated into 3 ml of nutrient broth with varying concentrations of ZnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO (0.25 to 5 mg/mL). Following incubation, the growth and resistance of each culture were evaluated by separate culturing on nutrient agar[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The isolates demonstrating the highest level of resistance were identified as potential candidates for ZnO nanoparticle production.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Bacterial production of the zinc oxide nanoparticles\u003c/h2\u003e \u003cp\u003eThe utilization of a bacterial extract for the production of nanoparticles involved several precise steps. Initially, a suspension containing 2.0g of cell biomass (dry weight) in 100 ml of deionized water was incubated on a rotating incubator (150 rpm) for 48 hours. Following this period, the suspension underwent centrifugation (7000 rpm, 15 minutes), and the resulting supernatant was filtered. Subsequently, 20ml of ZnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO (0.10 M) was added to the supernatant, and the mixture was incubated at 30˚C on a shaker incubator (120 rpm) for 48 hours under dark conditions. The resulting solution was then utilized with positive and negative controls, utilizing the cell extract and the salt solution, respectively [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Ultimately, after undergoing centrifugation (10000 rpm, 20 minutes), washing with distilled water and ethanol, the ZnO nanoparticles (ZnO Nps) were obtained.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. ZnO nanoparticles characterization\u003c/h2\u003e \u003cp\u003eThe biosynthesized ZnO nanoparticles (ZnO Nps) underwent a comprehensive evaluation process utilizing various analytical techniques. The UV-vis spectrophotometer (UV-2150; Unico, US) was employed to examine the nanoparticles in the range of 200\u0026ndash;600 nm with a resolution of 1nm. To further analyze the crystalline phases of the ZnO NPs, X-ray diffraction analysis (Rigaku) was conducted using Cu Kα radiation (λ\u0026thinsp;=\u0026thinsp;1.541874 A˚) within the range of 2Ѳ of 0\u0026ndash;80˚. Fourier-transform infrared (FTIR) spectroscopy (Bruker, Germany) was implemented to investigate the functional groups and bioactive molecules that played a significant role in the reduction, stabilization, or capping of the ZnO NPs, utilizing a wavenumber range of 400\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at a resolution of 4cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In addition, dynamic light scattering (DLS) (Malvern Instruments, UK) was utilized to identify the polydispersity index (PDI), hydrodynamic diameter, and zeta potential of the biosynthesized nanoparticles[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e][\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e][\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, the surface characteristics of the nanoparticles were analyzed by a field-emission scanning electron microscope (FESEM; S-4800, Hitachi, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Comparative study on different bacterial methods of ZnO NPs production\u003c/h2\u003e \u003cp\u003eIn this study, the production of ZnO NPs from four different protocols involving bacterial culture, extract, biomass, and supernatant were compared via UV-vis spectroscopy.\u003c/p\u003e \u003cp\u003eTo obtain NPs from bacterial biomass and supernatant, the overnight culture of N1.5.2 was centrifuged at \u0026times;5000, and the biomass and supernatant were collected. The biomass underwent washing with phosphate-buffered saline and was then suspended in 50 mL of Zn\u003csup\u003e2+\u003c/sup\u003e solution (0.5M). Followed by incubation at 30˚C and 150 rpm agitation. After centrifugation, the ZnO NPs were obtained via ultrasonic disruption. For the supernatant route, 100 mL of filtered supernatant was added to 100 mL of ZnSO4.H2O solution (0.1M) and incubated on a shaker incubator for 24 hours. The formation of white precipitation indicated the production of ZnO NPs in the solution [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To synthesize nanoparticles directly from bacterial culture, 20 mL of ZnSO4.H2O (0.1M) was added to the overnight culture and incubated at 30˚C with 120 rpm agitation for 72 hours. The final product was collected after centrifugation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSubsequently, the produced nanoparticles were collected, purified, and dried, after which UV-vis spectroscopy was utilized to evaluate and compare these different synthetic methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Bacterial identification\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Staining of bacteria\u003c/h2\u003e \u003cp\u003eIn order to investigate the morphological and physiological features of the chosen isolate, Gram and spore staining were performed using established protocols outlined in Laboratory Techniques In Microbiology And Biotechnology [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These techniques are widely recognized as key tools for characterizing microbial isolates and provide valuable insights into their cellular structure and metabolic properties.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. DNA extraction, PCR, and electrophoresis\u003c/h2\u003e \u003cp\u003eFor the amplification of the 16S-rRNA gene, genomic DNA was extracted using the boiling protocol, and the universal primers 8F (5\u0026acute;-AGAGTTTGATCCTGGCTCAG-3\u0026acute;) and 1492R (5\u0026acute;-CACGGATCCTACGGGTACCTTGTTACGACTT-3\u0026acute;) were utilized. To prepare the PCR reaction, a mixture with a total volume of 25 \u0026micro;L was prepared, including 1.25 \u0026micro;L each of forward and reverse primers, 0.5 \u0026micro;L of dNTP (10 mM), 2.5 \u0026micro;L of PCR buffer, 0.75 \u0026micro;L of MgCl2 (50 mM), 1 \u0026micro;L of template DNA, 1.25 \u0026micro;L of DMSO, 0.5 \u0026micro;L of SmartAq DNA polymerase (Cinnagen, Iran), and 16 \u0026micro;L of dH2O[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe PCR was conducted under the following conditions: an initial denaturation step at 95\u0026deg;C for 5 minutes, followed by 35 cycles of denaturation at 95\u0026deg;C for 45 seconds, annealing at 55\u0026deg;C for 1 minute, extension at 72\u0026deg;C for 1.5 minutes, and a final extension at 72\u0026deg;C for 10 minutes. To verify the results of the PCR analysis, gel electrophoresis was used to separate and visualize DNA fragments based on their size.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. Sequencing and phylogenic tree\u003c/h2\u003e \u003cp\u003eThe forward sequencing of the PCR product was carried out by Niagene Noor, Iran. Subsequently, the resulting sequence was subjected to analysis using BLAST, and the phylogenetic tree of life was constructed using MEGA11. To ensure proper documentation and accessibility, the obtained sequence (669 bp) was deposited in GeneBank under the accession number OM185305.1.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Photocatalytic studies\u003c/h2\u003e \u003cp\u003eThe photocatalytic activity of biosynthesized nanoparticles on polystyrene microplastics (1cm\u0026times;1cm) was investigated using a photoreactor equipped with a 6-watt UV-C Hg vapor lamp. The reaction was conducted for a period of 96 hours in a reaction vessel containing the biosynthesized ZnO nanoparticles and deionized water, with the vessel positioned at a distance of 10cm from the UV lamp.\u003c/p\u003e \u003cp\u003eTo prepare a suspension of ZnO NPs for microplastic degradation, a concentration range of 1\u0026ndash;3 mg/L in distilled water was utilized. In order to enhance the likelihood of effective nanoparticle-microplastic collisions, a magnetic stirrer was employed to circulate the contents of the reaction vessel. The reaction was initiated upon placement of the vessel under the UV lamp.\u003c/p\u003e \u003cp\u003eEvery 48 hours, 2 \u0026micro;l of the suspension were extracted for further analysis. The surface morphology of polystyrene microplastics and the variation of their surface functional groups were studied using scanning electron microscopy (GEMINI Ultra 55, Carl Zeiss, AG, Germany) and Fourier transform infrared spectroscopy over the range of 400 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eThe community of bacteria present in marine environments is characterized by its extensive diversity in terms of composition, genetic materials, and metabolisms. Of particular note is the fact that bacteria are responsible for 98% of primary production [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. As such, researchers have focused on exploring the potential applications of marine bacteria in both industrial and clinical settings, with the goal of harnessing their secondary metabolite production capabilities [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. One particularly promising source of bacterial diversity is the Caspian Sea, which is considered the world's largest inland body of water. This unique ecosystem contains a multitude of distinct bacterial communities [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], making it an attractive target for screening efforts aimed at identifying nanoparticle-producing bacteria. Through targeted sampling and culturing techniques, we have successfully isolated 22 distinct bacterial strains from this environment. These isolates represent a valuable resource for further study and exploration of potential biotechnological applications.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Studying the resistance of bacterial isolates against zinc salt\u003c/h2\u003e \u003cp\u003eEnvironmental stressors, such as the presence of toxic compounds or excessive concentrations of heavy metals, have been found to significantly impact bacterial communities. Chen et al. conducted research indicating that the presence of heavy metals, such as zinc compounds within bacterial habitats, triggers the expression of metal resistance genes (MRGs). This response may ultimately lead to the effective remediation of heavy metals from polluted areas[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, bacterial resistance to heavy metals like zinc is one of the main key factors for the bioremediation of a polluted area, and the production of metallic nanoparticles like ZnO by bacteria is not an exception to this rule [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In line with this research, 22 bacterial isolates were cultured in media containing varying concentrations of zinc, ranging from 0.25 to 5.0 mg/L. The goal of this approach was to identify the most resistant isolates capable of producing nanoparticles. Through this investigation, four highly resistant isolates were identified and subsequently utilized for nanoparticle production, namely: N0.5.2, N1.1, N1.5, and N1.5.2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. The production of zinc oxide nanoparticles\u003c/h2\u003e \u003cp\u003eBacteria, as eco-friendly catalysts, play an important role in the bioremediation of natural environments through redox reactions. Their bioactive molecules alter the ionic state of heavy metals which may affect their solubility and bioavailability which might lead to the alteration of their movements in the natural environment. Microbial active molecules and enzymes can reduce toxic metal ions like zinc salts to less toxic compounds like zinc nanoparticles [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This process of nanoparticle production can take place inside or outside of the cell [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, the bioactive molecules of bacterial extract converted the aqueous zinc ions to ZnO NPs. Based on Yousof et al. the first sign of ZnO NPs production is the appearance of white precipitation. Zinc sulfate monohydrate was added to the bacterial extract of four selected isolates (\u003cem\u003eN0.5.2\u003c/em\u003e, \u003cem\u003eN1.1\u003c/em\u003e, \u003cem\u003eN1.5\u003c/em\u003e, and \u003cem\u003eN1.5.2\u003c/em\u003e), just the Erlenmeyer containing the extract of N1.5.2 became turbid. Sambalova et al. produced Silver nanoparticles by using bacteria. They suggested carboxylate functional groups mediate this reaction [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn order to confirm the production of ZnO NPs, the obtained white precipitation was studied via UV-vis spectroscopy in the range of 300\u0026ndash;600 nm. Spectral analysis revealed a prominent peak at 360nm in the spectrum of the nanoparticles produced by the N1.5.2 bacterial isolate, thus providing further confirmation of successful ZnO NP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a)) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b)).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. ZnO NPs Characterization\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. X-Ray diffraction\u003c/h2\u003e \u003cp\u003eX-ray diffraction (XRD) analysis is a widely-used technique for confirming the phases of ZnO nanoparticles (NPs). The XRD pattern of ZnO NPs obtained in this study is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c), which indicates that the observed diffraction peaks at 31.84˚, 34.44˚, 36.28˚, 47.60˚, 56.66˚, 62.90˚, 68.06˚, and 72.62˚ can be characterized as (100), (002), (101), (012), (110), (013), (112), and (001) reflections, respectively.\u003c/p\u003e \u003cp\u003eBy comparing these results with the data from Match 3 software (database reference: COD-Inorg 2021.12.14), it was confirmed that the ZnO NPs exhibited a hexagonal phase with a Zincite structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c)). This finding provides further evidence to support the successful production of ZnO NPs using the bioactive molecules present in the bacterial extract from the N1.5.2 isolate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Particle size distribution by DLS\u003c/h2\u003e \u003cp\u003eDynamic Light Scattering (DLS) was employed to determine the size distribution profile and hydrodynamic diameter of Zinc Oxide Nanoparticles (ZnO NPs) in an aqueous solution, as reported by reference [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The study revealed that the resulting particles were dispersed in an aqueous medium with a size range of 171-262.6 nm. The Polydispersity Index (PDI) value of the biosynthesized nanoparticles was 0.444, with a Z-average of 232.5 nm, as presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(d)). Notably, the DLS analysis showed that the biosynthesized nanoparticles had medium dispersion in the aqueous solution, which contributed to their PDI value. It is pertinent to mention that the measured size obtained via DLS was larger than what was estimated by SEM. Such a difference can be attributed to the tendency of water molecules in the aqueous solution to surround the nanoparticles, forming hydrodynamic shells.\u003c/p\u003e \u003cp\u003eThe PDI is a characteristic feature of the homogeneity of nanoparticles, as referenced in [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This finding is distinct from previous research conducted by Yedurkar et al., using Ixora Coccinea Leaf Extract to produce monodisperse ZnO NPs with a size range of 78 to 145 nm, and Yusof et al., who applied the bacterial extract of Lactobacillus Plantarum to synthesize ZnO NPs with a size of 292.6\u0026thinsp;\u0026plusmn;\u0026thinsp;83.2 nm. These results underscore the notion that the characteristics of the produced nanoparticles are highly dependent on the source utilized, as outlined in [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e][\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3. Zeta potential analysis\u003c/h2\u003e \u003cp\u003eThe primary objective of the utilization of zeta potential is to characterize the surface charge potential of biosynthesized nanoparticles, which serves as an important factor indicative of their stability in an aqueous solution. Electrostatically stable nanoparticles typically possess a zeta potential value exceeding\u0026thinsp;+\u0026thinsp;30 mV or falling below \u0026minus;\u0026thinsp;30 mV. In this regard, (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(e)) demonstrates the obtained zeta potential graph of biosynthesized nanoparticles.\u003c/p\u003e \u003cp\u003eNotably, the zeta potential measurement of the produced nanoparticles was found to be -5.60 mV, as depicted in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(e)). Such a relatively low value implies the low stability of Zinc Oxide Nanoparticles (ZnO NPs) in an aqueous solution. It is pertinent to mention that deionized water served as a dispersant for the analysis conducted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4. Raman Spectroscopy\u003c/h2\u003e \u003cp\u003eThe Raman spectrum of the produced nanoparticles is presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(f)), showing distinct peaks observed at 341.7, 438, 638.3, 708.5, 807.2, and 999.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Among these observed peaks, those appearing at 341.7 and 438 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are attributed to the ZnO NPs [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.3.5. FTIR spectroscopy\u003c/h2\u003e \u003cp\u003eThe Fourier Transform Infrared (FTIR) test that provides information on the chemical bonding between Zinc (Zn) and Oxygen (O), as elaborated in reference [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. A comparison of the FTIR spectra of the biosynthesized ZnO NPs with those obtained from bacterial extract clearly reveals specific absorption peaks in both cases, as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(g)). However, there are several modifications concerning the location, shape, and number of peaks in the nanoparticle samples relative to those observed in the extract. For instance, the peaks within the range of 1400 to 1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to the stretching mode of the acetate group (-COOH). Additionally, the emergence of peaks around 3400 cm-1 suggests the presence of hydroxyl groups (-OH), as outlined in [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Furthermore, according to the study cited in, [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] the peak located at 457 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 545 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be attributed to the ZnO NPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.3.6. Field Emission Scanning Electron Microscope (FESEM)\u003c/h2\u003e \u003cp\u003eIn order to investigate the surface morphology, topography, and size of the biosynthesized nanoparticles, this study is carried out. The analysis revealed that the particles exhibited an average size range of 130 nm, with hexagonal shapes visible in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a-d)). However, some particles appeared to be aggregated, forming larger structures, which could potentially be attributed to solvent evaporation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These findings provide valuable insight into the physical characteristics of the synthesized nanoparticles, which have significant implications for their potential applications in various fields.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.3.7. Energy Dispersive X-Ray (EDX)\u003c/h2\u003e \u003cp\u003eIn order to determine the composition of the synthesized nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(e)), EDX analysis was employed as described in reference [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The results revealed the presence of both zinc and oxygen in the structure, indicating that ZnO nanoparticles had been produced, as depicted in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(f)). Specifically, the sharp peak observed at 1 Kev suggests that zinc is the major element in the composition, representing 52.57% of the total content. Additionally, the resulting peak at 0.5 Kev corresponds to oxygen, which accounts for 36.62% of the overall composition, as shown in\u003c/p\u003e \u003cp\u003e(Fig.\u0026nbsp;(2f)). These findings provide valuable information regarding the chemical makeup of the synthesized nanoparticles, which has important implications for their potential applications in various fields.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Investigation of different bacterial ZnO NPs production methods\u003c/h2\u003e \u003cp\u003eThere are two types of intracellular and extracellular methods for the production of nanoparticles by bacterial cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In order to produce nanoparticles extracellularly, the bacterial supernatant or the cultured bacteria can be used. The production of nanoparticles by these methods relies on extracellular enzymes and active biomolecules. However, intracellular methods like bacterial Biomass depend on intracellular biomolecules and enzymes. In this process, metal ions are absorbed and then reduced to metallic nanoparticles inside the bacterial cells [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. So, the cell should be ruptured and purified to get access to the biosynthesized nanoparticles. Intracellular nanoparticles are usually smaller than extracellular ones.\u003c/p\u003e \u003cp\u003eOne of our goals for doing this study is to use the process of nanoparticle production to bioremediate zinc compounds in natural habitats, like in the study of Ando et al. In their study, the dead biomass of \u003cem\u003eRhodotorula mucilaginosa\u003c/em\u003e was used to remove copper pollutants from the wastewater and produce copper nanoparticles [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Moreover, our second goal was the production of nanoparticles, the same as the study of Yusof et al. using bacterial supernatant and biomass to produce ZnO NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)). They compare these two methods of nanoparticle production and found the UV-vis peak value of ZnO nanoparticles produced by cell biomass was lower than the one produced by bacterial supernatant [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The same result was achieved in our study. As shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b)), the UV-vis peak value of nanoparticles produced with bacterial biomass is lower than the rest of the methods, including the bacterial supernatant. So, using \u003cem\u003eN1.5.2\u003c/em\u003e biomass for the production of nanoparticles does not seem to be a suitable method.\u003c/p\u003e \u003cp\u003eThe cultured bacteria were used in the study of Kundu et al. to directly reduce zinc salt ions and produce hexagonal nanoparticles with a size range of 100\u0026ndash;120 nm, and UV-vis peak at 340nm [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In our research, we used the same protocol to produce ZnO nanoparticles. The UV-vis peak of produced nanoparticles was located at 371 nm.\u003c/p\u003e \u003cp\u003eThe study of Yusof et al proved that the nanoparticles produced by different methods have different morphologies and characteristics. For example, nanoparticles produced by cell-free supernatant had flower shapes, while those produced by cell biomass had irregular- shapes. This is because the active biomolecules and enzymes of supernatant are different from those present in biomass. They also proved that other features of nanoparticles can be affected by the method of production, like the wavelength of the UV-vis peak in the spectrum[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In our study, the shape and wavelength of peaks contributed to the method of nanoparticles produced by \u003cem\u003eN1.5.2\u003c/em\u003e. This can be attributed to different available biomolecules and enzymes that are involved in the process of nanoparticle production.\u003c/p\u003e \u003cp\u003e \u003cem\u003eN1.5.2\u003c/em\u003e has the potential to produce ZnO NPs from all of these routes. All these methods can be used for nanoparticle production in the natural environment, each of which produces a nanoparticle with a certain shape and special properties[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Bacterial identification\u003c/h2\u003e \u003cp\u003eThe morphological and physiological characteristics of pure colonies were studied by Gram and spore staining. The selected isolate is gram-positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a)), motile, bacilli, and white in Nutrient agar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b)). It has the ability to produce spores in an undesirable situation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (b)).\u003c/p\u003e \u003cp\u003eThe result was deposited at GeneBank (Acc. No. OM185305.1). the Blast showed that this sequence had the highest level of similarity (100%) with \u003cem\u003eBacillus cereus strain L14\u003c/em\u003e (Acc. No. LN890010.1). then the phylogenetic tree of life is depicted in (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (c)).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Microplastics degradation\u003c/h2\u003e \u003cp\u003eZinc oxide nanoparticles (ZnO NPs) have been identified as a promising tool for various environmental applications, including bioremediation and photocatalysis. These nanoparticles are activated by exposure to ultraviolet (UV) radiation, resulting in the generation of highly reactive hydroxyl and superoxide radicals. This photochemical process has been found to be effective in the degradation of pollutants such as microplastics, through mechanisms such as branching, chain breaking, and oxidation. In light of their demonstrated efficacy, ZnO NPs are increasingly being explored as a feasible solution for the mitigation of environmental pollution. The potential of these nanoparticles extends beyond environmental remediation, as they have also been shown to possess antimicrobial properties, thus presenting opportunities for application in the fields of disinfection and sterilization.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (a-c)) indicate that an increase in cracks and spots on the surface occurred over time, providing evidence of the effective functioning of the photocatalyst. These observations are consistent with prior research conducted by Razali et al., who employed SEM micrographs to demonstrate the photocatalytic activity of ZnO NPs via the amplification of both the number and size of cracks on the surface of microplastics [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, it is notable that some cracks were present on the control sample surface; these may be attributed to defects introduced during the manufacturing process of the plastics. Such manufacturing flaws and weak bonds can act as initiation points for the oxidation process, ultimately leading to the degradation of microplastics and the initiation of the photocatalytic reaction [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Studying chemical changes by FTIR\u003c/h2\u003e \u003cp\u003ePhotocatalysts have the capacity to break down molecular bonds and modify functional groups, which can be readily detected through Fourier Transform Infrared Spectroscopy (FTIR). (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) displays several modifications in chemical structure and functional groups before and after treatment. A comparison with the control revealed various modifications that peaked at 1500\u0026ndash;2000 cm-1, which corresponded to the stretching of aromatic (C-H) and (C-C) vibrations. Additionally, absorption peaks were observed at 1600.8 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1492.7 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1452.2 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating aromatic (C\u0026thinsp;=\u0026thinsp;C) stretching vibration. The presence of (C-H) was also confirmed by the absorption peaks at 756.0 and 698.2 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It is worth noting that the study conducted by Bandyopadhyay and Basak in 2007 reported similar alterations at 3300\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1706 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to (O-H) and (C\u0026thinsp;=\u0026thinsp;O) vibrations [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. These observations demonstrate that FTIR is a powerful technique for characterizing photocatalytic reactions and provides insight into the specific chemical changes brought about by photochemical processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, the production of nanoparticles through the isolation of bacteria from the Caspian Sea presents a promising opportunity for bioremediation of natural environments that are contaminated with heavy metal pollutants and microplastics. The confirmation of ZnO nanoparticle production was achieved through the appearance of a peak in the UV-vis spectrum. The resulting nanoparticles were found to exhibit a hexagonal shape and possess a crystalline structure in the zincite phase, with a size distribution ranging from 171\u0026ndash;216 nm when dispersed in water. Additionally, the surface characteristics of the ZnO nanoparticles were analyzed using FTIR spectroscopy, which revealed the presence of carboxyl and hydroxyl groups.\u003c/p\u003e \u003cp\u003eBacterial extract of N1.5.2 was identified as a highly effective protocol for the production of nanoparticles, as evidenced by a greater UV-vis peak compared to other methods. These produced nanoparticles also demonstrated photocatalytic activity, enabling them to degrade microplastics over time. The degradation process was further investigated through SEM analysis, which revealed the formation of cracks and holes on the surface of the nanoparticles. Finally, changes in the surface functional groups of the nanoparticles were tracked using FTIR, highlighting the chemical transformations that occurred during the degradation process.\u003c/p\u003e \u003cp\u003eTogether, these findings underscore the potential of bacterial nanoparticle production as an effective and environmentally sustainable approach to bioremediation. Further research is warranted to explore the full range of applications for these nanoparticles in environmental remediation and beyond.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge Biotechnology Department, Alzahra University laboratory for support this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Biotechnology, Faculty of Biological Science, Alzahra University, Tehran, Iran\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Motahare Haghighatjoo, Parichehr Hanachi\u003c/p\u003e\n\u003cp\u003eDepartment of Microbiology, Faculty of Biological Science, Alzahra University, Tehran, Iran.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Ali Mohammadi\u003c/p\u003e\n\u003cp\u003eDepartment of Analytical Chemistry, Faculty of Chemistry, Alzahra University, Tehran, Iran.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Omran Moradlou\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMotahare Haghiaghatjoo was the first author and she performed the research, collected and analyzed the results. Parichehr Hanachi supervised and designed the research. Ali Mohammadi, and Omran Moradlou advised the research. All authors contributed to the revision of the manuscript. All the authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Parichehr Hanachi\u003c/p\u003e\n\u003cp\u003eDepartment of Biotechnology, Faculty of Biological Science, Alzahra University, Tehran, Iran\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eP. B. Tchounwou, C. G. Yedjou, A. K. Patlolla, and D. J. Sutton, \u0026ldquo;Molecular, clinical and environmental toxicicology Volume 3: Environmental Toxicology,\u0026rdquo; \u003cem\u003eMol. Clin. Environ. Toxicol.\u003c/em\u003e, vol. 101, pp. 133\u0026ndash;164, 2012, doi: 10.1007/978-3-7643-8340-4.\u003c/li\u003e\n\u003cli\u003eJ. Briffa, E. Sinagra, and R. Blundell, \u0026ldquo;Heavy metal pollution in the environment and their toxicological effects on humans,\u0026rdquo; \u003cem\u003eHeliyon\u003c/em\u003e, vol. 6, no. 9, p. e04691, 2020, doi: 10.1016/j.heliyon.2020.e04691.\u003c/li\u003e\n\u003cli\u003eS. Karbalaei, P. Hanachi, G. Rafiee, P. Seifori, and T. R. \u0026lrm;Walker, \u0026ldquo;Toxicity of polystyrene microplastics on juvenile Oncorhynchus mykiss (rainbow trout) after individual and combined exposure with chlorpyrifos\u0026lrm;,\u0026rdquo; \u003cem\u003eJ. Hazard. Mater.\u003c/em\u003e, vol. 403, no. June 2020, 2021, doi: 10.1016/j.jhazmat.2020.123980.\u003c/li\u003e\n\u003cli\u003eJ. P. McClung and A. G. Scrimgeour, \u0026ldquo;Zinc: An essential trace element with potential benefits to soldiers,\u0026rdquo; \u003cem\u003eMil. Med.\u003c/em\u003e, vol. 170, no. 12, pp. 1048\u0026ndash;1052, 2005, doi: 10.7205/MILMED.170.12.1048.\u003c/li\u003e\n\u003cli\u003eS. Hussain \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Zinc Essentiality, Toxicity, and Its Bacterial Bioremediation: A Comprehensive Insight,\u0026rdquo; \u003cem\u003eFront. Microbiol.\u003c/em\u003e, vol. 13, no. May, 2022, doi: 10.3389/fmicb.2022.900740.\u003c/li\u003e\n\u003cli\u003eH. Kaur and N. Garg, \u0026ldquo;Zinc toxicity in plants: a review,\u0026rdquo; \u003cem\u003ePlanta\u003c/em\u003e, vol. 253, no. 6, pp. 1\u0026ndash;28, 2021, doi: 10.1007/s00425-021-03642-z.\u003c/li\u003e\n\u003cli\u003eA. Selvi \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Integrated remediation processes toward heavy metal removal/recovery from various environments-A review,\u0026rdquo; \u003cem\u003eFront. Environ. Sci.\u003c/em\u003e, vol. 7, no. May, 2019, doi: 10.3389/fenvs.2019.00066.\u003c/li\u003e\n\u003cli\u003eM. Kumar, A. Seth, A. K. Singh, M. S. Rajput, and M. Sikandar, \u0026ldquo;Remediation strategies for heavy metals contaminated ecosystem: A review,\u0026rdquo; \u003cem\u003eEnviron. Sustain. Indic.\u003c/em\u003e, vol. 12, no. December 2020, p. 100155, 2021, doi: 10.1016/j.indic.2021.100155.\u003c/li\u003e\n\u003cli\u003eI. Michalak, K. Chojnacka, and A. Witek-Krowiak, \u0026ldquo;State of the art for the biosorption process - A review,\u0026rdquo; \u003cem\u003eAppl. Biochem. Biotechnol.\u003c/em\u003e, vol. 170, no. 6, pp. 1389\u0026ndash;1416, 2013, doi: 10.1007/s12010-013-0269-0.\u003c/li\u003e\n\u003cli\u003eR. T. Kapoor, M. R. Salvadori, M. Rafatullah, M. R. Siddiqui, M. A. Khan, and S. A. Alshareef, \u0026ldquo;Exploration of Microbial Factories for Synthesis of Nanoparticles \u0026ndash; A Sustainable Approach for Bioremediation of Environmental Contaminants,\u0026rdquo; \u003cem\u003eFront. Microbiol.\u003c/em\u003e, vol. 12, no. June, 2021, doi: 10.3389/fmicb.2021.658294.\u003c/li\u003e\n\u003cli\u003eS. Ghosh, R. Ahmad, M. Zeyaullah, and S. K. Khare, \u0026ldquo;Microbial Nano-Factories: Synthesis and Biomedical Applications,\u0026rdquo; \u003cem\u003eFront. Chem.\u003c/em\u003e, vol. 9, no. April, 2021, doi: 10.3389/fchem.2021.626834.\u003c/li\u003e\n\u003cli\u003eH. Yu, Y. Zhang, W. Tan, and Z. Zhang, \u0026ldquo;Microplastics as an Emerging Environmental Pollutant in Agricultural Soils: Effects on Ecosystems and Human Health,\u0026rdquo; \u003cem\u003eFront. Environ. Sci.\u003c/em\u003e, vol. 10, no. March, pp. 1\u0026ndash;18, 2022, doi: 10.3389/fenvs.2022.855292.\u003c/li\u003e\n\u003cli\u003eH. Park and B. S. Park, \u0026ldquo;Review of microplastic distribution, toxicity, analysis methods, and removal technologies,\u0026rdquo; \u003cem\u003eWater (Switzerland)\u003c/em\u003e, vol. 13, no. 19, 2021, doi: 10.3390/w13192736.\u003c/li\u003e\n\u003cli\u003eA. Amobonye, P. Bhagwat, S. Raveendran, S. Singh, and S. Pillai, \u0026ldquo;Environmental Impacts of Microplastics and Nanoplastics: A Current Overview,\u0026rdquo; \u003cem\u003eFront. Microbiol.\u003c/em\u003e, vol. 12, no. December, 2021, doi: 10.3389/fmicb.2021.768297.\u003c/li\u003e\n\u003cli\u003eR. Ahmed, A. K. Hamid, S. A. Krebsbach, J. He, and D. Wang, \u0026ldquo;Critical review of microplastics removal from the environment,\u0026rdquo; \u003cem\u003eChemosphere\u003c/em\u003e, vol. 293, no. January, p. 133557, 2022, doi: 10.1016/j.chemosphere.2022.133557.\u003c/li\u003e\n\u003cli\u003eM. T. Sturm, H. Horn, and K. Schuhen, \u0026ldquo;Removal of microplastics from waters through agglomeration-fixation using organosilanes\u0026mdash;effects of polymer types, water composition and temperature,\u0026rdquo; \u003cem\u003eWater (Switzerland)\u003c/em\u003e, vol. 13, no. 5, pp. 1\u0026ndash;15, 2021, doi: 10.3390/w13050675.\u003c/li\u003e\n\u003cli\u003eM. B. Tahir, M. Sohaib, M. Sagir, and M. Rafique, \u0026ldquo;Role of Nanotechnology in Photocatalysis,\u0026rdquo; \u003cem\u003eEncycl. Smart Mater.\u003c/em\u003e, no. January, pp. 578\u0026ndash;589, 2021, doi: 10.1016/B978-0-12-815732-9.00006-1.\u003c/li\u003e\n\u003cli\u003eM. Faezi, S. Javanbakht, F. A. Mohseni, and F. Elham, \u0026ldquo;International Journal of Molecular and Clinical Microbiology Isolation and characterization of bacterial strains from shoreline waters of Caspian Sea with novel antibacterial activity,\u0026rdquo; vol. 8, no. 2, pp. 982\u0026ndash;992, 2018.\u003c/li\u003e\n\u003cli\u003eA. Makhdoumi, \u0026ldquo;Bacterial diversity in south coast of Caspian Sea: Culture-dependent and culture-independent survey,\u0026rdquo; \u003cem\u003eCasp. J. Environ. Sci.\u003c/em\u003e, vol. 16, no. 3, pp. 259\u0026ndash;269, 2018.\u003c/li\u003e\n\u003cli\u003eR. Esmail, A. Afshar, M. Morteza, A. Abolfazl, and E. Akhondi, \u0026ldquo;Synthesis of silver nanoparticles with high efficiency and stability by culture supernatant of Bacillus ROM6 isolated from Zarshouran gold mine and evaluating its antibacterial effects,\u0026rdquo; \u003cem\u003eBMC Microbiol.\u003c/em\u003e, vol. 22, no. 1, pp. 1\u0026ndash;10, 2022, doi: 10.1186/s12866-022-02490-5.\u003c/li\u003e\n\u003cli\u003eD. Kundu, C. Hazra, A. Chatterjee, A. Chaudhari, and S. Mishra, \u0026ldquo;Extracellular biosynthesis of zinc oxide nanoparticles using Rhodococcus pyridinivorans NT2: Multifunctional textile finishing, biosafety evaluation and in vitro drug delivery in colon carcinoma,\u0026rdquo; \u003cem\u003eJ. Photochem. Photobiol. B Biol.\u003c/em\u003e, vol. 140, pp. 194\u0026ndash;204, 2014, doi: 10.1016/j.jphotobiol.2014.08.001.\u003c/li\u003e\n\u003cli\u003eM. Yusof \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Sustainable microbial cell nanofactory for zinc oxide nanoparticles production by zinc ‑ tolerant probiotic Lactobacillus plantarum strain TA4,\u0026rdquo; \u003cem\u003eMicrob. Cell Fact.\u003c/em\u003e, pp. 1\u0026ndash;17, 2020, doi: 10.1186/s12934-020-1279-6.\u003c/li\u003e\n\u003cli\u003eB. Balraj \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Synthesis and characterization of Zinc Oxide nanoparticles using marine Streptomyces sp. with its investigations on anticancer and antibacterial activity,\u0026rdquo; \u003cem\u003eRes. Chem. Intermed.\u003c/em\u003e, vol. 43, no. 4, pp. 2367\u0026ndash;2376, 2017, doi: 10.1007/s11164-016-2766-6.\u003c/li\u003e\n\u003cli\u003eT. Tiwari, Hoondal, \u003cem\u003eLaboratory_Techniques_In_Microbiology__amp__Biotechnology.pdf\u003c/em\u003e. 2009.\u003c/li\u003e\n\u003cli\u003eR. Rohban, M. A. Amoozegar, and A. Ventosa, \u0026ldquo;Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran,\u0026rdquo; \u003cem\u003eJ. Ind. Microbiol. Biotechnol.\u003c/em\u003e, vol. 36, no. 3, pp. 333\u0026ndash;340, 2009, doi: 10.1007/s10295-008-0500-0.\u003c/li\u003e\n\u003cli\u003eN. A. RAZALI, A. N. , WAN RAFIZAH WAN ABDULLAH, and M. ZIKIR, \u0026ldquo;EFFECT OF THERMO-PHOTOCATALYTIC PROCESS USING ZINC OXIDE ON DEGRADATION OF MACRO/MICRO-PLASTIC IN AQUEOUS ENVIRONMENT,\u0026rdquo; \u003cem\u003eJ. Sustain. Sci. Manag.\u003c/em\u003e, vol. 15, pp. 1\u0026ndash;14, 2020.\u003c/li\u003e\n\u003cli\u003eM. L. Sogin \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Microbial diversity in the deep sea and the underexplored \u0026lsquo;rare biosphere,\u0026rsquo;\u0026rdquo; \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e, vol. 103, no. 32, pp. 12115\u0026ndash;12120, 2006, doi: 10.1073/pnas.0605127103.\u003c/li\u003e\n\u003cli\u003eP. Velmurugan, C. K. Venil, A. Veera Ravi, and L. Dufoss\u0026eacute;, \u0026ldquo;Marine Bacteria Is the Cell Factory to Produce Bioactive Pigments: A Prospective Pigment Source in the Ocean,\u0026rdquo; \u003cem\u003eFront. Sustain. Food Syst.\u003c/em\u003e, vol. 4, no. November, pp. 1\u0026ndash;7, 2020, doi: 10.3389/fsufs.2020.589655.\u003c/li\u003e\n\u003cli\u003eN. Mahmoudi \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Microbial community composition and diversity in Caspian Sea sediments,\u0026rdquo; \u003cem\u003eFEMS Microbiol. Ecol.\u003c/em\u003e, vol. 91, no. 1, pp. 1\u0026ndash;11, 2015, doi: 10.1093/femsec/fiu013.\u003c/li\u003e\n\u003cli\u003eJ. Chen, J. Li, H. Zhang, W. Shi, and Y. Liu, \u0026ldquo;Bacterial heavy-metal and antibiotic resistance genes in a copper tailing dam area in northern China,\u0026rdquo; \u003cem\u003eFront. Microbiol.\u003c/em\u003e, vol. 10, no. AUG, pp. 1\u0026ndash;12, 2019, doi: 10.3389/fmicb.2019.01916.\u003c/li\u003e\n\u003cli\u003eS. Iravani and R. S. Varma, \u0026ldquo;Bacteria in Heavy Metal Remediation and Nanoparticle Biosynthesis,\u0026rdquo; \u003cem\u003eACS Sustain. Chem. Eng.\u003c/em\u003e, vol. 8, no. 14, pp. 5395\u0026ndash;5409, 2020, doi: 10.1021/acssuschemeng.0c00292.\u003c/li\u003e\n\u003cli\u003eV. Pande, S. C. Pandey, D. Sati, P. Bhatt, and M. Samant, \u0026ldquo;Microbial Interventions in Bioremediation of Heavy Metal Contaminants in Agroecosystem,\u0026rdquo; \u003cem\u003eFront. Microbiol.\u003c/em\u003e, vol. 13, no. May, pp. 1\u0026ndash;16, 2022, doi: 10.3389/fmicb.2022.824084.\u003c/li\u003e\n\u003cli\u003eO. Sambalova \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Carboxylate Functional Groups Mediate Interaction with Silver Nanoparticles in Biofilm Matrix,\u0026rdquo; \u003cem\u003eACS Omega\u003c/em\u003e, vol. 3, no. 1, pp. 724\u0026ndash;733, 2017, doi: 10.1021/acsomega.7b00982.\u003c/li\u003e\n\u003cli\u003eP. Hanachi, S. Karbalaei, T. R. Walker, M. Cole, and S. V. Hosseini, \u0026ldquo;Abundance and properties of microplastics found in commercial fish meal and cultured common carp (Cyprinus carpio),\u0026rdquo; \u003cem\u003eEnviron. Sci. Pollut. Res.\u003c/em\u003e, vol. 26, no. 23, pp. 23777\u0026ndash;23787, 2019, doi: 10.1007/s11356-019-05637-6.\u003c/li\u003e\n\u003cli\u003eZ. Gharari, P. Hanachi, and T. R. Walker, \u0026ldquo;Green synthesized Ag-nanoparticles using Scutellaria multicaulis stem extract and their selective cytotoxicity against breast cancer,\u0026rdquo; \u003cem\u003eAnal. Biochem.\u003c/em\u003e, p. 114786, 2022, doi: 10.1016/j.ab.2022.114786.\u003c/li\u003e\n\u003cli\u003eH. Mohd Yusof, N. A. Abdul Rahman, R. Mohamad, U. H. Zaidan, and A. A. Samsudin, \u0026ldquo;Biosynthesis of zinc oxide nanoparticles by cell-biomass and supernatant of Lactobacillus plantarum TA4 and its antibacterial and biocompatibility properties,\u0026rdquo; \u003cem\u003eSci. Rep.\u003c/em\u003e, vol. 10, no. 1, pp. 1\u0026ndash;13, 2020, doi: 10.1038/s41598-020-76402-w.\u003c/li\u003e\n\u003cli\u003eS. Yedurkar, C. Maurya, and P. Mahanwar, \u0026ldquo;Biosynthesis of Zinc Oxide Nanoparticles Using Ixora Coccinea Leaf Extract\u0026mdash;A Green Approach,\u0026rdquo; \u003cem\u003eOpen J. Synth. Theory Appl.\u003c/em\u003e, vol. 05, no. 01, pp. 1\u0026ndash;14, 2016, doi: 10.4236/ojsta.2016.51001.\u003c/li\u003e\n\u003cli\u003eK. Handore \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Novel green route of synthesis of ZnO nanoparticles by using natural biodegradable polymer and its application as a catalyst for oxidation of aldehydes,\u0026rdquo; \u003cem\u003eJ. Macromol. Sci. Part A Pure Appl. Chem.\u003c/em\u003e, vol. 51, no. 12, pp. 941\u0026ndash;947, 2014, doi: 10.1080/10601325.2014.967078.\u003c/li\u003e\n\u003cli\u003eA. Sharma, B. P. Singh, S. Dhar, A. Gondorf, and M. Spasova, \u0026ldquo;Effect of surface groups on the luminescence property of ZnO nanoparticles synthesized by sol-gel route,\u0026rdquo; \u003cem\u003eSurf. Sci.\u003c/em\u003e, vol. 606, no. 3\u0026ndash;4, 2012, doi: 10.1016/j.susc.2011.09.006.\u003c/li\u003e\n\u003cli\u003eL. F. A. Anand Raj and E. Jayalakshmy, \u0026ldquo;Biosynthesis and characterization of zinc oxide nanoparticles using root extract of Zingiber officinale,\u0026rdquo; \u003cem\u003eOrient. J. Chem.\u003c/em\u003e, vol. 31, no. 1, pp. 51\u0026ndash;56, 2015, doi: 10.13005/ojc/310105.\u003c/li\u003e\n\u003cli\u003eN. G\u0026uuml;rsoy, B. Yilmaz \u0026Ouml;zt\u0026uuml;rk, and İ. Dağ, \u0026ldquo;Synthesis of intracellular and extracellular gold nanoparticles with a green machine and its antifungal activity,\u0026rdquo; \u003cem\u003eTurkish J. Biol.\u003c/em\u003e, vol. 45, no. 2, pp. 196\u0026ndash;213, 2021, doi: 10.3906/biy-2010-64.\u003c/li\u003e\n\u003cli\u003eS. Ghosh, R. Ahmad, K. Banerjee, and M. F. Alajmi, \u0026ldquo;Mechanistic Aspects of Microbe-Mediated Nanoparticle Synthesis,\u0026rdquo; vol. 12, no. May, pp. 1\u0026ndash;12, 2021, doi: 10.3389/fmicb.2021.638068.\u003c/li\u003e\n\u003cli\u003eA. Ando, A. Oller, and B. Corre, \u0026ldquo;Intracellular Biosynthesis and Removal of Copper Nanoparticles by Dead Biomass of Yeast Isolated from the Wastewater of a Mine in the Brazilian Amazonia,\u0026rdquo; vol. 9, no. 1, pp. 1\u0026ndash;9, 2014, doi: 10.1371/journal.pone.0087968.\u003c/li\u003e\n\u003cli\u003eT. S. Tofa, K. L. Kunjali, S. Paul, and J. Dutta, \u0026ldquo;Visible light photocatalytic degradation of microplastic residues with zinc oxide nanorods,\u0026rdquo; \u003cem\u003eEnviron. Chem. Lett.\u003c/em\u003e, vol. 17, no. 3, pp. 1341\u0026ndash;1346, 2019, doi: 10.1007/s10311-019-00859-z.\u003c/li\u003e\n\u003cli\u003eA. Bandyopadhyay and G. C. Basak, \u0026ldquo;Studies on photocatalytic degradation of polystyrene,\u0026rdquo; no. March 2007, 2016, doi: 10.1179/174328407X158640.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Footnotes","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacterial Nanoparticle Production, Bioremediation, Microplastics, Photocatalytic Activity, Zinc Oxide Nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-2851887/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2851887/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe high levels of environmental pollutants like heavy metals and microplastics have prompted researchers to invent new remediation strategies, which might be accompanied by drawbacks, including the risk of secondary pollution, being costly, and inefficient. So, the aim of this study is the reduction of these environmental pollutants by the application of nanoparticle-producing bacteria.\u003c/p\u003e \u003cp\u003eFor this purpose, from 22 screened bacterial isolates of the Caspian Sea, the isolate (\u003cem\u003eN1.5.2\u003c/em\u003e) was used to produce ZnO NPs, which were characterized by techniques like FTIR, XRD, Raman, SEM, EDX, UV-vis spectroscopy, Zeta potential, and DLS. Subsequently, different protocols of bacterial nanoparticle production (using supernatant, extract, biomass, and dilution) were compared by UV-vis spectroscopy. Then, the produced nanoparticles were applied to polystyrene microplastics to degrade them. This process was monitored by SEM and FTIR. The selected bacterial isolate (\u003cem\u003eN1.5.2\u003c/em\u003e) was identified and its phylogenetic tree of life was depicted.\u003c/p\u003e \u003cp\u003eThe results of the blast illustrated 100% similarity of \u003cem\u003eN1.5.2\u003c/em\u003e to \u003cem\u003eBacillus cereus\u003c/em\u003e. The produced ZnO NPs peaked at 360 nm in their UV-vis spectrum. They showed a crystalline structure in the zincite phase and dispersed in the range of 171\u0026ndash;262 nm in an aqueous environment with a PDI of 0.444 and zeta potential of -5.60 meV. Moreover, the comparative study between different production protocols has shown that ZnO nanoparticles produced by bacterial extract have higher UV-vis peak values. Furthermore, the photocatalytic activity of ZnO NPs and microplastic degradation has been proved by SEM and FTIR.\u003c/p\u003e","manuscriptTitle":"Bioremediation of zinc metals and microplastics by biosynthesizing zinc oxide nanoparticles from isolated bacteria of the Caspian Sea, Iran","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-12 14:52:52","doi":"10.21203/rs.3.rs-2851887/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d00a0f63-5273-4f9a-9e56-1098b0517ddd","owner":[],"postedDate":"June 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-23T09:56:19+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-12 14:52:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2851887","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2851887","identity":"rs-2851887","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}