The emergence of antibiotic resistance is considered a major threat, and this problem is exacerbated due to the excessive use of antibiotics. Therefore, it is necessary to find new antimicrobials that are effective against pathogenic bacteria resistant to existing antimicrobials. This study presents a novel antimicrobial approach for the bio-control of multi-antibiotic-resistant (MAR) bacteria. Ten antibiotic discs were used to evaluate the bacterial sensitivity. Five bacterial strains showed resistance to all the tested antibiotic groups. The following MAR bacterial strains were selected, and their identification was confirmed by 16S rRNA with an accession number on the NCBI: Shigella flexneri MZ350855.1; Klebsiella aerogenes MZ352107.1; Serratia marcescens MZ618709.1; Yersinia enterocolitica MZ673567.1; and Achromobacter denitrificans OK560350.1. Star anise extract (SAE) gives the highest inhibitory efficiency against isolated MAR bacteria between three local plant extracts. SAE was used to synthesize magnetite nanoparticles (MNPs) using a simple and eco-friendly green biosynthesis technique. Synthesized MNPs were characterized by X-ray diffraction, ultraviolet–visible spectroscopy, Fourier transform infrared, and transmission electron microscopy techniques. The effect of synthesized magnetic nanoparticles for wastewater treatment in bacterial portion demonstrated remarkable bactericidal performance against total bacterial count with a percentage reduction of 84% using 0.05 g, and the efficiency of decreasing heavy metals was estimated.

  • Elucidate the antibacterial properties of three plant crude water extracts against different multi-antibiotic-resistant (MAR) isolated bacteria.

  • The green synthetic approaches for MNPs using SAE as an alternative source to the conventional chemical method.

  • Using this technique as an approach for wastewater treatment.

Graphical Abstract

Graphical Abstract
Graphical Abstract

Patients receiving antibiotic therapy harbor a large number of antibiotic-resistant bacteria in their intestinal tracts, where they are excreted in large numbers in feces and eventually can contaminate the aquatic environment through fecal materials. Consequently, multi-antibiotic-resistant (MAR) bacteria have been detected in raw sewage, rivers, and other polluted environments (Kessie et al. 1998).

MAR microorganisms (i.e., viruses, bacteria, and fungi) are resistant to multiple antimicrobial agents, making it extremely difficult for healthcare workers to treat patients and ultimately resulting in increased morbidity and mortality rates (Santos et al. 2013). MAR is a significant issue whose effects on public health and socioeconomic sectors are imminent. MAR bacteria are expected to be responsible for the death of many people. The resistance spectrum varies considerably from region to region (World Health Organization 2014). MAR bacteria pose a significant problem in high-income countries, while these data are unavailable for African countries (Tadesse et al. 2017).

Returning to nature has endowed us with vast botanical wealth. Some natural plants have been a source of novel natural bioactive compounds for a very long time, including natural microbial inhibitors and new biologically active compounds that can be used directly as antibacterial compounds; these compounds can be used to treat MAR bacteria. The significance of natural plant extracts is due to their low toxicity, economic viability, and eco-friendliness (Kloy et al. 2020), and they represent one of the alternative methods in wastewater treatment and render it suitable for human consumption.

In an attempt to discover new antimicrobial agents, many studies and surveys have been carried out worldwide on many plants according to the plant flora of a respective region (Sarg et al. 1990). According to Muralikrishna et al. (2014), aloe vera (AV) is a succulent plant species used in herbal medicine since the beginning of the first century. Aloe vera extracts (AVEs) are widely used as an alternative medicine. Previous studies reported that star anise extract (SAE; Illicium verum) was assessed for antimicrobial activity against 25 different genera of bacteria, including animal and plant pathogens, food, and spoilage bacteria. Results showed that the SAE inhibited the growth of 81.1% of the test organisms (Dorman & Deans 2000; Aly et al. 2016). This plant received significant attention after shikimic acid was extracted from star anise, which was used to manufacture Tamiflu, which was used as a drug to treat influenza (Wang et al. 2011). A study was conducted to evaluate using the extracts of rosemary (Rosmarinus officinalis) in folk medicine, which were screened for their activity in vitro against some Gram-positive and Gram-negative pathogenic bacteria (César de Souza Vasconcelos et al. 2003). Several studies reported that fresh and dried rosemary leaves have many potential effects, including antibacterial, antifungal, antiviral, and other activities (Devatkal et al. 2013; Houston et al. 2017).

Nanomolecules represent an alternative to conventional antimicrobial agents, where many researchers reported the ability to use some metal nanoparticles for wastewater treatment problems. Due to their large surface-to-volume ratio, they retain potent antibacterial properties (Klnight et al. 2003; Savage & Diallo 2005). In addition to its antimicrobial applications for water disinfection, it has a significant effect on the adsorption of various heavy metals such as Pb2+, Cd2+, Cu2+, and Ni2+ (Giraldo et al. 2013). Nanomolecules may be made using polymers, lipids, and metals (Kim et al. 2012). Nanoparticles are nano-sized molecules (less than 100 nm in diameter) with a large surface area compared to volume ratio, allowing them to penetrate bacterial cells relatively easily (Chen et al. 2012). Nanoparticles can be attached to microbial surfaces and cause a decrease in both cell mobility and nutrient flow. Different sizes and shapes of nanomaterials can be exploited for developing nanocomposites with antimicrobial activity (Pinto et al. 2012).

Chemical, physical, and biological methods can be used to synthesize magnetite nanoparticles (MNPs). The chemical reduction method is used, as it is a simple, quick, and inexpensive method. Using reducing agents in the synthesis of MNPs by a chemical reduction method may result in a toxic effect and a lack of environmental friendliness (Demchenko et al. 2020). Consequently, the reducing agent derived from plants may become one of the alternative options due to its lower toxicity and environmental friendliness. Bioreductors from plants can be obtained since their secondary metabolites, such as flavonoids, saponins, tannins, and terpenoids give antioxidant activity (Handayani et al. 2020).

There are successful studies in synthesizing MNPs using plant extracts (Sri et al. 2015). For instance, the fruit extract of Artemisia annua (Basavegowda et al. 2014a), the leaf extract of Perilla frutescens (Basavegowda et al. 2014a), Tridax procumbens (Senthil & Ramesh 2012), peel extract of plantain (Venkateswarlu et al. 2013), and also seed extract of grape proanthocyanidin (Narayanan et al. 2012). Based on several review studies, no specific research was done on magnetite synthesis by star anise, and this motivates us to synthesize this. Hence, this research proposes a novel green technique for synthesizing Fe3O4–NPs using star anise.

Concerning the above scope, the present study aims to investigate (i) the antibacterial activity of the crude water extract of three plants against the isolated MAR bacteria, (ii) the green synthetic approaches for MNPs using star anise water extract, which is an alternative source to the conventional chemical method, (iii) further application of synthesized magnetite by star anise in wastewater treatment with determining the antibacterial activity as well as efficiency in trace metal removal.

Sampling, isolation, and purification of bacterial isolates

The outlet points of three different drain resources have been selected to carry out the study. Agricultural drainage water from Sahl El-Husseiniya drain, Sharqia Governorate; industrial wastewater from Al-Khadrawiya drain, Menufia Governorate; and sewage-contaminated water from Bahr El-Baqr drains, Port Saied Governorate were collected according to the standard methods for the examination of water and wastewater (APHA 2017).

Bacterial strains from each water resource were isolated from the total viable bacterial count plates throughout the study. Growing colonies of each plate were differentiated and categorized according to the cultural characteristics, then purified and maintained for further identification.

Antibiotic sensitivity test for bacterial isolates

According to the Clinical and Laboratory Standards Institute (2019), the susceptibility behavior of the purified bacterial isolates from the wastewater samples to the antibiotics was determined on Mueller–Hinton agar by the disc diffusion method using 10 commercially available antibiotic discs belonging to eight different antibiotic groups, and discs were obtained from Pasteur lab and Oxoid. The following antibiotic discs were used: amoxicillin/clavulanate AG (30 μg); ampicillin AM (10 μg); vancomycin VA (30 μg); neomycin N (30 μg); tetracycline TE (30 μg); clindamycin DA (30 μg); erythromycin E (10 μg); cefotaxime CTX (15 μg); cefepime C (10 μg); and ofloxacin OFX (30 μg). Control Mueller–Hinton agar plates were incubated without antibiotic discs. Inhibition zone diameters (IZDs) were measured in millimeters (mm) (Bauer 1966). When a microorganism becomes resistant to more than one antimicrobial medication in three or more antimicrobial classes, MAR occurs (Sawatzky et al. 2015). The results were categorized as R (resistant), I (intermediate sensitive), and S (sensitive).

Identification of most MAR bacterial isolates

The selected purified isolate MAR bacteria exhibiting acquired antibiotic resistance patterns were identified utilizing culture characteristics, Gram's stain, and biochemical characterization as described by Holt & Krieg (1984) and Sneath (1986).

Finally, the pre-identified isolates were confirmed by the genetic analysis of 16S rRNA. Bacterial DNA was extracted and purified using the protocol of Gene Jet Genomic DNA Purification Kit (K0721; Thermo Fisher Scientific) and then used as a template to amplify the 16S rDNA gene by polymerase chain reaction (PCR). The 16S gene was amplified using universal forward primer 27F of (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-CGG YTA CCT TGT TAC GAC TT-3′) was used to amplify the 16S gene. The PCR cycles were run as follows: initial denaturation for 5 min at 94 °C, denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C and extension for 2 min at 72 °C, and the final extension for another 1 mL of first and 10 min at 72 °C. The previous cycles were repeated 30 times. The amplification products were sequenced utilizing Biomolecular Research Services (GATC Biotech, Konstanz, Germany) using ABI (Applied Bio systems) 3730 X DNA sequencer (Hitachi) with the same primers. The 16S rRNA gene sequences obtained were aligned to the NCBI Gen Bank database through BLAST analysis with the maximum similarity and identity using gene alignment database (http://blast.ncbi.nlm.nih.gov/Blast.cgi), sequences were aligned with the CLUSTAL W program and analyzed with the MEGA-X software. The phylogenic tree was carried out using the Maximum Likelihood method with bootstrap values calculated from 1,000 replicate runs (Megha et al. 2015).

Chemicals, reagents, and materials

The plants used in this study were purchased from the local market in Cairo, Egypt, and their inhibitory effect against MAR bacteria was examined. All chemicals used in this experiment were received from Al-Gomhorya Company for chemicals, Egypt. Materials used in the synthesis process were of analytical grade.

Preparation of crude plant extract against bacterial isolates

Star anise (I. verum) fruit, aloe vera (AV) leaves, and rosemary (R. officinalis) leaves extracts were studied for their inhibitory activity against MAR bacteria.

In order to prepare SAE, dried fruits were ground to a fine powder and stored. Rosemary and AV leaves were cleaned for extract preparation. After drying in shade at room temperature and then subjected to oven drying at 40 °C, the particles were crushed into smaller dimensions and then pulverized using a sterile mortar (Moulinex). The plant powders were kept in a sterile, airtight plastic container in the refrigerator. Twenty grams (20 g) of the ground plant samples were soaked in 100 mL of sterilized water then allowed to stand for about 72 h for extraction. After 72 h, the supernatant was collected and filtered using Whatman filter paper No. 1. The filtered samples were sterilized and filtered again by a 0.22 μm Millipore membrane filter (Whatman, Kent, UK). The filtrate was then stored in Eppendorf tubes at a temperature of −4 °C until the assessment of its inhibitory effect (Etemadifar et al. 2018).

The agar disc diffusion technique was utilized to determine the antibacterial activity of the crude plant extract against the selected MAR bacteria (Clinical and Laboratory Standards Institute 2019). The bacterial suspension was spread on a solid plate using a sterile swab aseptically and allowed to dry. Sterile filter paper discs (Whatman no. 5, 6 mm in diameter) were loaded with the plant extract at a concentration of 10 mg/disc; the discs were then placed on top of the seeded medium and gently pressed to ensure contact. The plates were incubated at 37 °C overnight. After the incubation period, inhibition zones on the medium were measured in millimeters. All the experiments were performed three independent times (Figure 1).

Statistical analysis

Each test was conducted in triplicate. The mean and standard deviation (SD) of the growth inhibition zone diameter in the agar disk diffusion method of the extracts were determined. Data were analyzed using Statistical Package for Social Sciences for Windows, version 19.0 (SPSS Inc.).

Purification and identification of the active component of star anise

Analytical thin layer chromatography (TLC) was carried out to purify the active components of star anise fruit extract (SAE), where TLC was placed in contact with the bacterial culture. For the separation of star anise constituents, first, leaving approximately 1 cm on the sides of the TLC plate (20 cm × 20 cm, 0.21 mm thickness, Whatman cellulose chromatography paper), which was marked with a pencil, and the prepared SAE was spotted on the marked line. About 5 μL of 10 mg/mL concentration of tested SAE was spotted with a band length of 10 mm chromatographic Whatman cellulose chromatography paper, and then allowed to dry for a few minutes. Afterward, the plate was developed with a running solvent system of ethyl acetate, methanol, and formic acid in a ratio (of 2:1:0.5 v/v). The paper was placed in an airtight chromatographic chamber saturated with the solvent mixture. Spots were separated by running on the TLC plate in the selected solvent system. Before the solvent reached the maximum point, the plate was removed. The retention factor (Rf) values of separated compounds were determined to quantify the distance each component of a mixture travels. Rf value is defined as the ratio of the spot's displacement above the baseline to the solvent front's displacement above the origin (Prakash et al. 2014). Bio-autography of different Rf values against tested MDR bacteria was performed using the agar well diffusion method when the plate was completely dry.

Gas chromatography–mass spectrometry analysis

Bioactive compounds of SAE extracted by TLC were analyzed by gas chromatography–mass spectrometry (GC–MS) using a TRACE GC Ultra Gas Chromatograph (Thermo Fisher Scientific Corp.) equipped with a thermo mass spectrometer detector (ISQ Single Qua drupole Mass Spectrometer). During analysis, an HP5MS UI (crosslinked 5% methyl phenyl Silox) capillary column was utilized (Moniruzzaman et al. 2015).

Biosynthesis and antibacterial activity of MNPs from star anise

Magnetite biosynthesis was carried out utilizing star anise fruit extract; Fe3O4–NPs were prepared to utilize the precursor salts ferrous sulfate (FeSO4·7H2O) and ferric chloride (FeCl3) at 1:2 M ratios as follows. Iron (II) sulfate and iron (III) chloride solutions were prepared by dissolving 3.73368 and 4.35695 g of the salts, respectively, in 500 mL of distilled water supplemented with nitrogen gas purging. Nitrogen gas was selected for purging because it is inert to solutions and readily available. After the complete dissolution (transparent solutions) of the salts, the two solutions were mixed and heated at 80 °C using a magnetic stirrer and under atmospheric pressure. After 10 min, different concentrations (2.5, 5, and 10%) of SAE were added to the reaction separately, named SAE1, SAE2 and SAE3 sequentially. After 5 min, 5% NaOH solution was added to the mixture dropwise with a rate of 5 mL min–1 to allow the magnetite precipitation uniformly. Once NaOH was added, the reddish-brown color of the mixture changed to brownish-black suspended particles, until complete precipitation of the black precipitate of Fe3O4 was achieved (the complete precipitation is indicated by testing the filtrate solution against drops of NaOH.

A conical flask containing 50 mL of 0.01 M Fe3O4 was prepared as a negative control (Fauzi et al. 2021) and testing the selected MAR bacteria against biosynthesis MNPs using the Kirby–Bauer disc-diffusion method (Clinical and Laboratory Standards Institute 2019). Changes in the color of the reaction mixtures were observed to determine nanoparticle formation characterized by a dark brown color (Figure 2).

Characterization techniques of MNPs

Further confirmation of synthesis MNPs was checked first using UV – a visible spectrum (Hitachi U-2910 Spectrophotometer (Tokyo, Japan). UV–Vis spectroscopic analysis measurements were performed via continuous scanning in the range of 200–800 nm.

The phase evaluation of the synthesized MNPs solution was performed using an X-ray diffractometer (Rotoflux, Model 10.61, Germany) with Ni-filtered Cu-Kα radiation (0.15406 Å). The voltage and current of X-ray tubes were 30 kV and 20 mA at room temperature of about 25 °C with a step size of 0.05 and diffraction range of 2θ = 10°–70°. The diffraction pattern of MNPs was compared with the reference data from the standards compiled by the Joint Committee on Powder Diffraction and Standards (JCPDS), which involved card No. 04-0829 for MNPs. According to Priyadarsini et al. (2020). Purification and concentration of MNPs from the final reaction mixture were assessed using Scherer's Equation (D = 0.9λ/β cosθ) to determine the size of the nanoparticles, where D is the nano-size, λ is the wavelength of X-ray, θ is the diffraction angle, and β is full width at half maximum of the peak (Dipankar & Murugan 2012).

Fourier transformer infrared spectroscopy (FTIR) analysis was carried out using Pelkin-Elmer FTIR 1650 spectrophotometer. Finally, the morphological characteristic of MNPS was examined via scanning electron microscope (SEM, JSM 8404, JEOL Limited, Japan) at a 10 kV with magnifications n of 50,000× and 100,000×. The size particle and morphology of samples were inspected in a transmission electron microscope using (TEM, JEM 2100Plus, JEOL Limited, Japan) which was performed at 100 kV.

Application of green-synthesized MNPs for wastewater treatment

A clean, pre-sterilized container was utilized for the collection of wastewater samples from the Al-Khadrawiya drain, Menufia Governorate (APHA 2017) and directly transported to Central Laboratory for Environmental Quality Monitoring (CLEQM). Five conical flasks were prepared, each containing 1,000 mL of the collected wastewater sample, then treated separately with different amounts of the tested concentration of biosynthesized MNPs (Fe3O4–NPs) by SAE (0.05, 0.04, 0.03, 0.02 and 0.01 g), along with a blank experiment, and were shaken for 30 min. The antibacterial effect of synthesized MNPs was examined using the colony forming unit (CFU) detection method where from the stirred solutions, 100 μL of the mixture was spread on nutrient agar plates and then incubated at 37 °C for 24 h, and the number of CFU/mL was counted. The percentage reduction of the bacterial count was calculated using Equation (1).
formula
(1)

The inductively coupled plasma (ICP-OES) with ultra sonic nebulizer (USN), Perkin-Elmer Optima 5300, USA, was used for estimating trace metals in the collected control sample and also the treated conical flasks with the examined above concentrations of synthesized nanoparticles in an attempt to investigate the ability of synthesized SAE–Fe3O4–NPs in absorption of the metal ions.

Antibiotic susceptibility patterns

About 50 bacterial isolates were isolated from the collected wastewater samples from the three selected locations. Ten antibiotics were used to evaluate the antibiotic susceptibility of the bacterial isolates. The resistance profile of these isolates is presented in Table 1, revealing multiple antibiotic resistances among bacterial isolates. Resistance was mainly directed to ampicillin, erythromycin, and cefepime, where all bacterial isolates (100%) were resistant, while the most active antibiotics were ofloxacin, and about (80%) of bacterial isolates were sensitive, followed by cefotaxime (72%), neomycin (64%), vancomycin (54%), amoxicillin/clavulanate (40%), and clindamycin (32%), and tetracycline (20%). Five of the most resistant bacterial isolates reported in our study were resistant to all groups of antibiotics discs with code no (4, 18, 22, 39, and 47). This finding demonstrates that these isolates are preliminarily to be MAR bacteria, so they were chosen for further studies. The increasing prevalence of MAR bacteria represents global health challenges (World Health Organization 2014). In this respect, Iversen et al. (2004) indicated a potential transmission route for pathogenic bacteria from patients in hospitals to hospital sewage, further to surface water, and possibly back to humans. Based on our findings, it was evident that water pollution may play a significant role in the dissemination of hazardous antibiotic-resistant bacteria. The morphological, physiological, and biochemical identification of the MAR bacteria was evaluated according to identification protocols. The sequences of MAR bacterial strains obtained were aligned to the BLAST analysis with the NCBI GenBank database and were identified under accession numbers Shigella flexneri MZ350855.1, Yersinia enterocolitica MZ673567.1, Klebsiella aerogenes MZ352107.1, Achromobacter denitrificans OK560350.1, and Serratia marcescens MZ618709.1 (Figure 3).
Table 1

Comparative susceptibility of bacterial isolates against different antibiotics

Group & scientific name of antibioticTrade nameAntibiotic codeDisc potency (μg/disc)Sensitive
Resistant
Resistant bacterial isolate numberCategoryNo%No%
1. Penicillins
Amoxicillin/Clavulanate 
Augmentin AG 30 1, 4, 6, (8–19), (21–27), 30, 33, 37, 39, 42, 44, 47, 50. 20 40 30 60 
ampicillin Ampicillin AM 10 (1–50) 100 50 
 2. Quinolones
ofloxacin 
Tarivid OFX 10 4, 14, 18, 22, 35, 39, 40, 44, 47,49 40 80 10 20 
3. Tetracyclines
Tetracycline 
Tetracycline TE 30 (1–23), (29–44), 47 10 20 40 80 
4. Aminoglycosides
Neomycin 
Neo-rx N 30 (1–7), 10, 18, 22, 28, 30, 31, 39, 40, 44, 46, 47. 32 64 18 36 
5. Lincosamides
Clindamycin 
lincocin DA 30 (1–9), (14–19), 23, 24, (29–35), 37, 38, (40–44), 47, 49, 50. 16 32 34 68 
6. Macrolides
Erythromycin 
Erythromycin 10 (1–50) 100 50 
7. Cephalosporins
cefotaxime 
Claforan CTX 30 4, 10, (17–22), 24, 27, 39, 42, 45, 47. 36 72 14 28 
Cefepime Maxipime 30 (1–50) 100 50 
8. Glycopeptides
Vancomycin 
Vancocin VA 30 (1–5), (10–13), (17–29), 32, 35, 39, 40, 47, 49. 27 54 23 46 
Group & scientific name of antibioticTrade nameAntibiotic codeDisc potency (μg/disc)Sensitive
Resistant
Resistant bacterial isolate numberCategoryNo%No%
1. Penicillins
Amoxicillin/Clavulanate 
Augmentin AG 30 1, 4, 6, (8–19), (21–27), 30, 33, 37, 39, 42, 44, 47, 50. 20 40 30 60 
ampicillin Ampicillin AM 10 (1–50) 100 50 
 2. Quinolones
ofloxacin 
Tarivid OFX 10 4, 14, 18, 22, 35, 39, 40, 44, 47,49 40 80 10 20 
3. Tetracyclines
Tetracycline 
Tetracycline TE 30 (1–23), (29–44), 47 10 20 40 80 
4. Aminoglycosides
Neomycin 
Neo-rx N 30 (1–7), 10, 18, 22, 28, 30, 31, 39, 40, 44, 46, 47. 32 64 18 36 
5. Lincosamides
Clindamycin 
lincocin DA 30 (1–9), (14–19), 23, 24, (29–35), 37, 38, (40–44), 47, 49, 50. 16 32 34 68 
6. Macrolides
Erythromycin 
Erythromycin 10 (1–50) 100 50 
7. Cephalosporins
cefotaxime 
Claforan CTX 30 4, 10, (17–22), 24, 27, 39, 42, 45, 47. 36 72 14 28 
Cefepime Maxipime 30 (1–50) 100 50 
8. Glycopeptides
Vancomycin 
Vancocin VA 30 (1–5), (10–13), (17–29), 32, 35, 39, 40, 47, 49. 27 54 23 46 

MAR, multi-antibiotic-resistant.

Figure 1

Photographs of three different plant extracts: (a) star anise (Illicium verum) fruit, (b) aloe vera leaves, and (c) rosemary (Rosmarinus officinalis) leaves.

Figure 1

Photographs of three different plant extracts: (a) star anise (Illicium verum) fruit, (b) aloe vera leaves, and (c) rosemary (Rosmarinus officinalis) leaves.

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Figure 2

Photograph showing green-synthesized magnetite nanoparticles (SAE–Fe3O4–NPs).

Figure 2

Photograph showing green-synthesized magnetite nanoparticles (SAE–Fe3O4–NPs).

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Figure 3

MAR bacterial strains.

Figure 3

MAR bacterial strains.

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Antibacterial activity of plant extracts

The antibacterial activity of some medicinal plants is higher than that of some antibiotics, where their bioactive components give them this potency. In our study, the inhibitory effect of three plants against MAR bacteria isolated from collected polluted water samples was proposed. The data of star anise fruit extracts (P1), AV leaves (P2), and rosemary (R. officinalis) leaves extracts (P3) are depicted in Table 2 and Figures 4 and 5. Star anise fruit extracts (SAE) produced the highest inhibitory activity against all selected bacteria, reaching 11–17 mm, followed by Rosemary leaves extract, with (4–10 mm). In contrast, AV did not affect MAR bacteria, as shown in Table 2. SAE was selected for purification by TLC and then analyzed by GC–MS as it gives the most and strongest inhibitory activity between the examined extracts against all the MAR-tested bacteria. The antimicrobial activity of SAE is attributed to the presence of a phenyl group which can damage proteins, so interacting in the cell membrane of the bacteria with the lipids lead to the death of bacterial cell by changing the permeability of the cell membrane (Qiao et al. 2018). Consistent with our results with Ismail et al. (2012), who reported that only three extracts (star anise, garlic juice, and clove) out of 12 plant extracts were tested for their inhibitory effect versus eight microbial strains showed a high antimicrobial effect against the bacteria under study.
Table 2

Antibacterial activities of SAEs against MAR bacteria

Plant codesPlant extractIZDs (mm) of bacterial isolates
Isolates codes
422183947
P1 Star anise fruit 12 11 13 16 17 
P2 Aloe vera leaf 
P3 Rosemary leaves 10 
Plant codesPlant extractIZDs (mm) of bacterial isolates
Isolates codes
422183947
P1 Star anise fruit 12 11 13 16 17 
P2 Aloe vera leaf 
P3 Rosemary leaves 10 

IZDs, inhibition zone diameters.

Figure 4

Antibacterial activities of different plant extracts against MAR bacteria.

Figure 4

Antibacterial activities of different plant extracts against MAR bacteria.

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Figure 5

Antibacterial activity of plant extracts against multidrug-resistant bacteria.

Figure 5

Antibacterial activity of plant extracts against multidrug-resistant bacteria.

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Identification of MAR bacteria

Using 16S rRNA gene sequencing for the identification of the most resistant bacteria during the study against different antibiotics were submitted to GenBank at the NCBI website under an accession number as follow: S. flexneri MZ350855.1; K. aerogenes MZ352107.1; S. marcescens MZ618709.1; Y. enterocolitica MZ673567.1; and A. denitrificans OK560350.1 Figure 6 demonstrates the phylogenetic tree of the isolated MAR and others from the NCBI.
Figure 6

Phylogenic tree of the MAR bacteria during the study with others from the NCBI.

Figure 6

Phylogenic tree of the MAR bacteria during the study with others from the NCBI.

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GC–MS analysis

On the TLC chromatogram, the preliminary partial purification of SAE by TLC autobiography revealed positive bands (Table 3). In the TLC assay, the results revealed a clear zone indicating the presence of antibacterial components against the MAR bacterial strains tested, with an Rf value of (0.909) in the TLC assay, compatible with Najdoska et al. (2010). Therefore, instrument GC–MS was used for detecting the bioactive compound of SAE, which was found in (0.909) Rf value by TLC (Figure 7).

Table 3

Antibacterial activity of SAE at different Rf values against the tested bacteria

Bacterial isolatesSolvent v/vRfIZDs (mm) of bacterial isolates
Shigella flexneri
MZ350855 
Ethyl acetate:methanol:formic acid (2:1:0.5) 0.2 10 
0.9 12 
Klebsiella aerogenes
MZ352107 
0.7 10 
0.9 15 
Serratia marcescens
MZ618709 
0.8 11 
0.9 17 
Yersinia enterocolitica
MZ673567 
0.9 13 
Achromobacter denitrificans
OK560350 
0.9 14 
Bacterial isolatesSolvent v/vRfIZDs (mm) of bacterial isolates
Shigella flexneri
MZ350855 
Ethyl acetate:methanol:formic acid (2:1:0.5) 0.2 10 
0.9 12 
Klebsiella aerogenes
MZ352107 
0.7 10 
0.9 15 
Serratia marcescens
MZ618709 
0.8 11 
0.9 17 
Yersinia enterocolitica
MZ673567 
0.9 13 
Achromobacter denitrificans
OK560350 
0.9 14 

The mass spectrum of 148 mz was obtained by GC–MS for trans-1-methoxy-4-[prop-1-enil] benzene (anethole), and the results of GC–MS represent that anethole is the primary compound in the star anise (I. verum) extract, approximately 98%, which is consistent with the results of (Marquez et al. 2008). Trans-1-methoxy-4-[prop-1-enil] benzene (anethole) with formula C10H12O is present as a major significant component that occurs widely in many natural plants, such as star anise (I. verum) (Ize-Ludlow et al. 2004). Natural anethole occurs in high concentrations in star anise (over 90%), according to Marinov & Valcheva (2015) (Figure 8).
Figure 7

(a) Gas chromatography–mass spectrometry analysis of (trans-1-methoxy-4-[prop-1-enil] benzene). (b) Thin-layer chromatography (TLC) scan of SAE extract at Rf = 0.9.

Figure 7

(a) Gas chromatography–mass spectrometry analysis of (trans-1-methoxy-4-[prop-1-enil] benzene). (b) Thin-layer chromatography (TLC) scan of SAE extract at Rf = 0.9.

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Figure 8

Chemical structure of trans-1-methoxy-4-[prop-1-enil] benzene (anethole) has the formula of C10H12O (molecular weight: 148.205).

Figure 8

Chemical structure of trans-1-methoxy-4-[prop-1-enil] benzene (anethole) has the formula of C10H12O (molecular weight: 148.205).

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Green biosynthesis of MNPs from SAE

MNPs were successfully synthesized by using the fruit star anise, as presented in Figure 1. The synthesized MNPs showed a dark black color in the aqueous solution and this is due to the collective oscillation of free electrons present in the reduced MNPs, according to Noginov et al. (2007). After adding ferric chloride solution to the plant extract containing the phenolic and flavonoid compounds used as alternative reducing agents (Rengga et al. 2017), the nanoparticles may be stabilized by the reduction of Fe3+ to Fe2+, as reported by Sonibare et al. (2017). The magnetic response of MNPs synthesized using star anise plant extract was confirmed by their migration in a few minutes toward a 0.5 T magnet placed in the proximity of the solution. This result indicates that MNPs with ferromagnetic properties were successfully synthesized and may contribute to cell separation.

UV–vis spectral analysis

UV–vis spectroscopy was first used to confirm the formation and stability of MNPs in an aqueous colloidal solution with different wavelengths ranging from 200 to 600 nm. The biosynthesized MNPs revealed a high peak at 250 nm (Figure 9). These results are in accordance with those recorded by Njagi et al. (2011), who have reported a similar absorbance peak for iron oxide magnetic nanoparticles synthesized using an aqueous sorghum bran extract leaf ranged from 240 to 360 nm. Based on UV–vis results, star anise fruit extract reduced Fe3+ to MNPs, and this finding was investigated further to confirm the star anise MNPs synthesis.
Figure 9

UV/Vis absorption spectra of the bio-MNPs produced using the SAE.

Figure 9

UV/Vis absorption spectra of the bio-MNPs produced using the SAE.

Close modal

Structural features of magnetic nanoparticles

XRD analysis

Figure 10(a) demonstrates the XRD patterns of synthesized MNPs using the extract of star anise, which was confirmed and proved via the characteristic peaks that appeared in the XRD spectrum. Six different peaks were virtually specified at scattering angles (2θ) of 30.22, 35.60, 45.60, 54.10, 57.68, and 63.10, corresponding to the hkl planes of (220), (311), (222), (400), (422) and (511), respectively. Readily, the diffraction peaks can be indexed to the pure cubic phase of magnetite according to comparison with the standard data from (Joint Committee on Powder Diffraction Standards, JCPDS file, PDF No. 19–0629) according to Basavegowda et al. (2014b). In addition, the absence of XRD-detectable impurities confirms that the prepared MNPs have a well-formed crystalline structure and high purity. Consequently, these diffraction peaks reveal the typical crystallite size of the particles.
Figure 10

Green biosynthesis of magnetite from SAE: (a) XRD patterns spectra, (b) SEM micrograph, and (c) TEM image.

Figure 10

Green biosynthesis of magnetite from SAE: (a) XRD patterns spectra, (b) SEM micrograph, and (c) TEM image.

Close modal
The particle size of MNPs (SAE–Fe3O4–NPs) was estimated using Scherrer's formula from the following Equation (2):
formula
(2)
where D is the nanoparticle size, k is the Scherrer constant which is 0.9 for magnetite; λ is the wavelength of radiation source Cu-kα (0.154056 nm), θ represents the diffraction angel, where β represents the full width at half maximum of the peak (FWHM) (Dipankar & Murugan 2012; Mahdavi et al. 2013). By applying the Scherrer equation, the average calculated crystallite sizes of the Fe3O4–NPs are in the range of 21–32 nm (Table 4).

Scanning and transmission electron microscopy

In order to evaluate the morphology and uniformity of the synthesized Fe3O4-MNPs by star anise plant extract, SEM analysis was performed, and the results are depicted in Figure 10(b). Additionally, the SEM image indicated that the morphology of synthesized SAE–Fe3O4–NPs appeared as roughly irregular spherical that aggregated in three-dimensional shape with relatively crystalline in their structure, and that the average particle size of the resultant nanoparticles was between 18 and 33 nm revealed that the resultant nanoparticles particles size was in the range of 18–33 nm in average. According to the SEM images, the prepared SAE–Fe3O4–NPs appeared in homogenous form, which confirms that the Fe3O4–NPs are cubic in shape with a slight difference in particle size that is ascribed to the presence of more faces in nano-crystallines due to its semi-uniform geometrical form (Sutradhar et al. 2011). Observation revealed that the crystalline structure of the prepared SAE–Fe3O4–NPs was supported by the correlation between the Scherrer equation and SEM particle sizes and that the morphology was predominantly porous and spongy (Uruş 2016). As observed in SEM images, the SAE–Fe3O4–NPs tend to aggregate, which may be attributed to the van der Waals force between particles. The agglomeration may be caused by the repulsive electrostatic forces between attracting particles closer to each other so they aggregate (Rosická & Šembera 2013).

A drop of the green-synthesized MNPs by SAE was deposited onto a TEM copper grid for TEM. After drying, the grid was imaged using TEM at 100 kV. Figure 10(c) reveals the TEM image. It is evident from the TEM image that the sizes of MNP are almost homogeneous crystalline structures, and all of the particles are highly cubic in shape, slightly equal to or larger than the calculated crystallite size from the XRD technique. Consequently, the XRD test can be used as a suitable method to evaluate the average size of crystallites; hence, the TEM technique can be appropriate for measuring particle size (Tamiji & Nezamzadeh-Ejhieh 2019).

The FTIR spectrum and absorption bands

FTIR analysis was done to define the functional groups of star anise that acted as bio-reduction of ferric chloride in the synthesis of MNPs. The FTIR spectrum of SAE is demonstrated in Figure 11(a). The spectra of the SAE revealed strong absorption bands at 3,312, 2,935, 1,614, 1,406, 1,255, 1,064, 800, 669, 600, and 547 cm−1 (Figure 11(a)), while the absorption bands of synthesized Fe3O4–NPs were observed at 3,402, 1,616, 680, 583 and 559 cm−1 (Figure 11(b)). FTIR analysis proved that the bio-reduction of ferric chloride into MNPs is due to the reduction by capping material of plant leaf extracts (Sulaiman et al. 2018). The strong absorption peak at 3,312 cm−1 in the SAE indicated the O–H stretching, proving the presence of alcohol and phenolic compounds or stretching of the –NH band of the amino group. In contrast, the presence peak at 2,935 cm−1 contributed to the C–H stretching vibrations of –CH3 and –CH2 functional groups (Awwad & Salem 2012). The absorption peaks at 1,614 and 1,406 cm−1 are due to the stretching vibration of C=C alkene and –CH3 stretching, respectively, where the peaks belonging to 1,255 and 1,064 cm−1 corresponded to the asymmetric stretching vibration of the COO group. The peaks at 800, 669, and 600 cm−1 revealed the existence of an aromatic C–H bending band. Venkateswarlu et al. (2014) recorded that all the bands were shifted after green-synthesized nanoparticles, indicating the participation and interaction of nanoparticles with plant extract. FTIR spectrum of green-synthesized MNPs is presented in 9 b, disclosed peaks at 3,402 cm−1 corresponding to –OH stretching, indicating that OH stretching of the water in the precursor, which disappeared in the nanoparticles caused by the adsorbed water molecules. Since the nano-crystalline materials exhibit a high surface-to-volume ratio and thus absorb moisture (Lu et al. 2010), 1,616 cm−1 corresponds to C=O stretching vibration, confirming the presence of the ester group. The formation of MNPs is characterized by absorption bands from 650 to 446 cm−1. This peak was absent in plant extract, indicating the formation of iron oxide nanoparticles, which aligns with the findings reported by Kumar Das et al. (2014).
Table 4

Calculation of MNPS size using the Scherrer equation

Peak numberPos. (2θ)FWHMSize (nm)
30.22 0.3825 21.72 
35.60 0.3825 23.89 
45.60 0.5510 23.99 
54.10 0.4723 29.32 
57.68 0.5723 31.44 
63.10 0.6723 32.73 
Peak numberPos. (2θ)FWHMSize (nm)
30.22 0.3825 21.72 
35.60 0.3825 23.89 
45.60 0.5510 23.99 
54.10 0.4723 29.32 
57.68 0.5723 31.44 
63.10 0.6723 32.73 

FWHM, full width at half maximum.

Figure 11

FTIR analysis where (a) shows the spectrum of fruit SAE and (b) shows the spectrum of Fe3O4-MNPs synthesized by SAE.

Figure 11

FTIR analysis where (a) shows the spectrum of fruit SAE and (b) shows the spectrum of Fe3O4-MNPs synthesized by SAE.

Close modal

Antibacterial effect comparisons

The antibacterial effect of green-biosynthesized MNPs using different concentrations from SAE (2.5, 5, and 10%) was determined by the disc diffusion method and was assessed by measuring IZDs around the discs, as shown in Table 5. Based on the results, SAE of 10% concentration was used in synthesizing MNPS revealed the highest antimicrobial activity against A. denitrificans OK560350, with IZDs reaching 25 mm, followed by Y. enterocolitica MZ673567 (20 mm IZDs), S. marcescens MZ618709 (17 mm IZDs), S. flexneri MZ350855 (17 mm IZDs), and K. aerogenes MZ352107 (15 mm IZDs) (Figure 12).
Table 5

Effect of different concentration percentages of SAE in biosynthesis of MNPs on isolated MAR bacteria

Bacterial isolatesIZD (mm)
2.5%5%10%
Shigella flexneri MZ350855 8 ± 0.85 12 ± 1.0 17 ± 1.0 
Klebsiella aerogenes MZ352107 6 ± 1.0 11 ± 1.0 15 ± 0.87 
Serratia marcescens MZ618709 6 ± 1.8 13 ± 0.85 17 ± 1.0 
Yersinia enterocolitica MZ673567 8 ± 1.0 16 ± 1.0 20 ± 1.0 
Achromobacter denitrificans OK560350 10 ± 1.0 17 ± 0.87 25 ± 1.0 
Bacterial isolatesIZD (mm)
2.5%5%10%
Shigella flexneri MZ350855 8 ± 0.85 12 ± 1.0 17 ± 1.0 
Klebsiella aerogenes MZ352107 6 ± 1.0 11 ± 1.0 15 ± 0.87 
Serratia marcescens MZ618709 6 ± 1.8 13 ± 0.85 17 ± 1.0 
Yersinia enterocolitica MZ673567 8 ± 1.0 16 ± 1.0 20 ± 1.0 
Achromobacter denitrificans OK560350 10 ± 1.0 17 ± 0.87 25 ± 1.0 
Figure 12

Screening inhibition zones of different concentrations of SAE in green biosynthesis for magnetite using the agar disc diffusion method: (a) Klebsiella aerogenes MZ352107.1; (b) Yersinia enterocolitica MZ673567.1; (c) against Serratia marcescens MZ618709.1; (d) against Achromobacter denitrificans OK560350.1; and (e) against Shigella flexneri MZ350855.1.

Figure 12

Screening inhibition zones of different concentrations of SAE in green biosynthesis for magnetite using the agar disc diffusion method: (a) Klebsiella aerogenes MZ352107.1; (b) Yersinia enterocolitica MZ673567.1; (c) against Serratia marcescens MZ618709.1; (d) against Achromobacter denitrificans OK560350.1; and (e) against Shigella flexneri MZ350855.1.

Close modal

Nanotechnology in wastewater treatment experiment

This study aimed to demonstrate the potential use of synthesized SAE–Fe3O4–NPs in the removal of contaminants from collected wastewater samples as applicable experiments. An overall schematic was set up, and employed results in the present study are shown in Table 6 and Figure 13. The antibacterial activity of biosynthesized magnetite by SAE was examined. The bacterial count was observed to be reduced to more than 20% in the treated flask with 0.01 g of SAE–Fe3O4–NPs compared with the wastewater sample that was not treated with any of the synthesized antibacterial agents. Based on the obtained CFU value and calculation from the percentage reduction of bacterial count in Equation (1), it is evident that the amount of bacteria colony in treated wastewater with synthesized MNPs was decreased by raising the added amounts of nanoparticles until reaching about 84% removal with 0.05 g of nanoparticles and appeared in Figure 13(c). From these observations, the antibacterial activity of SAE–Fe3O4–NPs against various types of water-borne bacteria was confirmed. Previous findings demonstrate that synthesized MNPs from pollen ethanolic extracts (PEEs) showed antimicrobial activity against a large antimicrobial spectrum, including Gram-positive, Gram-negative, and antifungal microorganisms (Spulber et al. 2018).
Table 6

Water quality parameters of raw wastewater and treated water after the application of SAE–Fe3O4–NPs, and their removal efficiency (%)

ParametersUnitesRaw wastewaterTreated water with SAE–Fe3O4–NPs
0.01 g%0.02 g%0.03 g%0.04 g%0.05 g%
PH  7.29 7.19 – 7.10 – 7.11 – 7.20 – 7.30 – 
Trace metal 
Al mg/L 0.008 >0.001 87 >0.001 87 >0.001 87 >0.001 87 >0.001 87 
Ba mg/L 0.056 0.031 44 0.022 60 0.017 69 0.015 73 0.014 75 
Cd mg/L 2.404 1.081 55 0.983 59 0.040 98 0.016 99 0.013 99 
Cu mg/L 0.055 0.027 50 0.025 54 0.023 58 0.022 60 0.022 60 
Fe mg/L 0.655 0.320 51 0.101 84 0.005 99 >0.001 99 >0.001 99 
Mo mg/L >0.001 >0.001 >0.001 >0.001 >0.001 >0.001 
Pb mg/L 3.021 2.001 33 1.043 65 0.026 99 0.019 99 0.017 99 
Se mg/L >0.001 >0.001 >0.001 >0.001 >0.001 >0.001 
Mn mg/L 0.240 0.230 0.121 49 0.065 72 0.013 94 0.010 95 
mg/L 0.011 0.011 0.011 0.011 0.011 0.011 
Sn mg/L > 0.004 >0.004 >0.004 >0.004 >0.004 >0.004 
Organic parameters 
BOD mg/L 90.00 70 22 50 44 20 77 11 87 10 88 
COD mg/L 857.0 600 29 500 41 200 76 190 77 140 83 
Microbiological parameters (count × 105) 
Total bacterial count at 37 °C (CFU/mL) 1,200 799 33 500 58 200 83 180 85 150 87 
Total bacterial count at 22 °C (CFU/mL) 2,250 1,800 20 800 64 600 73 400 82 350 84 
ParametersUnitesRaw wastewaterTreated water with SAE–Fe3O4–NPs
0.01 g%0.02 g%0.03 g%0.04 g%0.05 g%
PH  7.29 7.19 – 7.10 – 7.11 – 7.20 – 7.30 – 
Trace metal 
Al mg/L 0.008 >0.001 87 >0.001 87 >0.001 87 >0.001 87 >0.001 87 
Ba mg/L 0.056 0.031 44 0.022 60 0.017 69 0.015 73 0.014 75 
Cd mg/L 2.404 1.081 55 0.983 59 0.040 98 0.016 99 0.013 99 
Cu mg/L 0.055 0.027 50 0.025 54 0.023 58 0.022 60 0.022 60 
Fe mg/L 0.655 0.320 51 0.101 84 0.005 99 >0.001 99 >0.001 99 
Mo mg/L >0.001 >0.001 >0.001 >0.001 >0.001 >0.001 
Pb mg/L 3.021 2.001 33 1.043 65 0.026 99 0.019 99 0.017 99 
Se mg/L >0.001 >0.001 >0.001 >0.001 >0.001 >0.001 
Mn mg/L 0.240 0.230 0.121 49 0.065 72 0.013 94 0.010 95 
mg/L 0.011 0.011 0.011 0.011 0.011 0.011 
Sn mg/L > 0.004 >0.004 >0.004 >0.004 >0.004 >0.004 
Organic parameters 
BOD mg/L 90.00 70 22 50 44 20 77 11 87 10 88 
COD mg/L 857.0 600 29 500 41 200 76 190 77 140 83 
Microbiological parameters (count × 105) 
Total bacterial count at 37 °C (CFU/mL) 1,200 799 33 500 58 200 83 180 85 150 87 
Total bacterial count at 22 °C (CFU/mL) 2,250 1,800 20 800 64 600 73 400 82 350 84 
Figure 13

Images of bacterial cultures of (a) untreated sample; (b) treated sample with MNPs by SAE; (c) bacterial concentration with respect to the amount of added MNPs.

Figure 13

Images of bacterial cultures of (a) untreated sample; (b) treated sample with MNPs by SAE; (c) bacterial concentration with respect to the amount of added MNPs.

Close modal
The control and treated flasks were examined for trace heavy metal (HMs) as an experiment to discuss the adsorption efficiency of SAE–Fe3O4–NPs. Some trace elements were determined and some of those elements below method detection limits (MDL) of ICP-OES instrument that used in analysis and standard limits of the Egyptian law EGL (92/2013) for wastewater. The trace elements were Mo (>0.001), Se (>0.001), ST (>0.005) and Sn (>0.004). The recorded value of Al, Ba, Cu, Fe and Mn were within the permissible value of EGL (92 /2013) limits for drain water. Therefore, the values of Cd and Pb were higher the permissible value of EGL (92/2013). After standing MNPs in the treated flask, they were collected magnetically. The concentration of the remaining metal ions was determined by the ICP-OES with USN, where this nebulizer decreases the instrument's detection limit by 10%. It was found that the removal percentage of the Pb 2+, Cd 2+, Fe2+ ions reach 99% after 30 min of the sample treatment, as depicted in Table 6. Recently, MNPs have received attention due to their high surface area and superior magnetic properties as an emerging adsorbent. Magnetite efficiency was investigated as adsorbents for Cd2+, Pb2+, and Zn2+ from wastewater (Yong-Gang et al. 2019). In water treatment methods using nano-sorbent materials, separating nanoparticles from contaminated water is challenging due to its small size. However, MNPs can be easily separated and recovered from the system by an external magnetic field. The removal percent of HMs can be calculated from Equation (3).
formula
(3)
where Vo is the initial volume of the heavy metal and Ve is the final volume of the heavy metal.

The values of biochemical oxygen demand (BOD) and chemical oxygen demand (COD) concentration reach high levels because of direct discharge of factories in qusena on El Khadrawia drain; the results exceed the permissible value of EGL (92 /2013). A notable decrease in BOD and COD values was observed after using SAE–Fe3O4–NPs in treatment with 88 and 83%, respectively, as presented in Table 6.

Several researchers are currently focusing on biomimetic approaches, such as plant extracts and microorganisms, to synthesize the nanoparticles, a ‘green chemical or phytochemical’ approach. Based on the current study findings, it can be concluded the ability for successful green syntheses of iron oxide magnetic nanoparticles by SAE and its effectiveness in decreasing the microbial content in wastewater samples as well as toxic metal adsorptive. The study also revealed that increasing the concentration of SAE resulted in increased inhibition activity against some isolated MAR bacteria from wastewater samples. In addition, an active compound named trans-1-methoxy-4-[prop-1-enil] benzene (anethole) was identified from SAE by GCMS analysis of partially purified SAE at a Rf value of 0.9, which was detected by thin-layer chromatography which has an antimicrobial effect and was promising in reducing MAR bacteria and may be help in the progress of new safe bactericidal products having a natural base. The heavy metal removal results show that the synthesized MNPs appear to be very promising material for wastewater treatment application. Therefore, SAE–Fe3O4–NPs offer promise for environment-friendly components for successful water treatment and thus will lead to an increase in safe water resources. Future application of a study to treat wastewater using green-synthesized magnetite for agriculture sector in water-scarce regions is recommended.

M.E.-S. provided conception and design of research; acquisition, analysis, and interpretation of data; drafted the manuscript and substantively revised it. S.A.O. processed creation of new software used in the research and revised the manuscript. All the authors read and approved the final manuscript.

All data analyzed during our study are from our own work and it is our pleasure to be available publically.

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

The authors declare there is no conflict.

Aly
S. E.
,
Sabry
B. A.
,
Shaheen
M. S.
&
Hathout
A. S.
2016
Assessment of antimycotoxigenic and antioxidant activity of star anise (Illicium verum) in vitro
.
Journal of the Saudi Society of Agricultural Sciences
15
(
1
),
20
27
.
American Public Health Association (APHA), American Water Works Association, Water Pollution Control Federation, & Water Environment Federation
2017
Standard Methods for the Examination of Water and Wastewater
, Vol.
2
.
American Public Health Association, Washington, DC, USA
.
Awwad
A. M.
&
Salem
N. M.
2012
A green and facile approach for synthesis of magnetite nanoparticles
.
Nanoscience and Nanotechnology
2
(
6
),
208
213
.
Bauer
A. W.
1966
Antibiotic susceptibility testing by a standardized single disc method
.
American Journal of Clinical Pathology
45
,
149
158
.
César de Souza Vasconcelos
L.
,
Sampaio
M. C. C.
,
Sampaio
F. C.
&
Higino
J. S.
2003
Use of Punica granatum as an antifungal agent against candidosis associated with denture stomatitis
.
Mycoses
46
(
5-6
),
192
196
.
Chen
D.
,
Love
K. T.
,
Chen
Y.
,
Eltoukhy
A. A.
,
Kastrup
C.
,
Sahay
G.
&
Anderson
D. G.
2012
Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation
.
Journal of the American Chemical Society
134
(
16
),
6948
6951
.
Clinical and Laboratory Standards Institute
2019
Performance Standards for Antimicrobial Susceptibility Testing
, 29th edn. Washington, DC, USA.
CLSI document
, p.
M100
.
Demchenko
V.
,
Riabov
S.
,
Sinelnikov
S.
,
Radchenko
O.
,
Kobylinskyi
S.
&
Rybalchenko
N.
2020
Novel approach to synthesis of ssilver nanoparticles in interpolyelectrolyte complexes based on pectin, chitosan, starch and their derivatives complexes based on pectin, chitosan, starch and their derivatives
.
Carbohydrate Polymers
242, 116431
Devatkal
S. K.
,
Jaiswal
P.
,
Jha
S. N.
,
Bharadwaj
R.
&
Viswas
K. N.
2013
Antibacterial activity of aqueous extract of pomegranate peel against Pseudomonas stutzeri isolated from poultry meat
.
Journal of Food Science and Technology
50
(
3
),
555
560
.
Dorman
H. D.
&
Deans
S. G.
2000
Antimicrobial agents from plants: antibacterial activity of plant volatile oils
.
Journal of Applied Microbiology
88
(
2
),
308
316
.
Etemadifar
R.
,
Kianvash
A.
,
Arsalani
N.
,
Abouzari-Lotf
E.
&
Hajalilou
A.
2018
Green synthesis of superparamagnetic magnetite nanoparticles: effect of natural surfactant and heat treatment on the magnetic properties
.
Journal of Materials Science: Materials in Electronics
29
(
20
),
17144
17153
.
Fauzi, N. N., Hamdan, R. H., Mohamed, M., Ismail, A., Zin, A. A. & Mohamad, N. F. 2021 Prevalence, antibiotic susceptibility, and presence of drug resistance genes in Aeromonas spp. isolated from freshwater fish in Kelantan and Terengganu states, Malaysia. Veterinary World 14 (8), 2064.
Giraldo
L.
,
Erto
A.
&
Moreno-Piraján
J. C.
2013
Magnetite nanoparticles for removal of heavy metals from aqueous solutions: synthesis and characterization
.
Adsorption
19
(
2
),
465
474
.
Handayani
W.
,
Ningrum
A. S.
&
Imawan
C.
2020
The role of pH in synthesis silver nanoparticles using Pometia pinnata (Matoa) leaves extract as bioreductor
.
Journal of Physics: Conference Series
1428
(
1
),
1
5
.
Holt
J. C.
&
Krieg
N. R.
1984
Bergeys' Manual of Systemic Bacteriology
, Vol.
9
, 4th edn.
William and Wilkins
, Baltimore, MD, USA, pp.
40
97
.
Ismail
M.
,
Essam
T.
,
Mohamed
A.
&
Mourad
F.
2012
Screening for the antimicrobial activities of alcoholic and aqueous extracts of some common spices in Egypt
.
Microbiological Research
3
,
200
207
.
Iversen
A.
,
Kühn
I.
,
Rahman
M.
,
Franklin
A.
,
Burman
L. G.
,
Olsson-Liljequist
B.
, Torell, E. &
Möllby
R.
2004
Evidence for transmission between humans and the environment of a nosocomial strain of Enterococcus faecium
.
Environmental Microbiology
6
(
1
),
55
59
.
Ize-Ludlow
D.
,
Ragone
S.
,
Bruck
I. S.
,
Bernstein
J. N.
,
Duchowny
M.
&
Peña
B. M. G.
2004
Neurotoxicities in infants seen with the consumption of Star anise tea
.
Pediatrics
114
(
5
),
e653
e656
.
Kessie
G.
,
Ettayebi
M.
,
Haddad
A. M.
,
Shibl
A. M.
,
Al-Shammary
F. J.
,
Tawfik
A. F.
&
Al-Ahdal
M. N.
1998
Plasmid profile and antibiotic resistance in coagulase-negative staphylococci isolated from polluted water
.
Journal of Applied Microbiology
84
(
3
),
417
422
.
Kim
Y.
,
Lee Chung
B.
,
Ma
M.
,
Mulder
W. J.
,
Fayad
Z. A.
,
Farokhzad
O. C.
&
Langer
R.
2012
Mass production and size control of lipid–polymer hybrid nanoparticles through controlled microvortices
.
Nano Letters
12
(
7
),
3587
3591
Klnight
V.
,
Sanglier
J. J.
,
DiTullio
D.
,
Braccili
S.
,
Bonner
P.
,
Waters
J.
, Hughes, D. &
Zhang
L.
2003
Diversifying microbial natural products for drug discovery
.
Applied Microbiology and Biotechnology
62
(
5
),
446
458
.
Kloy
A.
,
Ahmad
J.
,
Yusuf
U.
&
Muhammad
M.
2020
Antibacterial properties of rosemary (Rosmarinus officinalis)
.
South. Asian. Res. J. Pharm. Sci
2
(
1
), 4–7.
Kumar Das
A.
,
Marwal
A.
&
Verma
R.
2014
Bio-reductive synthesis and characterization of plant protein coated magnetite nanoparticles
.
Nano Hybrids
7 (2),
69
86
.
Lu
W.
,
Shen
Y.
,
Xie
A.
&
Zhang
W.
2010
Green synthesis and characterization of superparamagnetic Fe3O4 nanoparticles
.
Journal of Magnetism and Magnetic Materials
322
(
13
),
1828
1833
.
Marinov
V.
&
Valcheva-Kuzmanova
S.
2015
Review on the pharmacological activities of anethole
.
Scripta Scientifica Pharmaceutica
2
(
2
),
14
19
.
Megha
V.
,
Meenakshi
S.
&
Rai
J. P. N.
2015
Optimization of different parameters on synthetic dye decolorization by free and immobilized Mucor hiemalis MV04 (KR078215)
.
Research Journal of Chemical Sciences
5
(
6
),
20
27
.
Moniruzzaman
M.
,
Shahinuzzaman
M.
,
Haque
A.
,
Khatun
R.
&
Yaakob
Z.
2015
Gas chromatography mass spectrometry analysis and in vitro antibacterial activity of essential oil from Trigonella foenum-graecum
.
Asian Pacific Journal of Tropical Biomedicine
5
,
1033
1036
.
Muralikrishna
T.
,
Pattanayak
M.
&
Nayak
P. L.
2014
Green synthesis of gold nanoparticles using (aloe vera) aqueous extract
.
World Journal of Nano Science & Technology
3
(
2
),
45
51
.
Najdoska
M.
,
Bogdanov
J.
&
Zdravkovski
Z.
2010
TLC and GC-MS analyses of essential oil isolated from Macedonian Foeniculi fructus
.
Macedonian Pharmaceutical Bulletin
56
(
1
),
2
.
Narayanan
S.
,
Sathy
B. N.
,
Mony
U.
,
Koyakutty
M.
,
Nair
S. V.
&
Menon
D.
2012
Biocompatible magnetite/gold nanohybrid contrast agents via green chemistry for MRI and CT bioimaging
.
ACS Applied Materials & Interfaces
4
(
1
),
251
260
.
Njagi
E. C.
,
Huang
H.
,
Stafford
L.
,
Genuino
H.
,
Galindo
H. M.
,
Collins
J. B.
, Hoag, G. E. &
Suib
S. L.
2011
Biosynthesis of iron and silver nanoparticles at room temperature using aqueous sorghum bran extracts
.
Langmuir
27
(
1
),
264
271
.
Noginov
M. A.
,
Zhu
G.
,
Bahoura
M.
,
Adegoke
J.
,
Small
C.
,
Ritzo
B. A.
, Drachev, V. P. &
Shalaev
V. M.
2007
The effect of gain and absorption on surface plasmons in metal nanoparticles
.
Applied Physics B
86
(
3
),
455
460
.
Pinto
R. J.
,
Neves
M. C.
,
Neto
C. P.
&
Trindade
T.
2012
Composites of cellulose and metal nanoparticles
.
Nanocomposites–New Trends and Developments
2 (3), 1–25.
Prakash
D.
,
Ramesh
K.
,
Gopinath
N.
,
Shantha Kumar
S. S.
&
Varuvelil
G. J.
2014
Antibacterial efficacy of Syzygium aromaticum extracts on multi-drug resistant streptococcus mutans isolated from dental plaque samples
.
Journal of Biochemical Technology
3
(
5
),
155
157
.
Priyadarsini
S.
,
Mukherjee
S.
,
Bag
J.
,
Nayak
N.
&
Mishra
M.
2020
Application of nanoparticles in dentistry: current trends
. In: Shukla, A. (ed.)
Nanoparticles in Medicine
.
Springer
,
Singapore
, pp.
55
98
.
Santos
T.
,
Silva
N.
,
Igrejas
G.
,
Rodrigues
P.
,
Micael
J.
,
Rodrigues
T.
, Resendes, R., Gonçalves, A., Marinho, C., Gonçalves, D., Cunha, R. &
Poeta
P.
2013
Dissemination of antibiotic resistant Enterococcus spp. and Escherichia coli from wild birds of Azores Archipelago
.
Anaerobe
24
,
25
31
.
Sarg
T. M.
,
Salem
S. A.
,
Farrag
N. M.
,
Abdel-Aal
M. M.
&
Ateya
A. M.
1990
Phytochemical and antimicrobial investigation of Hemerocallis fulva L. grown in Egypt +
.
International Journal of Crude Drug Research
28
(
2
),
153
156
.
Savage
N.
&
Diallo
M. S.
2005
Nanomaterials and water purification: opportunities and challenges
.
Journal of Nanoparticle Research
7
(
4
),
331
342
.
Sawatzky
P.
,
Liu
G.
,
Dillon
J. A. R.
,
Allen
V.
,
Lefebvre
B.
,
Hoang
L.
, Tyrrell, G., Van Caeseele, P., Levett, P. &
Martin
I.
2015
Quality assurance for antimicrobial susceptibility testing of Neisseria gonorrhoeae in Canada, 2003 to 2012
.
Journal of Clinical Microbiology
53
(
11
),
3646
3649
.
Senthil
M.
&
Ramesh
C.
2012
Biogenic synthesis of Fe3O4 nanoparticles using tridax procumbens leaf extract and its antibacterial on Pseudomonas Aeruginosa
.
Digest Journal of Nanomaterials & Biostructures (DJNB)
7
(
4
), 212–230 .
Sonibare
M. A.
,
Ayoola
I. O.
&
Elufioye
T. O.
2017
Antioxidant and acetylcholinesterase inhibitory activities of leaf extract and fractions of Albizia adianthifolia (Schumach) WF wright
.
Journal of Basic and Clinical Physiology and Pharmacology
28
(
2
),
143
148
.
Spulber
R.
,
Chifiriuc
C.
,
Fleancu
M.
,
Popa
O.
&
Băbeanu
N.
2018
Antibacterial activity of magnetite nanoparticles coated with bee pollen extracts
. In
Agriculture for Life, Life for Agriculture’ Conference Proceedings
, pp.
579
585
.
Sri
P. U.
,
Leelavathi
V.
,
Sree
N. V.
&
Kumar
M. A.
2015
Antihelmenthic and antimicrobial activity of green synthesized silver nanoparticles from Illicium Verum Hook. F. Fruit
.
IOSR Journal of Pharmacy and Biological Sciences
10
(
6
),
61
65
.
Sutradhar
N.
,
Sinhamahapatra
A.
,
Pahari
S. K.
,
Pal
P.
,
Bajaj
H. C.
,
Mukhopadhyay
I.
&
Panda
A. B.
2011
Controlled synthesis of different morphologies of MgO and their use as solid base catalysts
.
The Journal of Physical Chemistry C
115
(
25
),
12308
12316
.
Venkateswarlu
S.
,
Rao
Y. S.
,
Balaji
T.
,
Prathima
B.
&
Jyothi
N. V. V.
2013
Biogenic synthesis of Fe3O4 magnetic nanoparticles using plantain peel extract
.
Materials Letters
100
,
241
244
.
Venkateswarlu
S.
,
Kumar
B. N.
,
Prasad
C. H.
,
Venkateswarlu
P.
&
Jyothi
N. V. V.
2014
Bio-inspired green synthesis of Fe3O4 spherical magnetic nanoparticles using Syzygium cumini seed extract
.
Physica B: Condensed Matter
449
,
67
71
.
Wang
G. W.
,
Hu
W. T.
,
Huang
B. K.
&
Qin
L. P.
2011
Illicium verum: a review on its botany, traditional use, chemistry and pharmacology
.
Journal of Ethnopharmacology
136
(
1
),
10
20
.
World Health Organization
2014
Antimicrobial Resistance Global Report on Surveillance: 2014 Summary (No. WHO/HSE/PED/AIP/2014.2)
.
World Health Organization
, Geneva, Switzerland.
Yong-Gang
S. U. I.
,
Si-Yong
T. E. N. G.
,
Jie
Q. I. A. N.
,
Yuan
W. U.
,
Ke-Fei
D. O. U.
,
Yi-Da
T. A. N. G.
&
Yong-Jian
W. U.
2019
Invasive versus conservative strategy in consecutive patients aged 80 years or older with non-ST-segment elevation myocardial infarction: a retrospective study in China
.
Journal of Geriatric Cardiology
16
(
10
),
741
748
.
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