Green synthesis and characterization of iron nanoparticles synthesized from bioflocculant for wastewater treatment: A review

Abstract

Nanotechnology is a rapidly expanding field with diverse healthcare, agriculture, and industry applications. Central to this discipline is manipulating materials at the nanoscale, particularly nanoparticles (NPs) ranging from 1 to 100 nm. These NPs can be synthesized through various methods, including chemical, physical, and biological processes. Among these, biological synthesis has gained significant attention due to its eco-friendly nature, utilizing natural resources such as microbes and plants as reducing and capping agents. However, information is scarce regarding the production of iron nanoparticles (FeNPs) using biological approaches, and even less is available on the synthesis of FeNPs employing microbial bioflocculants. This review aims to provide a comprehensive examination of the synthesis of FeNPs using microbial bioflocculants, highlighting the methodologies involved and their implications for environmental applications. Recent findings indicate that microbial bioflocculants enhance the stability and efficiency of FeNP synthesis while promoting environmentally friendly production methods. The synthesized FeNPs demonstrated effective removal of contaminants from wastewater, achieving removal rates of up to 93 % for specific dyes and significant reductions in chemical oxygen demand (COD) and biological oxygen demand (BOD). Additionally, these FeNPs exhibited notable antimicrobial properties against both Gram-positive and Gram-negative bacteria.

This review encompasses studies conducted between January 2015 and December 2023, providing detailed characterization of the synthesized FeNPs and underscoring their potential applications in wastewater treatment and environmental remediation.

Highlights

• •Water scarcity is a major global issue driven by population growth, urbanization, and climate change.
• •Traditional FeNPs production methods face high energy use, low yields, and harmful substances.
• •Green synthesis methods using plants, bacteria, and fungi offer environmentally friendly alternatives.
• •This review focuses on producing FeNPs with microbial flocculants that are sustainable and cost-effective.
• •Synthesized FeNPs are highlighted for their application in wastewater treatment, antimicrobial effects and tixicity concerns.

1. Introduction

Wastewater treatment is a crucial process that protects public health and the environment by removing impurities from wastewater before it reaches natural bodies of water.¹ With the increasing demand for clean water and growing concern over water pollution, there is a pressing need for better treatment systems that effectively remove pollutants from wastewater. Traditional sedimentation tanks have been widely employed to allow solid particles to settle out.² However, the use of chemical coagulants and flocculants has proven beneficial in enhancing the removal of suspended solids.³ Coagulation and flocculation are essential components of both drinking water and wastewater treatment, enabling a reduction in suspended solids and organic loads. Despite their effectiveness, these conventional methods face limitations in addressing emerging contaminants such as pharmaceuticals, microplastics, and certain chemical compounds,² Therefore, there is an urgent need to explore alternative and innovative technologies to achieve more comprehensive and effective wastewater treatment solutions.

Green synthesis refers to environmentally friendly methods that utilize natural biological systems such as microorganisms, plants, and their metabolites to produce nanomaterials.^4,5 This approach contrasts with conventional chemical and physical synthesis methods that often involve toxic substances and high energy consumption, leading to significant environmental concerns.⁶ Green synthesis not only reduces environmental impact but also enhances the safety and cost-effectiveness of nanoparticle production.⁷

Nanomaterials have emerged as a fascinating subject in wastewater treatment, holding the potential to enhance treatment efficiency. These materials are characterized by their manipulation at the atomic and molecular levels, typically ranging between 1 and 100 nm.⁶ The unique properties of nanomaterials such as increased surface area-to-volume ratios facilitate their application across various fields including agriculture, biotechnology, environmental remediation, and medicine.^5,8

The synthesis of nanoparticles can be achieved through both physical and chemical processes. However, these methods often require significant energy inputs and involve hazardous substances.^9,10 In response to these challenges, there is a growing interest in developing green processes that utilize microorganisms as sustainable precursors for nanoparticle production.¹¹ These methods not only offer safety benefits but also present cost-effective alternatives for synthesizing nanoparticles.¹²

In recent years, bioflocculants have gained recognition as promising alternatives for synthesizing iron nanoparticles for wastewater treatment. Bioflocculants are polymeric substances derived from living organisms that are environmentally friendly, non-toxic, and biodegradable.¹³ They provide several advantages over traditional chemical flocculants, including reduced environmental impact and waste generation.¹⁴ The synthesis of iron nanoparticles using bioflocculants follows a green approach by utilizing metal precursors such as iron sulfate and natural sources like activated sludge or plant extracts. Researchers have demonstrated the efficacy of bioflocculants-synthesized iron nanoparticles in removing pollutants from wastewater,¹⁵ demonstrating their potential as an effective treatment method.

Iron nanoparticles (FeNPs) possess remarkable properties such as dimensional stability, non-toxicity, and a strong magnetic behavior due to their high surface area and conductivity.¹⁶ Their superparamagnetic characteristics make them particularly valuable in various applications including biomedical fields.¹⁷ Despite numerous studies on the plant-based synthesis for FeNPs,18, 19, 20, 21, 22, 23 research on the synthesis of nanoparticles using bioflocculants remains limited.^13,24, 25, 26 There are even fewer reports on the synthesis of FeNPs using microbial bioflocculants.^15,26,27

Therefore, this review aims to elucidate the current research on the green production of Fe nanoparticles using microbial bioflocculants from January 2015 to December 2023. The review will cover characterization techniques and environmental applications of the FeNPs. The uniqueness of microbial bioflocculants in FeNP synthesis will be emphasized throughout the discussion. By providing insights into environmentally sustainable practices for producing FeNPs, this review seeks to inform readers about innovative approaches that leverage local resources while reducing reliance on conventional chemical flocculants.

2. Iron nanoparticle synthesis methods

Iron nanoparticles can be synthesized using various physicochemical and biological methods, as indicated in Fig. 1²⁸ These methods can be categorized into two main approaches: top-down and bottom-up procedures.²⁹

Fig. 1.

Strategies developed for iron nanoparticle synthesis.¹⁶ Created with Biorender.com.http://www.biorender.com (accessed on May 11, 2024).

Top-down procedures involve reducing bulk materials through techniques such as ball milling, etching, laser ablation, sonochemical production, thermal breakdown, and sputtering in a vacuum.³⁰ These methods often require sophisticated equipment and considerable energy to create nanoparticles from bulk substances.

In contrast, bottom-up techniques utilize metal precursors to produce NPs that can exist in solid, liquid, or gas stages.²⁹ This category includes methods like aerosol procedure, atomic condensation, precipitation, hydrothermal production, hydrolysis, microemulsion, sol-gel analysis, thermal decomposition, and chemical vapor formation.³¹

The biological synthesis of iron nanoparticles (FeNPs) is environmentally benign and creates stable nanoparticles without harmful additives.³² This natural production strategy is considered an ecological route for the production of FeNPs.³³ Various biological techniques have been employed to produce iron nanoparticles using algae, bacteria, diatoms, fungi, and extracts from plants.³⁴ These methods are preferred over synthetic production because they lack toxic substances and can enhance colloidal stability by preventing particle aggregation and degradation.¹³ A review of the literature reveals that many studies have reported the fabrication of iron nanoparticles using plants extract.35, 36, 37, 38, 39 However, there are few studies focusing on the synthesis of iron nanoparticles using bioflocculants, and even fewer on the use of microbial bioflocculants for producing FeNPs. With that being said, this review aims to highlight recent advancements in the biological method of utilizing bioflocculants as reducing and stabilizing agents for the production of iron nanoparticles.

2.1. Plant-based iron nanoparticles synthesis

Plant-derived extracts include a variety of water-soluble substances such as polyphenols, sucrose, alkaloids, phenolic acids, proteins, and coenzymes. These compounds can function as stabilizing and reducing agents in the biosynthetic process.⁴⁰ To synthesize nanoparticles, numerous iron salts like FeCl₃·6H₂O, FeCl₂·4H₂O, FeSO₄, or Fe (NO₃)₃, can be utilized as precursors as shown in Fig. 2.

Fig. 2.

(a) Biological method of iron nanoparticle synthesis and (b) Possible mechanism reaction for iron nanoparticle synthesis.⁴⁸ Created with Biorender.com., http://www.biorender.com (accessed on May 11, 2024).

⁴⁰ Reported the synthesis of iron nanoparticles, where the plant-extracted product and iron precursor were mixed and allowed to interact at room temperature for 24 h. The reaction speed increased when heat was raised to between 25 and 80 °C. The successful production of FeNPs was shown by the alteration in the color of the mixture from light brown to dark brown. The formed FeNPs were gathered, rinsed multiple times with purified water, and dried out nightly in a heated oven at 80 °C.

The author also noted that this synthesis approach could be used to fabricate various nanoparticles, like ZVI, iron oxides, iron oxide hydroxide (FeOOH), and Iron mineral complex nanoparticles. The FeNPs synthesized utilizing various plant forms and precursors are shown in Table 1.

Table 1.

Shows the formation of FeNPs utilizing different extracts of plants and precursors.

Plant extract	Precursor	Shape	Size (nm)	Citation
Omani mango tree	FeSO4	Nanorods	3–15	41
Eucalyptus	FeCl3·6H2O	Spherical	7 ± 10	42
Moringa oleifera	FeCl3·6H2O	Rod	10–90	18
Barberry	FeCl3	Irregular	20–240	19
Green tea and grapes	FeCl3·6H2O	Spherical	120–160	20
Olive	Fe (NO3)3	Spherical	7.58–11.6	21
Mikania mikrantha	FeSO4·7H2O	Rhomboidal	15–20	43
Piper betel	FeCl3	Cubic	22–35	44
Prunus serotine	FeCl3·6H2O	Spherical	7.9±11.9	45
Vaccinium floribundum	FeCl3·6H2O	Spherical	13.2±5.9	45
Fenugreek	FeCl3·6H2O	Spherical	10.61–43.5	46
Musa paradisiaca	FeCl3	Cylindrical	60	47

2.2. Synthesis of iron nanoparticles using bacteria

Microbial production is an effective method for producing Fe nanoparticles utilizing microorganisms like fungi, bacteria, and yeasts (Fig. 3).⁴⁹ The two primary synthesis methods are extracellular and intracellular.⁵⁰ In extracellular production, enzymes reductase, proteins, peptides, and other substances such as reducing substances work as coverings to produce NPs with greater strength and less accumulation.⁵¹ The cells are cultivated in the medium under the proper conditions before being collected by centrifuge or filtering to eliminate the cells of bacteria. The supernatant containing the cell-free mix is then combined with the appropriate quantity of Fe precursor and kept in a dark environment for a set amount of time. Ions are carried inside the microbial cell to produce NPs in the presence of enzymes during intracellular production.⁵² The biochemical pathways comprising microorganism-mediated nanoparticle formation might be viewed as part of microbial resistance processes for cellular purification. This entails altering the solubility of inorganic and hazardous ions by reducing enzymatic and/or precipitation in the manner of nanostructures.⁵² Recent studies have shown that a co-factor Nicotinamide adenine dinucleotide hydrogen (NADH) and its dependent enzymes could possess a function in NP fabrication, with the NADH reductase enzyme assisting in the electron transfer from NADH, leading to the reduction of Fe precursors to form FeNPs.⁵³ Some microorganisms used for intracellular synthesis of nanoparticles include Ralstonia pickettii sp., Saccharomyces cerevisiae, and Cryptococcus humicola, which were cultivated in a Fe precursor-having broth under normal heat environments.⁵⁴ Sonication was utilized to produce FeNPs on the cell wall of Xanthomonas campestris without disrupting the cell bacteria. Because of its fewer downstream processing steps and ease of product recovery, extracellular microbial NP production has garnered more attention than intercellular fabrication.⁵⁵ synthesized quasi-spherical iron oxide nanoparticles (IONPs) using Streptomyces sp. (SRT12) having a medium size of 65.0–86.7 nm. These nanoparticles displayed significant antioxidant and bacterial activities.

Fig. 3.

The use of bacteria in the creation of iron NPs is depicted graphically.⁶⁵ Created with Biorender.com., http://www.biorender.com (accessed on May 11, 2024).

⁵⁶ conveyed the production of iron NPs using Staphylococcus warneri with a medium length of 34 nm. The synthesized FeNPs were spherical and exhibited high biocompatibility, making FeNPs suitable for target therapies. Another study by⁵⁷ utilized a cytoplasmic extract of Lactobacillus casei to produce spherical FeNPs with a normal size of 15 nm. These nanoparticles were also biocompatible and could be used for biomedical applications such as cancer therapy. Table 2 Summarized some microorganisms utilized in the formation of iron NPs.

Table 2.

Numerous bacteria are used in the synthesis of iron nanoparticles.

Organism	Location of synthesis	Size and shape	Reaction time	Citation
Bacillus cereus (HMH1)	Extracellular	18.8–28.3 nm and spherical	1 h at 25 °C	58
Ralstoniapickettii sp.	Intracellular	1.2–2 nm and spherical shaped	10 days at 25 °C and anaerobic	54
Lactobacillus fermentum	Intercellular	10–15 nm and spherical	3 weeks at 37 °C	59
Lactobacillus casei	Nicotinamide riboside	10–15 nm and spherical	3 weeks at 37 °C	57
Proteus mirabilis	Nicotinamide riboside	1.44–1.92 nm an d spherical	50 h aerobic and 71 h anaerobic 37 °C	60
Paenibacillus polymyxa	Nicotinamide riboside	26.65 nm and spherical	24 h at 37 °C	61
Bacillus subtilis	Nicotinamide	12–32 nm and spherical	48 h at 35 °C	62
Pseudomonas stutzeri	Nicotinamide riboside	10–20 nm and NR	12 h at 25 °C	63
Magnetospirillum gryphiswaldense	Extracellular	25–55 nm and polydisperse	120 h at 28 °C	64

2.3. Synthesis of iron nanoparticles using fungi

The generation of FeNPs utilizing fungal species is known to be a sustainable production, which is an environmentally favourable technique for nanoparticle production.⁶⁶ The formation of iron NPs might be extracellular or intercellular (Fig. 4), depending on the type of microbial species used.⁶⁷ Aspergillus flavus and Aspergillus terreus are among the microbial species that have been used for the extracellular synthesis of iron nanoparticles. For example, Aspergillus flavus was used to synthesize spherical iron nanoparticles with an average size of 28–33 nm,⁶⁸ while Aspergillus terreus was utilized to produce iron nanoparticles in size of 40–100 nm.

Fig. 4.

Graphical representation of Iron nanoparticle synthesis using fungal isolate.⁷⁶ Created with Biorender.com., http://www.biorender.com (accessed on May 11, 2024).

Asperellum Trichoderma, Phrialemoniopsis ocularis, and Fusarium incarnatum have been also reported to be utilized in the production of iron nanoparticles.⁶⁹ It has been discovered that Aspergillus niger was utilized in the production of magnetite iron nanoparticles, which were assessed utilizing XRD and SEM and found to be spherical having a median dimension of 15–18 nm.⁷⁰

Iron nanoparticles produced show good hyperthermia phenomena in cancer. Rhizopus stolonifera was also employed to create iron nanoparticles that were supported by additional metabolites encompassing thiol, carboxylic acid, hydroxyl, and alkyl groups.⁷¹ Fungi that live inside plants Penicillium oxalicum were utilized in the developing of spherical FeNPs capable of catalyzing the breakdown of methylene blue color.⁷² A summary of other fungal species used in the synthesis of FeNPs, their characterization, and potential applications is provided in Table 3.

Table 3.

Various fungal species were reported for the production of iron NPs.

Organisms	Location of synthesis	Size and shape	Reaction time	Citation
Aspergillus niger BSC-1	Extracellular	20–40 nm, orthorhombic	3 h, 28 °C	73
Fusarium uncinatum	Extracellular	15–55 nm, spherical	5 min, 30 °C	69
Alternaria alternate	Extracellular	75–650 nm, quasi-spherical as well as rectangular	24 h, 30 °C	74
Alternaria alternata	Extracellular	∼9 nm nm cubical	72 h, 28 °C	75

2.4. Synthesis of iron nanoparticles using microbial bioflocculant

The synthesis of iron nanoparticles using microbial bioflocculants offers significant advantages over traditional chemical flocculants, particularly in terms of yield, purity, effectiveness, stability, and environmental impact.²⁷ Traditional chemical flocculants, such as aluminum sulfate and polyacrylamide, are effective but often generate harmful residues and non-biodegradable sludge that can contaminate water bodies and pose risks to human health.⁷⁷ In contrast, bioflocculants derived from natural sources do not leave toxic residues in effluents, contributing to a safer environment.⁷⁸

The cost of using alum for drinking water treatment varies significantly across different studies. For example,⁷⁸ reported that some studies indicated a cost of approximately USD 0.05 per cubic meter (m³) of treated water, while other studies noted a higher cost of USD 0.10 and a cost of USD 1.50 in some cases. For wastewater treatment, alum costs around USD 0.10 per m³ for treating meat processing effluent, whereas ferric chloride costs about USD 0.15.⁷⁹ In contrast, treating palm oil mill effluent with alum can reach USD 19 per m³, and using polyaluminium chloride (PAC) for leachate treatment can climb to USD 1.80.⁸⁰

Many studies have indicated that the production of bioflocculants is generally more costly than that of conventional chemical flocculants.81, 82, 83 However, the long-term benefits of bioflocculants especially regarding sludge management, non-toxic, and operational efficiency make them an increasingly attractive option for both drinking water and wastewater treatment. Moreover, bioflocculants can lead to lower operational costs in wastewater treatment due to their effectiveness at lower dosages.⁸⁴

Bioflocculants exhibit lower yields compared to chemical flocculants. For instance,.⁸⁵ Reported a yield of 2.7 g/L for bioflocculant production in the synthesis of FeNPs. Chemical methods generally yield more nanoparticles due to their synthetic nature; however, they often require careful management to mitigate environmental impacts. Thus, ongoing research is focused on optimizing the bioflocculant production processes to enhance yield and reduce costs.⁸⁶ Furthermore, researchers are now exploring the combination of bioflocculants with nanoparticles to increase both the yield and efficiency of their application. However, there has been limited research on the use of bioflocculants for the synthesis of iron nanoparticles. To the best of our knowledge, a review of recent literature reveals only three studies conducted between 2015 and December 2023 that focused on the green synthesis of iron nanoparticles using bioflocculants.^26,87,88 For instance,⁸⁸ employed iron sulfate (FeSO₄) as the metal precursor for FeNP synthesis. The authors dissolved 0.5 g of undiluted bioflocculant Alcaligenes faecalis in a 0.2 M solution of FeSO₄ along with 10 mL of 5.0 M sodium hydroxide (NaOH) to prevent nanoparticle aggregation (Fig. 5). The mixture was maintained at ambient temperature overnight, with nanoparticle formation confirmed by visual observation of color changes and subsequent analysis. The resulting solution was centrifuged at 5000 rpm for 15 min at 4 °C to collect the nanoparticles, which were then air-dried under vacuum at 25 °C for two days. This method illustrates the potential for optimizing bioflocculant production to enhance FeNP synthesis while minimizing environmental impact.

Fig. 5.

Graphical illustration of iron nanoparticle synthesis using microbial bioflocculant.⁸⁵

Shende and Mitra (2021)²⁶ reported the production of FeNPs utilizing purified Okra bioflocculant. The formation process involved washing and cutting Okra fruits into fine pieces, macerating them in a combination of purified water and methanol, and centrifuging the liquefied mixture to obtain the supernatant. The supernatant was then employed as a reducing agent in the creation of the FeNPs. Under the magnetic shaking circumstances, 5 mL of the bioflocculant was introduced in drops into the ferric NO₃ solution, and the resulting solution was put on a spinning orbital mixer for 2 h at 30 °C in the dark area. The decrease in the concentration of Fe ions was recorded every 24 h, and the spectra of UV–Vis rays were measured with a spectrophotometer. The synthesized iron nanoparticles were confirmed by physical observation and characterization.

Pullabhotla and co-workers (2024)⁸⁷ utilized the green procedure to produce FeNPs by employing iron sulfate (FeSO₄) as a metal precursor. The authors mixed 0.5 g of bioflocculant Pichia kudriavzevii and a dosage of 0.2 as well as 10 mL of 0.2 concentration of FeSO₄. To avoid nanoparticle aggregation, 10 mL of 5.0 sodium hydroxide were poured. The combination was let to remain in an empty dark place for 24 h before vacuum drying. The development of FeNPs was verified via examination, especially as color change as well as characterization.

3. Comparative analysis of bio-based synthesis methods for FeNPs

The synthesis of iron nanoparticles through biological methods has gained significant attention due to its eco-friendly nature and potential applications in various fields, including environmental remediation, medicine, and catalysis.⁷ This process utilizes different organisms such as plants, bacteria, fungi, and bioflocculants, each offering distinct advantages and disadvantages in terms of synthesis efficiency, size range, morphology, and stability. Understanding these differences is essential for optimizing synthesis processes tailored to specific applications.

Plants are a prominent source for synthesizing FeNPs, utilizing extracts that serve as reducing agents to transform iron salts into nanoparticles.⁸⁹ The presence of phytochemicals such as phenols and flavonoids plays a crucial role in this process. Plant-based synthesis is cost-effective and environmentally friendly, typically resulting in stable nanoparticles due to natural capping agents.⁹⁰ For instance, FeNPs synthesized from Phoenix dactylifera have demonstrated antimicrobial properties with significant efficacy against pathogens like Escherichia coli.⁹¹ However, the production rate can be slower compared to microbial methods, and variability in phytochemical concentrations due to seasonal changes can influence the properties of the synthesized nanoparticles.

Bacterial synthesis is characterized by rapid production and high yields. Bacterial strains such as Bacillus subtilis can reduce metal ions through enzymatic processes or metabolic activities, resulting in nanoparticles as small as 5 nm.⁹² This method allows for precise control over nanoparticle size and morphology, making it particularly advantageous for targeted drug delivery applications. Nevertheless, concerns regarding potential pathogenicity and variability in nanoparticle characteristics pose challenges that need to be addressed.

Fungi also play a significant role in the biogenic synthesis of FeNPs. Fungi utilize their enzymatic systems to reduce metal ions into nanoparticles, often resulting in irregularly shaped particles with high biocompatibility.⁹³ However, the longer cultivation times required for fungal growth compared to plants or bacteria can limit rapid production needs.⁹⁴

Bioflocculants, which are natural polymeric substances produced by microorganisms, facilitate the aggregation of particles in wastewater treatment and can also stabilize FeNPs during synthesis.⁸⁵ They are non-toxic and biodegradable, making them suitable for environmental applications such as enhancing the removal of contaminants from wastewater. However, their effectiveness can vary based on environmental conditions such as pH and ionic strength.⁹⁵

Stability is a critical factor influencing the application of FeNPs synthesized through these biological methods. Generally, plant-derived nanoparticles exhibit high stability due to natural capping agents that prevent aggregation.⁹⁶ Bacterial extracts can provide good sedimentation stability under certain pH conditions; however, stability may vary based on environmental factors.⁹⁷ Fungal-derived nanoparticles often require additional stabilizers to enhance their performance, while bioflocculant-stabilized FeNPs are influenced by pH and ionic strength.⁹⁸ Some of these factors are summarized in Table 4.

Table 4.

Shows a comparison of biological agents for FeNP synthesis.

Biological agent	Types of FeNPs produced	Size range (nm)	Shape	Advantages	Disadvantages	Stability factors	Citation
Plants	Zero-valent iron (ZVI), Iron oxide nanoparticles (IONPs)	10–100	Spherical	Eco-friendly; easy to source; no toxic chemicals involved; simple extraction process, high stability due to natural capping agents	Slower production rate compared to microbes; variability due to seasonal changes in phytochemical concentrations	Generally high stability due to natural capping agents from plant extracts	91
Bacteria	ZVI	5–50	Rod-like	High yield; rapid synthesis; specific targeting for applications like drug delivery	Potential pathogenicity; variability in nanoparticle characteristics	Stability can vary; extract-stabilized FeNPs exhibit good sedimentation stability at specific pH levels	92
Fungi	IONPs	20–200	Irregular	High biocompatibility; scalable production; produce uniform shapes; efficient enzyme systems for nanoparticle formation	Longer cultivation time; complex growth requirements	Moderate stability; often requires additional stabilizing agents	99
Bioflocculant	ZVI, IONPs	Varies widely	Varies	Non-toxic; biodegradable; effective in wastewater treatment; enhances removal of contaminants	Production may require specific conditions; effectiveness varies with environmental factors	Stability influenced by pH and ionic strength of the medium	100

4. Factors that affect the synthesis of iron nanoparticles

There are various factors affecting the synthesis of metal nanoparticles (Fig. 6). For instance, the nature of the plant species involved, the type and concentration of biomolecules present in it, and the reaction conditions such as pH, temperature, the concentration of reactants, and reaction time.¹⁰¹ Variation in any of these parameters can alter the morphology and yield of nanoparticles. Therefore, understanding these physico-chemical properties is crucial for optimizing the synthesis process and achieving the desired nanoparticle attributes. Some of these factors are discussed in detail below.

Fig. 6.

Factors affecting the synthesis of iron nanoparticles.

4.1. The effect of temperature and time

Temperature is a crucial factor that significantly influences the biological methods of the formed nanoparticles.¹⁰² It also plays an important role in determining the morphology and yield of nanoparticles. The optimal temperature range for the green production of FeNPs utilizing bioflocculants is generally between 25 and 100 °C.¹⁰³ However, several challenges arise from temperature variations, particularly due to the volatility of secondary metabolites produced by microbial bioflocculants, which are essential for the bioreduction of iron ions. Consequently, many researchers prefer conducting syntheses at ambient temperatures to preserve these metabolites' stability.¹⁰³

In the study by,¹⁰⁴ the synthesis of hematite iron nanoparticles (α-Fe₂O₃) from Bacillus cereus SVK1 was observed to be temperature-dependent. The researchers noted that the synthesis was completed in 48 h at 37 °C. However, they also reported that increasing temperatures above 40 °C resulted in a significant decline in nanoparticle production due to the inactivation of biomolecules responsible for reducing iron precursors. Nanoparticles of different shapes such as triangular, pentagonal, spherical, and rod-shaped were mostly formed at 25 °C, while at higher temperatures mainly spherical nanoparticles were obtained.¹⁰⁵ The size of the produced FeNPs from Bacillus cereus SVK1 was found to be 15–40 nm with a hexagonal structure.

Bibi et al. (2019)¹⁰⁶ reported successful fabrication of iron nanoparticles using Punica granatum seed extract at 70 °C, achieving effective synthesis within 24 h. The spherical shape with uniformly distributed and particle size range of 25–55 nm was observed for the produced Fe₂O₃ nanoparticles.

Radini et al. (2018)¹⁰⁷ indicated that increasing temperatures from 40 °C to 80 °C correlated with enhanced antioxidant activity, which is beneficial for the reduction process during nanoparticle synthesis. This increase in antioxidant activity can facilitate improved synthesis rates but must be balanced against potential biomolecule degradation.¹⁰⁷

Other studies showed that the flocculating activity of bioflocculants decreases with increasing temperature. For instance,¹⁰⁸ reported a decrease in flocculating activity from 77.7 % at 50 °C to approximately 70 % at 80 °C. The bioflocculant maintained about 70 % of its flocculating activity at this temperature due to its polysaccharide composition, which contributes to its thermal stability.¹⁰⁸

In another study, it was found that FeNPs retained over 86 % of their flocculation activity even at 100 °C, highlighting their superior thermal stability compared to the bioflocculant that was used to synthesize FeNPs.⁸⁵

The decline in flocculation activity for bioflocculants at elevated temperatures emphasizes the need for careful temperature management during synthesis. For example, studies have documented that bioflocculants produced by various bacterial strains maintained their effectiveness only up to certain temperatures, with significant drops in performance noted beyond.^109,110

Therefore, It can be seen that the synthesis time for nanoparticles can vary significantly with temperature changes. For instance, Dodonaea viscosa extract enabled nanoparticle production in just 5 min at ambient temperatures, while other extracts may require up to 24 h for optimal formation.¹¹¹ This variability indicates the need for careful optimization based on specific extract properties.

4.2. The effect of concentration of the precursor salt

The concentration of the precursor salt is a critical variable that significantly influences the production and characteristics of iron nanoparticles. Research indicates that variations in precursor concentration can lead to notable changes in both the rate of nanoparticle synthesis and their physical properties.¹¹²

Identifying the optimal concentration of bioflocculant used to synthesize nanoparticles is also crucial for maximizing flocculation efficiency. Both low and high concentrations of bioflocculants can lead to poor flocculation performance. There may be insufficient coverage for effective flocculation at low concentrations, while at high concentrations, the inability of bioflocculant molecules to stretch across the liquid medium can limit their effectiveness.^112,113 This decline could be due to increased viscosity, which reduces particle interaction¹⁰⁹.¹⁰⁸ found that optimal flocculation activity was achieved at a concentration of 0.1 mg/mL in the bioflocculant produced by a consortium of Halomonas sp. Okoh and Micrococcus sp. Leo. Beyond this threshold, high viscosity inhibited particle settling.

Pullabhotla and co-workers (2023)²⁷ further noted that high concentrations of bioflocculants increase solution viscosity, hampering nanoparticle movement and effective flocculation. Excess bioflocculant molecules create a thick medium that obstructs flocs settling, ultimately reducing synthesis efficiency. The researcher reported a highly efficient flocculating activity of 85 % achieved at 0.6 mg/mL dosage in the synthesized Fe nanoparticles using a bioflocculant Pichia kudriavzevii.

On the other hand, Guo et al. (2017)¹¹⁰ stated that the concentration of precursor salts also plays a vital role in maintaining nutrient balance within the reaction medium, which influences the size. High concentrations can lead to nutrient saturation, inhibiting microbial growth and reducing bioflocculant production. An optimal carbon-to-nitrogen ratio is essential for maximizing bioflocculant yield; deviations from this ratio due to high precursor concentrations can negatively impact microbial health and flocculant production efficiency.¹¹⁰ Moreover, higher precursor concentrations tend to produce larger nanoparticles due to increased nucleation events followed by aggregation.¹¹⁴ This size variation can significantly affect the reactivity and application of FeNPs in various fields, including wastewater treatment and catalysis. A rise in precursor dosage accelerates production rates, while a decrease can delay the bio-reduction process during iron nanoparticle synthesis.¹¹⁵ This delay occurs when there are insufficient biomolecules in the extract to interact with the precursor for nanocrystal development.¹¹⁵

Khalil et al. (2017)¹¹⁶ highlighted that the dosage of plant extract used in synthesizing iron nanoparticles significantly influences particle size and dispersion. The dosage correlates with the number of metabolites available as stabilizing agents. For example, increasing the dosage of Tangerine peel extract from 2 to 6 % reduced Fe nanoparticles size from 200 to 50 nm, however, further increases led to agglomeration.¹¹⁷

The anti-oxidant-reducing capacity of plant extracts plays an important role in the green production of iron nanoparticles.¹¹⁸ The more active the antioxidant properties, the more productive the synthesis process becomes. Various assays can determine the antioxidant ability of these extracts, including ferric-reducing antioxidant power (FRAP), the Foline-Ciocalteu method, and DPPH radical scavenging tests.¹¹⁹

4.3. Effect of pH

The influence of pH on the synthesis of iron nanoparticles using microbial bioflocculants has been extensively studied.²⁶ Research indicates that variations in pH significantly affect both the size and morphology of synthesized nanoparticles. For instance,⁸⁵ reported that at a pH of 6, the average size of synthesized iron nanoparticles was approximately 30 nm, while at pH 8, this size increased to about 60 nm due to enhanced aggregation.

Maintaining an optimal pH during synthesis is challenging due to the inherent buffering capacity of bioflocculants, which can lead to fluctuations that complicate the process.¹²⁰ found that even minor pH variations (±0.5) could result in significant changes in nanoparticle size and distribution, with larger particles being formed at higher pH levels. The researcher observed that at pH 7, FeNPs exhibited a narrow size distribution with an average diameter of 40 nm, while at pH 9, the size distribution broadened significantly, leading to particles exceeding 100 nm.

Such fluctuations not only introduce variability in nanoparticle characteristics but also impact their effectiveness in applications such as wastewater treatment. Zúñiga-Miranda et al. (2023)¹²¹ quantified this effect by demonstrating that iron nanoparticles synthesized at optimal pH levels (6–7) showed up to a 75 % removal efficiency of contaminants in wastewater, compared to only a 50 % removal rate at higher pH levels (8–9). Therefore, precise regulation and control of pH are crucial for achieving desired outcomes in nanoparticle synthesis.

5. Characterization of FeNPs

Nanoparticles possess properties that make them useful in a variety of applications. Biomedical applications, in particular, need extensive characterization to establish if they are acceptable for the intended use. This is performed by using a variety of methods and equipment that can offer the necessary information.¹²²

5.1. Fourier transform infrared (FT-IR) spectrometry

Fourier transform infrared spectroscopy (FT-IR) was also used to confirm the potential role of nanoparticle production. It determines the functional groups on the surface of nanoparticles by detecting chemical bond excitations. The molecular data obtained give structural and conformational changes. Wavenumbers showed the interaction between the capping agent and the FeNPs.¹²³ However, in biosynthesized FeNPs, the presence of biomolecules from the biological source can lead to overlapping absorption bands. This overlap complicates the interpretation of spectra, making it difficult to distinguish between functional groups associated with the nanoparticles, particularly FeNPs in this study and those from the bioflocculants themselves.^124,125

For instance, Maaza and co-workers (2017)¹¹⁶ analyzed the functional groups of iron oxide nanoparticles synthesized from Sageretia thea extract using FT-IR spectroscopy. Their funding indicated that while the FT-IR spectrum recorded in the 4000-400 cm⁻¹ revealed significant absorption features, the broad O–H stretching observed around 3400 cm⁻¹ was attributed to phenolic compounds present in the extract. This overlapping signal complicated the identification of specific vibrational modes related to Fe–O bonds, which were detected at approximately 500 cm⁻¹. Other peaks corresponding to C–O, C O, and CN functional groups were also noted around 1100-1200 cm⁻¹, 1600 cm⁻¹, and 2200 cm⁻¹, respectively, further illustrating how biomolecular contributions can interfere with accurate spectral analysis.

Hussain et al. (2023)¹²⁶ used Ficus palmata extract to study the production of FeNPs and discovered three FT-IR peaks at 1087.29 cm⁻¹, 520.34 cm⁻¹, and 435.70 cm⁻¹. These peaks revealed that the phytochemicals in the plant extract encapsulated the FeO NPs. The peaks at 520.34 cm⁻¹ and 435.70 cm⁻¹ indicated the presence of Fe–O linkages, while the peak at 1087.29 cm⁻¹ was attributed to phytochemicals containing C–O linkages. The peaks occurrence suggest that phytochemicals in the plant extract encapsulated the iron oxide nanoparticles. However, the peaks could also overlap with similar vibrational modes from other biomolecules present in the extract.

The data provided were similar to Darwish et al. (2019)¹²⁷ in the synthesis of iron oxide by Papa ver somniferum L. where a peak at 108.29 cm⁻¹ was found which might be related to the existence of phytochemicals with C–O linkages.

Majeed et al. (2021)¹²⁸ reported the functional groups of the produced Fe oxide nanoparticles from Proteus vulgaris ATCC-29905 in the infrared range of 500–4000 cm⁻¹. The researchers observed significant peaks at 3418, 1643, 1556, 1404, and 1072 cm⁻¹. The peak at 3418 cm⁻¹ indicates the O–H stretch of hydroxyl groups, which are alkane C–H stretches. The N–H bend of amines corresponds to the peak at 1643 cm⁻¹, the N–H bend of amides to the peak at 1556 cm⁻¹, the C C stretch to the spike at 1404 cm⁻¹ and the C–O stretch of alcohols to the peak at 1072 cm⁻¹. These functional groups have a role in the formation and stability of FeNPs.¹²⁹

Adeleye et al. (2020)⁷¹ utilized Rhizopus stolonifera extract to produce Fe nanoparticles with maxima ranging from 617.4 cm⁻¹ to 3908.6 cm⁻¹. The fingerprint region at 750 cm⁻¹ and 1100 cm⁻¹ donates the CH (alkane and out of the planned link) and C–O (carboxylic) groups, respectively. The chemical structures responsible for nanosizing, according to the researchers, include carboxylic acid (C O), mercaptans (S–H), and free hydroxyl (OH), which are seen at 1700 cm⁻¹, 2500 cm⁻¹, and 3750 cm⁻¹, respectively. The C OH and S–H compounds are connected with the fungus employed and hence serve to cap the NPs following reduction. Carboxyl, sulfhydryl, and mercaptans (phenols and alcohols) are believed to be implicated in the transition of metallic ions to NPs.

Mathur et al. (2021)⁷² found absorption peaks in the FT-IR spectrum of FeNPs at various wavelengths for the synthesized Fe nanoparticles utilizing Penicillium oxalicum fungal. According to the authors, the signal at 3430.70 cm⁻¹ suggested the probable O–H vibration during the stretching of phenolic categories, that could be implicated in the production and stability of NPs. Another significant signal, 1627.71 cm⁻¹, was detected, showing C C aromatic band interactions. A small peak at 2923.17 cm⁻¹ was also discovered by the researchers, which indicated the C–H motion of aliphatic hydrocarbons. Other small peaks at 1121.46 cm⁻¹ and 619.00 cm⁻¹ suggested C–O bonds and Fe–O stretching bonds, respectively. The existence of these various molecules and functionalities implies that developed FeNPs might form and stabilize.

Sarkar et al. (2017)⁷⁴ reported the use of Fourier transform infrared (FT-IR) spectroscopy to analyze the absorption spectra of biosynthesized Fe₂O₃ nanoparticles by Alternaria alternata. The FT-IR spectra showed several peaks that correspond to different chemical bonds and functional groups. The peaks at 3430 and 2920 cm⁻¹ were brought about by pyranoid ring O–H stretching and asymmetric aldehydic stretching vibration of C–H, respectively. The values measured at 1415 and 1610 cm⁻¹ suggested C–C compounds formed from aromatic ring structure and the inverted carbonyl (-C O) chain expanding movement, respectively, which possibly have entered the culture filtrate from the fungal cell. The attachment of a C O group with the NPs was linked to the displacement of the maxima around the 1600 cm-1 spectrum. The amide III band of the random coil of protein was identified by the FT-IR intensities between 1240 and 1260 cm⁻¹. The Fe oxide NPs’ FT-IR spectra revealed the existence of absorbing bands located near 522 and 627 cm⁻¹, and these were indicative of Fe–O vibrational patterns. The soluble components in the microbial filtrate might have worked as protective coatings, limiting NPs clumping in solution and so performing an important role in the extracellular production and shape of the quasi-spherical magnetite NPs. The research also reveals that microscopic fungi can produce various extracellular NPs via a mechanism containing an enzyme called NADH-reductase.

Priya et al. (2017)¹³⁰ utilized the Actinodapne madraspatna Bedd leaves extract to synthesize Fe nanoparticles. The FT-IR measurements were done to investigate the involvement of functional groups in the synthesis of FeNPs. Prominent peaks were obtained by the formed FeNPs at 3422, 1645, 1404, and 1091 cm⁻¹. The prominent band at 3422 cm-1 reflected the vibration caused by the stretching of O–H group of phenolic substances. The strong signal at 1645 cm⁻¹ is attributable to polyphenol C C ring stretching. The in-plane bending vibrations of the –OH group in the phenol and ester link of tannin caused the peaks at 1404 and 1091 cm⁻¹, respectively.

The presence of these functional groups suggested the crucial of the synthesized FeNPs during applications. The synthesized FeNPs showed a minor shift, showing that the Actinodaphne madraspatna Bedd leaves extract served as both a reducing and protecting agent in the natural synthesis process. The nanoparticles' stability was evaluated utilizing zeta potential measurements. The zeta potential of the produced FeNPs was 21.3 mV, showing acceptable stability, which was due to the phytochemoities bound to the NPs’ surface. Furthermore, even after a week, no appreciable flocculation or sedimentation was found, indicating that the polyphenol-capped NPs were resistant to agglomeration.

Tsilo et al. (2023)²⁷ reported the FT-IR functional groups of the produced FeNPs using microbial bioflocculant from Pichia kudriavzevii. The authors observed the signal at 3244 cm⁻¹ reflects the hydroxyl (OH) and amide (-NH₂) groups. The occurrence of these functionalities proves the production of as-synthesized iron NPs. The minor signal of the aliphatic linkages is explained by the peak at 1587 cm⁻¹. The detection of a signal at 978 cm⁻¹ suggests the development of an amide group. The presence of oscillatory maxima at 567 cm-1 confirms the presence of the –OH group, which agrees with observed C–O stretching. The occurrence of vibrational peaks at 567 cm⁻¹ confirms the presence of the OH group, which is compatible with the C–O expansion reported in alcohols.

5.2. Ultraviolet–visible spectrophotometry

Ultraviolet–visible spectrophotometry is a reliable method for characterizing produced metal NPs. This approach is critical in the production of FeNPs because it provides the first confirmation of nanoparticle biosynthesis, characterizes their optical characteristics, monitors synthesis and stability, and provides qualitative information on the nanoparticles.¹³¹

Jamzad and colleagues performed an experiment to synthesize iron oxide nanoparticles using a biogenic synthesis method, specifically employing the aqueous extract of the plant Laurus nobilis. The initial characterization of the synthesized Fe nanoparticles was conducted using UV–Vis spectroscopy, which revealed a maximum absorption peak at approximately 285 nm, indicating successful synthesis of FeNPs.¹³²

According to a study, the UV spectrum of iron nanoparticles (FeNPs) synthesized using the bulb extract of Murraya koenigii exhibited broad absorption peaks between 275 and 500 nm. This range indicates the successful formation of FeNPs, as the absorption characteristics are indicative of their optical properties.¹³³

Adeleye et al. (2020)⁷¹ utilized ultraviolet–visible spectrophotometry to confirm the formation of the synthesized FeNPs from Rhizopus stolonifera. The researchers revealed that the synthesized FeNPs had the highest point of absorbance around 325 nm, which accords with earlier findings suggesting the spectrum interval of absorbency of FeNPs identified by UV–Vis existed at 200 and 900 nm.¹³⁴

Demirezen et al. (2019)¹³⁵ observed the UV absorption peaks detected at 205 and 291 nm in the synthesized Fe nanoparticles from Ficus carica. These peaks were attributed to the hydrolysis products of ferric ions that had precipitated on the surface of the nanoparticles.¹³⁶ The peaks confirmed the creation of Fe oxide NPs.

The findings from¹³⁷ indicate that the peaks at 210 nm and 289 nm in the FeNPs synthesized from Ageratum conyzoides correspond to Fe(H₂O)3 + 6 and Fe (OH) (H₂O)2 + 5 species in the FeCl₃ solution. The emission spectrum showed significant changes upon adding plant extract to Fe³⁺ ions, with a decrease in the first peak at 220 nm and a disappearance of the peak at 298 nm, indicating FeNP formation. The color change from bright yellow to dark reflects surface plasmon resonance activation, confirming the synthesis of Fe nanoparticles with a characteristic peak around 200 and 300 nm.

5.3. Morphology analysis of FeNPs using SEM

SEM is another procedure used to characterize the morphology of nanoparticles through direct visualization. This technique is based on electron microscopy and offers several advantages for morphological and size analysis; however, it is also associated with several disadvantages, such as the ability to provide only limited information about the size distribution and true population average.¹³⁸ The shape of iron NPs is important during flocculation.¹³⁹ Materials having a higher surface area have a stronger propensity to bind suspended particles, resulting in greater elimination efficiency and flocculating activity.

Önal et al. (2019)¹⁴⁰ reported the successful synthesis of iron nanoparticles via a green procedure employing loquat leaf extract as a reducing renewable agent, which was further confirmed by SEM images. By using the image J software program, the mean particle size of the synthesized FeNPs was found to be 89 nm. The SEM images indicated that the formed nanoparticles have an irregular spherical and porous morphology. Additionally, the nanoparticles were observed to be quite agglomerated.

Bhutto et al. (2023)¹⁴¹ reported the synthesized iron nanoparticles from Ixoro coccinea leaf extract to have a quasi-spherical shape with clusters. The particles were observed to be agglomerated, with a rough and porous surface.¹⁴² described the SEM and the designed FeNPs were found to possess nest-like spheres exhibiting a typical diameter of around 40 nm in TEM images.¹⁴³ stated they successfully synthesized iron nanoparticles using Aesculus hippocastanum seed extract. The formation of iron nanoparticles gives a spherical form with a typical size of 9 ± 7 nm.

Zafar et al. (2022)¹²⁹ reported the formed Fe nanoparticles from the biomass of Enterobacter to reveal the hexagonal and rectangular morphology that was confirmed by the SEM micrograph with an average diameter of 23 nm.

According to the study by Amutha et al. (2018),¹⁴⁴ SEM analysis of the synthesized iron oxide nanoparticles exhibited the size of FeNPs in the range of 58–79 nm, while the morphology of the produced FeNPs was spherical. Another example includes SEM analysis of FeNPs with a diameter of 7.7 nm synthesized by using Passiflora foetida extract.¹⁴⁵

Pullabhotla and co-workers (2020)¹⁵ revealed that the synthesized FeNPs from a bioflocculant have a granular-like morphology with an average size of 28–32 nm and are agglomerated together which might be owing to stresses among the fragments.

5.4. A typical X-ray diffraction analysis

Employing X-ray diffraction (XRD) for the characterization of Fe nanoparticles is a crucial step in comprehending their properties and potential applications. XRD pattern confirms the crystalline nature of the Fe nanoparticles and offers valuable information about their crystalline structure and composition.

The XRD shape of the synthesized iron nanoparticles from Moringa oleifera displays points with 2ɵ values at 24.16, 30.4, 33.24, 35.8, 43.5, 54.1, and 57.4, which correspond to 012, 104, 200, 311, 511, and 440 crystalline peaks.¹⁴⁶ The broadening peak in the XRD pattern is related to the size and purity of the nanoparticles and can be predicted using the Debye-Scherrer equation. The particle size ‘d’ can be calculated using the equation d = KI/(b cosq), where k represents the Sherrer constant (0.9), I represent the X-ray wavelength (0.15406 nm), q represents the Bragg diffraction angle, and b represents the width of the XRD peak at half-height, which is determined as the full width at half-maximum peak (FWHM) by Gaussian fitting.¹⁴⁶

Bhutto et al. (2023)¹⁴¹ detailed the XRD spectrum of Fe₃O₄ NPs from Ixoro coccinea which revealed six distinct diffraction peaks location at the point of diffraction (2ɵ): 30.22, 35.60, 45.06, 54.10, 57.68, and 63.10, corresponding to the reflection planes (220), (311), (222), (400), (422), and (511), respectively. These diffraction peaks indicate the highly crystalline nature of the particles and confirm the presence of magnetite's pure cubic phase. The diffraction peaks are well-matched with the Joint Committee on Powder Diffraction Standards (JCPDS) card No. 19–0629. The authors found that no XRD peak was detected for impurities.

Önal et al. (2019)¹⁴⁰ observed the XRD pattern peaks of the formed iron NPs from loquat at 2ɵ = 25.9, 28.3, and 35.6 correspond to iron oxyhydroxide, maghemite (y-Fe₂O₃), and magnetite (Fe₃O₄), respectively. Additionally, an intensity peak at 2ɵ = 17.56 was identified as organic matter in polyphenols, as reported in the literature. The relatively weak peaks in the XRD diagram indicate that the synthesized nanoparticles have amorphous grains.

Shende and Mitra (2021)²⁶ reported that the X-ray diffraction (XDR) analysis of produced Fe nanoparticles from bioflocculant okra displayed distinct maxima at 2ɵ angles of 28.28 and 64.54, corresponding to the 311 and 564 planes, accordingly. The intensity of these peaks indicated a substantial amount of crystallization in the Fe NPs. These findings are consistent with those published by¹⁴⁷ who discovered maxima at 2ɵ numbers of 25 and 78.

Zafar et al. (2022)¹²⁹ evaluated the microscopic structure of the formed FeNPs from Enectobacter using the EDX profile. The XRD findings of the produced iron NPs, which consist of the JCPDS file 019–0629 are summarized as follows. The XRD profile displays individual points at 2ɵ of 20.75, 31.71, 36.59, 38.89, 45.45, 53.49, 56.44, and 61.11, matching reflections of the centered-face cubic point of (111), (220), (311), (222), (400), (422), (511), and (440) lattice planes, correspondingly. The researchers revealed that the presence of these peaks confirmed that the synthesized FeNPs are crystalline in nature. The mean size of the crystals of the made FeNPs at (111) was calculated using the Debye-Scherrer formula which proved to be 20.7 nm.

5.5. SEM-EDX analysis

The EDX analysis of the produced Fe nanoparticles from the Aesculus hippocastanum extract revealed the atomic percentages of different elements present in the sample. The composition was determined to be 71.35 oxygen, 8.46 % iron, 19.19 % chlorine, and 1.01 % potassium (K). This high percentage of oxygen indicates that the nanoparticles are in the form of iron oxide. The presence of iron and oxygen signals in the EDX spectrum further confirmed the existence of elemental iron and oxygen in the nanoparticles. It is important to note that the sample's homogeneity and the degree to which of the oxide coating can also affect the analysis results.¹³⁵

Jeyasundari et al. (2017)¹⁴⁸ stated the produced FeNPs from the Psidium guajava plant revealed EDX elements which indicated the reduction of iron ions to elements iron. The EDX profile of the FeNPs demonstrates a strong signal to the Fe atom, indicating its crystalline properties. The signals for C and O are observed, which may originate from the biomolecules capped to the surface of the FeNPs.

Demirezen et al. (2018)¹⁴³ reported the elemental composition percentage of the produced FeNPS from Aesculus hippocastanum seed extract to give EDX of 56.18 % oxygen, 12.89 % iron, and 30.93 % chlorine. The presence of chlorine was due to the FeCl₃ that was utilized in the iron synthesis protocol. The appearance of oxygen content that was found at a larger amount than the metallic iron gives information about oxide iron forms which also confirmed the development of FeNPs.

Madivoli et al. (2019)¹³⁷ stated that the composition of the fabricated FeNPs from Ageratum conyzoides extracts was analyzed using the EDX spectrum, which showed that they are primarily composed of Fe, O, C, and Cl. The existence of C and O in the synthesis can be explained by polyphenols and other Carbon-having compounds in the extracts of plants employed, whereas Cl came from the precursor FeCl₃. Because of the other elements such as K and Ca, which are connected to impurities of the starting leaves, the researchers revealed that iron nanoparticles did not exhibit metal features like magnetism.¹⁴⁹

Hussein and colleagues stated the synthesis of magnetite nanoparticles (FeNPs) using extracts from onion, potato peels, tea waste, and moringa leaves. Energy-dispersive X-ray (EDX) analysis was performed on the surface of the FeNPs, revealing the elemental composition of the prepared nanoparticles. The EDX profile indicated intense peak signals of iron with Kα peaks at 6.5 keV, 6.2 keV, 0.9 keV, and 0.7 keV for moringa leaves and potato peels, onion, and tea waste. Additionally, signals for oxygen and carbon were observed; the presence of C and O peaks was attributed to polyphenols or other carbon- and oxygen-containing compounds in the natural materials' extracts. The existence of elemental iron and oxygen confirmed that the nanoparticles were predominantly present in oxide form. The detected elemental percentages were carbon (C) at 12 %, iron (Fe) at 52 %, and oxygen (O) at 28 %. These results indicated that the extracts from moringa leaves and potato peels were more efficient than those from onion and tea in forming magnetite iron nanoparticles.¹⁵⁰

5.6. TEM analysis

TEM was utilized to investigate the shape and size distribution of the formed Fe nanoparticles. The researchers reported the particles in the TEM picture of the produced FeNPs from Plantago major leaf extract to be spherical and have sizes ranging from 4.6 to 30.6 nm. According to the particle size analyzer previously determined by the particle size analysis (PSA) procedure, the hydrodynamic diameter of iron NPs ranged from 340 to 6000 nm, with an average particle size of 870 nm surrounded by a layer of biological material. Therefore, it can be seen that the particle size determined using the PSA technique differs greatly from the particle size reported by TEM microscopy.¹⁵¹

Shi et al. (2021)¹⁵² reported the synthesis of chitosan-modified iron oxide magnetic nanoparticles and investigated their potential anti-lung cancer effects. The study detailed the preparation and characterization of Fe immobilized on these nanoparticles, which were found to be roughly globular in shape, with sizes ranging from 15 to 20 nm, consistent with findings from field emission scanning electron microscopy (FESEM) analyses.

Pullabhotla and co-workers (2021)²⁴ show transmission electron micrographs of the synthesized Fe nanoparticles from microbial bioflocculant. The author described a chain-like aggregation with densely clustered particles, which might be related to the magnetic properties found in these chemicals. The coupling of the electrons of the linked particles might explain the agglomeration.⁸⁵

Shende and Mitra (2021)²⁶ conducted a study on the green synthesis of Fe nanoparticles utilizing bioflocculant extracted from Okra. The synthesized FeNPs revealed a mean diameter of 50 nm and agglomerated spherical particles.

6. Antimicrobial activity effect of iron nanoparticles

Microbial infections have increased dramatically in recent decades, and the rise of multidrug-resistant (MDR) bacteria has become a global health hazard.¹⁵³ To address this issue, nanotechnology-based therapeutics for illness diagnostics and the creation of innovative therapeutic medications against various diseases have been developed.¹⁵⁴ The use of green-produced iron nanoparticles (FeNPs) versus several infectious types of bacteria is one such treatment.¹⁵⁵ Researchers are interested in tiny particles as possible antibacterial agents due to their biodegradability, safety, and eco-friendliness.¹⁵⁶ Although the antibacterial efficacy of such NPs has not been shown, it is thought that they destroy germs in the same manner as their chemical counterparts do.¹⁵⁷ The inclusion of capping agents, which themselves have antimicrobial potency, as well as multiple mechanisms, such as membrane damage, organelle distraction, biomolecular deformation, and interference with protein formation in cells of bacterial, is an added benefit of biosynthesized NPs.¹⁵⁴

According to numerous research-based studies, FeNPs have evident antimicrobial activities against bacterial cultures including Escherichia coli, Staphylococcus aureus, Salmonella enteric, Pseudomonas aeruginosa, Streptococcus pyogenes, Aeromonas hydrophila, Klebsiella pneumonia, Bacillus cereus, and Enterococcus faecalis.^158,159

Batool et al. (2021)⁹¹ investigated the antimicrobial activity of iron nanoparticles (FeNPs) synthesized using an aqueous extract of Phoenix dactylifera. The study evaluated the efficacy of the synthesized FeNPs against several bacterial strains, including Escherichia coli, Bacillus subtilis, Micrococcus luteus, and Klebsiella pneumoniae. The results indicated that biosynthesized FeNPs exhibited varying levels of antimicrobial potential against the different bacterial strains tested. Notably, the maximum zone of inhibition recorded was 25 ± 0.360 mm against Escherichia coli, demonstrating the effectiveness of the green-synthesized FeNPs as potential antimicrobial agents.

Irshad et al. (2017)¹⁶⁰ reported on the antibacterial activity of iron oxide nanoparticles (IONPs) synthesized from Punica granatum peel extract are promising. The study indicates that these nanoparticles exhibited a significant zone of inhibition measuring 22 ± 0.5 mm against Pseudomonas aeruginosa, a notable pathogen known for its antibiotic resistance.

Another study by Elkhateeb et al. (2024)¹⁶¹ evaluated the antibacterial activity of the biosynthesized iron oxide nanoparticles prepared from the leaves of cabbage, turnips, and moringa leaves against Escherichia coli (ATCC 25922) and Staphylococcus aureus (PTCC 2592) using the penetration diffusion test. The researchers prepared a bacterial suspension (10⁸ cfu/mL) and spread it on nutrient agar plates. Wells were created in the agar, and 50 μL of ethanolic Fe oxide extract at concentrations of 25, 50, 100, and 200 ppm were added. After incubating at 37 °C for 24 h, the inhibition zones were measured. The results showed that the biosynthesized FeNPs exhibited the highest antibacterial activity against E. coli, with a maximum inhibition zone of 8.0 mm.

Jagathesan and Rajiv (2018)¹⁶² reported significant findings regarding the antibacterial properties of iron oxide nanoparticles synthesized using Eichhornia crassipes. The study revealed that the highest zone of inhibition was observed at a concentration of 100 μg/mL for the biosynthesized Fe oxide NPs against Staphylococcus aureus and Pseudomonas fluorescens. Some of the antimicrobial activities of dissimilar microbial species are given in Table 5 below.

Table 5.

Several species of bacteria were tested for resistance to microbe-mediated FeNPs.

S. no	Species	Inhibition method	Activity against	Citation
1	Proteus vulgaris	Disc diffusion procedure	Escherichia coli, Vibro cholera, Salmonella typhi, and Staphylococcus epidermidis	128
2	Streptomycetes (SRT12)	Disc diffusion procedure	Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Shigella flexneri, and Escherichia coli	55
3	Proteus mirabilis	Well-diffusion procedure	P. aeruginosa, Clostridium perfrigenes, Aspergillus brasiliensis, and Candida albicans	60
4	Alternaria alternata	Well-diffusion procedure	Bacillus subtilis	75
5	Aspergillus flavus	Diffusion agar method	Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus Fumigatus	68
6	NPs-penicillin G conjugates	Disc diffusion technique	Staphylococcus aureus	163

Bacterial cells are mostly killed by reactive oxygen species (ROS) such as superoxide radicals (O²⁻), hydroxyl radicals (⁻OH), hydrogen peroxide (H₂O₂), and singlet oxygen (O₂).⁶⁶ These ROS cause serious harm to the microbial cell's nucleic acids and proteins. Nanoparticles bind to proteins linked to membranes (thiol groups), causing oxidative harm, protein degradation, and lipid resistance.¹⁶⁴ This gradually kills the bacteria. Nanoparticles can impair the integrity of structures and the architecture of cells in addition to disrupting membranes.¹⁵⁷ Antibacterial Iron nanoparticles (FeNPs) can destroy both Gram-negative and Gram-positive bacteria. FeNPs, on the other hand, are more efficient against Gram-positive bacterial strains because of the complicated structure of Gram-negative bacteria.¹⁶⁵ As shown in Fig. 7, there are eight ways in which FeNPs can damage bacteria cells. These include (1) cell wall damage by intervening with regular homeostasis, (2) cell membrane destruction triggered by the disorientation of the lipid bilayer through ROS creation, (3) ion channel misconfiguration arises once the protein transporter remains injured, (4) physiological enzyme is disturbed through inhibition of their domains catalytic, (5), the nucleic acid is damaging resulting to fragmentation of DNA and RNA, (6) biomolecules interruption happens, particularly, in proteins and NPs, (7) denaturation of proteins through ROS, and (8) damage of organelles, especially, mesosomes.⁶⁶

Fig. 7.

Illustrates the antibacterial potential of iron nanoparticles (FeNPs).⁶⁶ Created with Biorender.com., http://www.biorender.com (accessed on May 11, 2024).

7. Anticancer action of FeNPs

Cancer is another greatest cause of mortality worldwide, after heart disease.¹⁶⁶ There is currently, no effective remedy for cancer, although the search for innovative anticancer drugs is ongoing.¹⁶⁷ Recently, nanoparticles have been used in a variety of disease management applications, including cancer treatment, given their high stability, biologic compatibility, and selectivity against different cancer cells.^168,169 Iron nanoparticles are currently utilized to treat a variety of diseases, including breast cancer, glioblastoma tumor, cancer of the liver, promyelocytic leukaemia, and prostate cancer.⁵⁵ Fig. 8 depicts FeNPs’ antitumor potential. FeNPs greatly increased antitumor efficacy when combined with other anticancer medicines.¹⁷⁰ Using their magnetic hyperthermia perspective, they can be employed to selectively destroy malignant cells. The therapeutic properties of microbe-mediated FeNPs are attributed to their ability to increase the stress response and damage cells by limiting the division of cells and disrupting the macromolecule structure, resulting in the death of cells through triggering apoptosis.¹²⁸ Although FeNPs have shown great cytotoxic activity towards various kinds of cancer cells, their harmful effect in humans must be addressed.

Fig. 8.

Illustration of the potential of microbes-mediated Fe nanoparticles to combat cancer. In 1, 2, 5, and 6, FeNPs produce reaction oxygen species (ROS) and induce death, interfering in organelles and enzyme activity, notably in mitochondria, endoplasmic reticulum, and Golgi bodies. 3 showed how ion channel blocking causes malignant cells to die. 4 indicated FeNPs attack malignant cells by degrading down nucleic acid, notably DNA. 8 showed that membrane polarity is disrupted.⁶⁶ Created with Biorender.com., http://www.biorender.com (accessed on May 11, 2024).

Digitale (2016)¹⁷¹ reported the utilization of Fe nanoparticles in the treatment of cancer. The researcher conducted the study on the mouse and observed that FeNPs stimulated macrophages causing them to assault cancer cells in the mice. They discovered unexpected results where tumor development was decreased in control rats that only received Fe nanoparticles, relative to other controls.

Soetaert et al. (2020)¹⁷² published information regarding the potential of Fe oxide NPs in cancer treatment, notably in thermal as well as immunological therapies. According to the report, the Food and Drug Administration approved the utilization of iron oxide nanoparticles including cancer diagnostics, cancer hyperthermia treatment, and iron deficient anaemia.

Nejad et al. (2024)¹⁷³ studied the synthesis of iron nanoparticles using an aqueous extract of Mentha spicata as the reducing agent. The synthesis process involved mixing 10 mL of iron (III) chloride hexahydrate (FeCl₃·6H₂O) at a concentration of 0.05 M with 40 mL of a 100 μg/mL aqueous extract solution. The mixture was refluxed at 50 °C for 1 h, during which a color change from yellow to black indicated the successful production of iron nanoparticles. The researchers further investigated the effects of Mentha spicata extract, FeNPs, and Mentha spicata-loaded FeNPs on LS174 t-colon cancer cells. The results demonstrated that all three treatments exhibited significant cytotoxic effects against LS174 t-cells, with Mentha spicata-loaded FeNPs showing the most pronounced activity.

The therapeutic effects of Cu-immobilized chitosan-modified iron oxide magnetic nanoparticles in lung cancer treatment present a significant advancement in nanomedicine. A review highlighted that iron oxide nanoparticles have been clinically approved for various applications, including cancer diagnosis and treatment through hyperthermia and drug delivery systems, demonstrating their versatility and effectiveness in oncology.¹⁵²

8. FeNPs in the treatment of gestational diabetes

Gestational diabetes mellitus (GDM) is a condition that arises during pregnancy, characterized by impaired glucose tolerance, and it poses risks for both mothers and their babies.¹⁷⁴ Women with GDM face complications such as high blood pressure, excessive amniotic fluid, and an increased likelihood of preterm delivery, while their children may be at risk for obesity, type 2 diabetes, and kidney disease later in life.¹⁷⁵ The rising prevalence of GDM is often linked to factors like obesity and advanced maternal age, making awareness and management crucial.¹⁷⁴ Exciting advancements in nanotechnology are paving the way for innovative treatments, such as using nanoparticles to deliver insulin non-invasively, which could significantly improve the quality of life for those managing diabetes.¹⁷⁶ However, there's still much to learn about the potential of iron nanoparticles as dietary supplements to prevent liver disorders associated with gestational diabetes, highlighting an important area for future research.

¹⁷⁷ conducted a study on the treatment of gestational diabetes using Acroptilon repens leaf aqueous extract to synthesize green-formulated iron nanoparticles. The research focused on the synthesis process, which involved combining the extract with iron chloride, resulting in a noticeable transition in color from yellow to black, indicating successful nanoparticle formation. This color change is attributed to the reduction of iron ions facilitated by the phytochemicals present in A. repens extract, which also contributes to the stabilization of the nanoparticles during synthesis.

In their investigation, the authors induced gestational diabetes in rats using streptozotocin and subsequently administered FeNPs at doses of 60 μg/kg and 120 μg/kg over a period of 25 days. The study revealed that treatment with FeNPs significantly reduced blood glucose levels in the diabetic rats. Furthermore, histological analysis indicated changes in liver tissue, including a decrease in the volume of sinusoids and hepatocytes after high-dose FeNP administration, while other structures such as bile ducts and portal veins remained unaffectedwound‐healing.

9. FeNPs in wound healing

The skin serves as the body's largest protective barrier, safeguarding against environmental threats. When physical, chemical, or thermal damage compromises the dermis, wounds form due to the loss of integrity.¹⁷⁸ Wound healing is a complex process involving overlapping phases: inflammatory, proliferative, maturation, and remodeling. This intricate mechanism requires various biochemical and cellular pathways to restore damaged tissues.¹⁷⁹ Key factors that promote faster wound healing include reducing oxidative stress and inflammatory cytokines, increasing enzymatic antioxidants, and enhancing neovascularization.¹⁸⁰ Delayed healing often results from elevated oxidative stress and pro-inflammatory cytokines, which hinder growth factors and cellular signaling; thus, agents with strong antioxidant and anti-inflammatory properties are essential.¹⁸¹ Recently, nanomaterial-based therapies have emerged as effective solutions for wound healing, offering protection against infections and enabling targeted treatment at the wound site.¹⁸² Among these, iron oxide nanoparticles are notable for their superparamagnetic properties, allowing them to be directed by external magnetic fields. Studies have shown that topically applied iron chelators and novel therapeutic agents can significantly benefit delayed wound healing.¹⁸³

Sathiyaseelan et al. (2021)¹⁸⁴ conveys the study of the synthesized iron oxide nanoparticles from Pinus densiflora (PD), focusing on their antimicrobial and wound healing properties when incorporated into a chitosan/polyvinyl alcohol (CS/PVA) nanocomposite sponge. The in vitro wound-healing assay revealed that the incorporation of PD-FeO NPs at a concentration of 0.01 % into the CS/PVA matrix significantly increased cell proliferation in HEK293 cells, indicating enhanced wound healing potential.

Nahari et al. (2022)¹⁸⁵ reported the synthesis of iron nanoparticles using the aqueous leaf extract of Vitex leucoxylon as a reducing agent. The researchers investigated the wound healing activity of both the FeNPs and the leaf extract by monitoring cell migration and conducting a wound closure study using a scratch assay with normal fibroblast L929 cells. The results showed increased cell migration in both the standard and FeNPs-treated cells compared to those treated with the aqueous leaf extract. Specifically, ascorbic acid, the standard drug, exhibited the highest cell migration activity, followed by FeNPs, the plant extract, and untreated cells. In the wound closure study, which analyzed a 24 h incubation period, ascorbic acid again demonstrated the highest percentage of wound closure, followed by FeNPs, the plant extract, and untreated cells. This suggests that FeNPs synthesized from V. leucoxylon may have significant potential as a dermal wound healing agent.

Another study by Zangeneh and Zangeneh (2020)¹⁸⁶ synthesized iron nanoparticles (FeNPs) using the extract of Allium eriophyllum Boiss and investigated their effects on cutaneous wound healing in rats. The study involved creating excisional wounds and dividing the rats into six treatment groups, including those treated with 0.2 % FeNPs@AE ointment, 0.2 % A. eriophyllum ointment, 0.2 % FeCl₃·6H₂O ointment, 3 % tetracycline ointment, Eucerin basal ointment, and an untreated control group. The results revealed that the FeNPs@AE ointment significantly enhanced wound healing, as evidenced by a substantial increase in wound contracture rates and improved vascularization.

Additionally, levels of hydroxyl proline and hexuronic acid were significantly elevated, indicating enhanced collagen synthesis and tissue regeneration. The treatment also resulted in a higher number of fibrocytes and an increased fibrocyte-to-fibroblast ratio, reflecting improved cellular activity at the wound site. Notably, the application of FeNPs@AE led to a significant reduction in wound area and inflammatory cell counts, including neutrophils and lymphocytes, suggesting a decrease in inflammation.

10. Application of iron nanoparticles in wastewater treatment

According to Pereao et al. (2021),¹⁸⁷ a high amount of both chemical oxygen demand (COD) and biochemical oxygen demand (BOD) is harmful to the aquatic environment. This condition causes a reduction in dissolved oxygen, resulting in anaerobic circumstances that are harmful to larger aquatic species.¹⁸⁸ Additionally, a substantial amount of BOD in water indicates an excessive level of nutrients, which could end up in a bloom of algae.¹⁸⁸ BOD is defined as the amount of oxygen consumed by the decomposition of the sample during incubation. BOD generally represents how much oxygen is needed to break down organic matter in water.¹⁸⁹ Table 6 shows the efficiency of the biosynthesized iron NPs in contrast to the bioflocculant in removing COD and BOD from different wastewaters.⁸⁵ The materials were examined using a UV–Vis spectrophotometer called Spectroquant® at an intensity of 680 nm. The FeNPs and bioflocculant were used to remove COD and BOD at a concentration of 0.4 and 0.8 mg/mL, accordingly. When contrasted with both the bioflocculant and ferric chloride, the biosynthesized FeNPs were more successful, with BOD removal exceeding 80 %, while COD removal was 76 % for coal mine effluent and 48 % for river water. In comparison to bioflocculant and ferric chloride, the authors determined that biosynthesized FeNPs were a more effective flocculant. The bioflocculant performed poorly in all tests for BOD deletion, with just 50 % effectiveness. Nevertheless, COD removal by the bioflocculant improved significantly with 72 % for coal mine effluent, while it continued unworthy in the river water sample.

Table 6.

Removal of COD and BOD in wastewater by the bioflocculant and FeNPs.⁸⁵

Flocculant	Types of wastewaters	Types of pollutants in water	Water Quality before treatment (mg/L)	Water Quality after treatment (mg/L)	Removal Efficiency (%)
FeNPs	Coal mine water	COD BOD	842 123.2	204 23	76 81
	Umzingazi river water	COD BOD	3.300 136	1.700 24	48 82
Bioflocculant	Coal mine water	COD BOD	842 123.2	208 77.88	72 59
	Umzingazi river water	COD BOD	3.300 136	1.68 72.08	51 53

Pullabhotla and co-workers (2023)²⁷ emphasize the elimination of pollutants utilizing the synthesized FeNPs from the Tendele coal mine in comparison to the microbial bioflocculant and chemical flocculant (FeCl₃). The pollutants such as COD, BOD, P, S, and L were tested and the outcomes are indicated in Table 7. The authors observed high removal efficiencies in all tested pollutants when the biosynthesized FeNPs were utilized in comparison with bioflocculant and FeCl₃. The synthesized Fe nanoparticles showed to be effective in the removal of COD with 77 %, BOD (87 %), P (85 %), S (82 %), and N (73 %). Furthermore, the produced Fe nanoparticles revealed a high flocculation efficiency of 98 % which is better than that of microbial bioflocculant and FeCl_3.

Table 7.

The potential of biosynthesized FeNPs in the removal of different contaminants from Tendele coal-mine effluent in contrast to the microbial bioflocculant.²⁷

Flocculants	Water quality	COD (mg/L)	BOD (mg/L)	P (mg/L)	S (mg/L)	N (mg/L)	Flocculating Activity (%)
Microbial bioflocculant	Before treatment	146.6	203	145	33.4	9.0	2.982
	After treatment	53.8	55	6.3	11.3	3.2	0.275
	Elimination rate (%)	63	73	57	66	64	91
FeNPs	Before treatment	146.6	203	14.5	33.4	9.0	2.982
	After treatment	33.2	25.4	2.2	6.1	2.4	0.053
	Elimination rate (%)	77	87	85	82	73	98
FeCl3	Before treatment	146.6	203	14.5	33.4	9.0	2.982
	After treatment	37.3	52	3.5	7.4	3.3	0.293
	Elimination rate (%)	75	74	76	78	63	90

An experimental study reported by³⁷ with the synthesized AI-FeNPs from different leaf extracts showed 98.1 %, 84.3 % and 82.4 % removal efficiency for total phosphates, ammonia nitrogen and COD.

Hussain and colleagues reported the synthesis of iron nanoparticles using an environmentally friendly method. It was observed that the removal efficiencies of the synthesized FeNPs from Opuntia extract were 87.4 %, 87.3 %, 96.28 %, 98.19 %, 96.28 %, 34.4 %, 86.4 %, and 100 % for chemical oxygen demand (COD), biochemical oxygen demand (BOD), total Kjeldahl nitrogen (TKN), total nitrogen (TN), ammonia (NH₄⁺), phosphate (PO₄³⁻), total suspended solids (TSS), and nitrate (NO₃⁻) respectively.¹⁹⁰ This confirmed that the biosynthesized FeNPs from opuntia serves as an environmentally friendly adsorbent that can be effectively utilized in environmental remediation efforts.

Devatha et al. (2016)³⁷ conducted a study on the synthesis of iron nanoparticles and their application in treating domestic wastewater. The researchers collected wastewater samples from the NIT-K campus, revealing concentrations of 448 mg/L for organic matter (OD), 92.50 mg/L for total phosphates, and 44.46 mg/L for ammonia nitrogen. To evaluate the effectiveness of the synthesized iron nanoparticles, experiments were performed in batch mode over 15 days, with no pH adjustments made to the leaf extract used in the synthesis. The results indicated that FeNPs achieved a phosphate removal efficiency of 91.89 % by the 14th day, while ammonia nitrogen and chemical oxygen demand (COD) removal efficiencies were recorded at 54.42 % and 78.57 %, respectively.

Jyotsana and co-workers (2017)¹⁹¹ reported the synthesis of iron nanoparticles using various methods. The initial concentrations of nitrate and phosphate in the wastewater were measured at 14.77 mg/L and 23.50 mg/L, respectively. A diluted suspension of the synthesized FeNPs was added to the wastewater in a 1:5 (v/v) ratio, with no alterations made to the wastewater characteristics. The results indicated that chemically synthesized iron nanoparticles achieved a removal efficiency of 85.27 % for nitrate and 67.98 % for phosphate. In comparison, FeNPs synthesized from extract removed up to 74.52 % of nitrate and 55.39 % of phosphate. These findings demonstrate the effectiveness of both chemically and biologically synthesized iron nanoparticles in reducing nutrient concentrations in wastewater.

In another study by Mehrotra et al. (2017)¹⁹² reported protein-capped zero-valent iron nanoparticles synthesized using yeast for the complete degradation of the organophosphorus insecticide dichlorvos. Under optimal experimental conditions, with a dosage of 2000 mg/L of Fe nanoparticles and 1000 μL of hydrogen peroxide (H₂O₂), the study achieved a remarkable degradation efficiency of 99.9 % within 60 min of reaction time.

Maham and co-workers (2016)¹⁹³ synthesized a copper-supported iron oxide nanocatalyst to study the reduction of nitroarenes in wastewater. The degradation process utilized sodium borohydride (NaBH₄) as a reducing agent. Experimental studies revealed that the prepared nanocatalyst, in conjunction with NaBH₄, achieved a maximum degradation efficiency of 90 % within 90 min. In contrast, no degradation was observed when NaBH₄ was used alone.

11. Heavy metal removal from wastewater with the biosynthesized FeNPs versus bioflocculant

Heavy metals are dumped into water from a variety of businesses, and they can be poisonous or dangerous in nature, posing serious hazards for individuals and aquatic life. Therefore, the elimination of toxic metals is crucial for a clean environment and human health.¹⁹⁴

Xiao et al. (2016)¹⁹⁵ reported the synthesized iron nanoparticles using leaf extracts from Syzygium jambos (SJA), Oolong tea (OT), and Acalypha moluccana (AMW) to remove chromium from water. Among these leaf extracts, SJA exhibited the strongest antioxidant properties in the synthesis of FeNPs. The results showed that just 1 mL of SJA-mediated iron nanoparticles achieved a remarkable 91.9 % removal of Cr (VI) within the first 5 min, with complete removal reached after 60 min. The synthesized nanoparticles had an average diameter of about 5 nm and were amorphous.

In a study by Sebastian et al. (2018),¹⁹⁶ magnetite nanoparticles were synthesized using coconut husk extract for the adsorption of calcium and cadmium at low concentrations. The results demonstrated that the removal efficiencies for cadmium and calcium exceeded 40 % and 50 %, respectively, after 120 min of contact time.

Ehrampoush et al. (2015)¹⁹⁷ utilized tangerine peel extract to prepare iron oxide nanoparticles for the removal of trace amounts of cadmium from water bodies. Experimental studies revealed that a maximum removal efficiency of 90 % was achieved at a pH of 4 within 90 min.

Similarly, Prasad et al. (2017)¹⁹⁸ synthesized a graphene oxide-based iron oxide nanocomposite to remove lead (II) ions from water. The experimental results indicated that approximately 96 % of Pb (II) was removed at a pH of 5, with rapid adsorption occurring within the first 40 min and equilibrium reached by 80 min.

Fazlzadeh et al. (2017)¹⁹⁹ reported the removal of chromium (Cr (VI)) using iron nanoparticles synthesized from three different plant extracts: Rosa damascena (RD), Thymus vulgaris (TV), and Urtica dioica (UD). Experimental studies demonstrated that these nanoparticles achieved greater than 90 % removal efficiency within just 10 min.

Another study by Xiao et al. (2016),¹⁹⁵ effectively utilized plant-mediated iron nanoparticles for the removal of chromium (Cr). The FeNPs were synthesized using various leaf extracts selected based on their reduction potential, including Syzygium jambos (L.) Alston (SJA) with strong reducing ability, Oolong tea (OT) with moderate reducing ability, and Aleurites moluccana (L.) Willd (AMW) with weak reducing ability. The study demonstrated that the removal efficiency of chromium (VI) correlated with the reducing capacity of the plant extracts, revealing that 1 mL of SJA-FeNPs colloidal solution was able to remove 91.9 % of Cr (VI) within 5 min and achieve 100 % removal in 60 min. However, this study lacks information on whether the removal of chromium depends on the reduction potential of the plants or on the size of the nanoparticles produced from the extracts.

Shende and Mitra (2021)²⁶ reported the flocculation efficiencies of FeNPs and purified bioflocculants that were tested using simulated wastewater containing heavy metals. The outcomes showed that the FeNPs were able to eliminate 59 % of Ni²⁺, 48 % of Zn²⁺, 70 % of Pb²⁺, 29 % of Cd²⁺, and 23 % of Cu²⁺. In comparison, bioflocculant was found to remove 50 % of Ni²⁺, 43 % of Zn²⁺, 65 % of Pb²⁺, 27 % of Cd²⁺, and 20 % of Cu²⁺. Table 8 also summarizes the heavy metals percentage (Pb²⁺, Ni²⁺, Zn²⁺, Cd²⁺, and Cu²⁺) removed from effluent. The authors stated that the highest removal percentages for Pb²⁺ were recorded using bioflocculant (66.22 %) and FeNPs (70.14 %). The results indicated that FeNPs were more efficient than the bioflocculant alone in removing heavy metals. The removal pattern was consistent in both cases, with Pb²⁺, being removed the most, followed by Ni²⁺, Zn²⁺, C d²⁺, and Cu²⁺.

Table 8.

The percentage removal of heavy metals from real wastewater.²⁶

Percentage Removal (%)
Heavy metals	Bioflocculant	FeNPs
Pb2+	66.22 ± 0.22∗∗	70.14 ± 0.11∗∗
Ni2+	50.42 ± 0.31	60.69 ± 0.48
Zn2+	48.10 ± 0.13	50.22 ± 0.04
Cd2+	33.19 ± 0.08	45.64 ± 0.47
Cu2+	30.68 ± 0.16	37.58 ± 0.28

∗∗∗ highest percentage removal for each metal (p < 0.05).

12. The use of Fe nanoparticles in the elimination of dyes

The textile and dye industry generates a significant amount of wastewater containing dyes and pigments that need to be treated before discharge to protect the environment and human health. Various methods are used to remove dyes from wastewater, including physical and chemical procedures. However, these procedures have been reported to be unfriendly to wastewater purification due to their chemical toxicity.²⁰⁰ Biological wastewater treatment is preferred because it is cost-effective, environmentally friendly, and effective at removing organic matter and nutrients from wastewater. However, biological wastewater purification is still in the experimental stage.²⁰¹

Iron nanoparticles (FeNPs) have a wide range of sorption characteristics for eliminating colors as well as organic and inorganic pollutants, making FeNPs good nano-adsorbents for environmental remediation²⁰².⁷² reported that FeNPs were effective against several dyes, including methylene blue, due to their photocatalytic activity. Because the subject of decolorization of colors utilizing iron NPs is quickly expanding, a search using the keywords “green synthesis of iron nanoparticles” in the EBSCO host database yielded 497 academic papers from 2015 to 2023. Utilizing the keywords “green synthesis of iron nanoparticles for the decolorization of dyes” yielded 32 scholarly papers featuring the concepts of “iron nanoparticles” and “dye” that are closely related. Anthraquinone color, azo color, triphenylmethane color, and reactive colors have all been decolorized using iron nanoparticles. Table 9 lists some of the dye-removal efficiency, mechanism, and other details of different dyes that have been decolorized using synthesized FeNPs.

Table 9.

Applications of FeNPs in the removal of dyes.

Dye	Size (nm)	Dosage of NP (g/L)	Initial concentration dye (mg/L)	Experimental parameter	Dye removal	Efficiency	Citation
Acid red 1114	<100	4.0	4.00	pH 5.00, embient temp, 500 rpm	–	94.96 % in 106 s	210
Basic Blue 41	<100	8.0	9.00	pH 9.00, ambient temp, 500 rpm	–	96.75 % in 205 s	210
Green Methyl orange	12-43 core, 3–12 shell	1.3	25	pH 8, 25 °C	Degradation by cleavage azo bond	99.52 % in 280 min	211
Methylene blue	<50	1	125	At the initial pH of the dye, 30 °C	Oxidation degradation	100 % in 420 min	212
Methylene blue	6–16	0.5	50	pH 10, 27 °C, 200 rpm	Adsorption	100 % in 2 h	34
Reaction blue 222	–	5.0	50	pH 3, 140 rpm	Adsorption	90.39 % in 180 min	213

The removal of methylene by FeNPs with the help of H₂O₂ was shown to be successful, with 99.17 % of the color decolorized after 6 h of treatment. Formed iron NPs have additionally been demonstrated to decolorize additional colors such as bromothymol blue, malachite green, and rhodamine B.203, 204, 205

Another study reported that the synthesized FeNPs decomposed the methylene blue within 10 s in 10 mg/L dye (98 %), however, the shade shift was reduced in 20 mg/L (87 %), and 30 mg/L (66 %) after 25 and 30 min of fermentation, correspondingly. Inoculating dye at 40 mg/L (27 %) and 50 mg/L (15 %) with 1 mL of FeNPs resulted in little decolorization.²⁰⁶ The experimental results show that decolorization increased considerably when switching on the electrochemical production of H₂O₂. Complete decolorization (>99 %) was achieved for all dyes under the applied experimental conditions, and partial mineralization (49–85 %) was obtained, which depends on the type of dye.

Sarma and co-workers (2017)²⁰⁷ investigated the removal of methylene blue dye utilizing green tea-mediated synthesized iron oxide nanoparticles. The results showed that a degradation efficiency of 95 % was achieved within 16 min.

Moreover, Dastkhoon et al. (2017)²⁰⁸ prepared Ni-doped Ferric Oxy-hydroxide FeO(OH) nanowires supported on AC for removing Indigo Carmine (IC) and Safranin-O (SO) dyes. The maximum adsorption capacities for IC and SO were found to be 37.85 mg/g and 29.09 mg/g for IC and SO, respectively at pH 5.0. Chemically prepared iron-based nanocomposites also proved to be efficient adsorbents for dye removal applications under small reaction times.

Shalaby et al. (2021)²⁰⁹ conducted a study on the green synthesis of iron oxide nanoparticles (SP-IONPs) utilizing Spirulina platensis microalgae. The biosynthesized Fe nanoparticles were evaluated for their effectiveness in removing cationic crystal violet (CV) and anionic methyl orange (MO) dyes from aqueous solutions. The results demonstrated impressive removal capacities, achieving 256.4 mg/g for crystal violet and 270.2 mg/g for methyl orange. The study highlights the potential of SP-IONPs as an efficient and eco-friendly solution for wastewater treatment, particularly in the context of dye pollutants, thereby contributing to sustainable environmental practices.

While effective, traditional methods often show lower efficiency compared to FeNPs. For example, lead removal using conventional adsorbents achieved a reduction of only 66.53 % in pharmaceutical effluent.

13. Other potential applications of FeNPs

Iron nanoparticles (FeNPs) have potential applications in a variety of sectors because of their distinctive features. In addition to their antimicrobial, anticancer, wastewater treatment, and dye removal potential, FeNPs have been used in drug delivery, antioxidant therapies, and catalysis.⁵⁵ Nevertheless, the present understanding of the catalytic process is insufficient, which must be addressed before it can be used as a catalytic agent in remediation procedures.⁶³ Microbial-mediated FeNPs have been tested in agriculture and have shown positive outcomes when contrasted with chemical substances.⁶¹ With such enormous potential, they are thought to have a promising prospect in agriculture and might be exploited to create innovative fertilizers, bio-control agents, and sophisticated sensing technologies.⁵⁵ Yet, some constraints like cytotoxicity and eutrophication must be solved before this technique can be applied in the field.⁶⁶

14. Cytotoxicity

IONPs as a therapeutic agent are becoming more common in the clinical setting. As a result, conducting a cytotoxicity experiment to assess the biocompatibility of IONPs both in vitro and in vivo is essential. Cell viability assay, LDH assay to examine cell membrane integrity, MTT assay to evaluate mitochondrial function, immunohistochemistry to detect apoptosis markers, hemolysis assay, microscopic analysis of intracellular localization, and genotoxicity assessment for specific cell expression are all commonly used techniques for investigating IONP toxicity and biocompatibility in vitro.214, 215, 216 The toxicity of IONPs is affected by several factors, including their size, morphology, surface coating, methods of administration, attached peptides, medication, and targeting agents.^217,218 Smaller IONPs (less than 10 nm) are often more dangerous than bigger ones because they have a larger surface area, which increases the chances of entering cells.²¹⁹

In an in vivo system, smaller IONPs are promptly removed by extravasation and renal clearance, whereas the spleen captures bigger ones via mechanical filtration.²²⁰ Furthermore, IONPs' cytotoxicity can be considerably influenced by their surface charges. A cell's membrane potential is normally negative, and the surface charge of IONPs is either negative or positive; their contact can be repulsive or attractive. Smaller NPs with a larger surface area would have a bigger change in ratio, enhancing the electrostatic contact between the cell and the IONPs and harming them. Furthermore, different coating materials can alter IONP toxicity. For example, prolonged exposure to IONPs may cause cytotoxicity due to the generation of free radicals. However, harmful effects can be mitigated by PEGylation.²²¹ PEG provides a hydrophilic barrier surrounding IONPs, decreasing their interactions with cell membranes. More significantly, PEG coating improves antibacterial action and biocompatibility. Furthermore, because Fe³⁺ naturally occurs in the human body, IONPs are less likely to create serious health concerns. Therefore, the release of the iron would not cause any major side effects in the in vivo system.222, 223, 224

Kahru and co-workers (2017)²²⁵ evaluated the toxicity of nanosized and bulk iron oxide nanoparticles on Daphnia magna (D. magna), a commonly used model organism in ecotoxicology. Their study found no significant differences in the biological effects between the two sizes of magnetite nanoparticles. Despite this, the iron oxide nanoparticles exhibited very low toxicity, with an effective concentration (EC₅₀) of less than 100 ppm for both D. magna and the aquatic plant Lemna minor in standard acute assays. Interestingly, at acutely subtoxic concentrations of magnetite (10 and 100 ppm), a decrease in the number of neonates hatching from D. magna ephippia was observed, indicating potential sub-lethal effects that warrant further investigation.

Pullabhotla and co-workers (2023)²⁷ revealed the effects of the produced Fe nanoparticles from microbial bioflocculant on HEK 293 cells. The cytotoxicity of the produced FeNPs reduced the viability of HEK 293 cells. The author observed 68 % cell survival when the produced material was utilized at a concentration of 25 g/L, but only 34 % were found when the concentration was raised to 200 g/L. The results presented here imply that iron NPs are harmless to utilize in small concentrations.

Pullabhotla and co-workers (2020)⁹⁸ demonstrated the biosafety of the bioflocculant synthesized Fe nanoparticles in HEK 293 cells. The cell survival was observed to be 56 % at a maximum concentration of 100 g/L. As a result, the FeNPs produced from biological sources could be used as an alternative in industrial applications.

15. Challenges and potential future developments of iron nanoparticles synthesized using eco-friendly methods

Future research and advancements are essential to fully explore the unique characteristics of green-synthesized nanoparticles, and it is crucial to adopt diverse accessible materials for such fabrication. Exploring the fundamental processes of these environmentally friendly production methods is critical for uncovering novel changes and enhancements. Furthermore, progress should strive for an economically viable technology that can readily grow and is more ecologically friendly than traditional procedures.

Future studies should incorporate the utilization of more local resources to enhance economic viability, and particular attention should be given to the stability issues of the synthesized nanoparticles to improve their biocompatibility for numerous uses. It is vital to emphasize the synthesis of these green nanoparticles with greater efficiency in tacking contaminants while minimizing eco-toxicological effects. Additionally, a significant focus should be directed toward addressing and managing the hazards of management, density, and neurotoxic impacts of biological-produced FeNPs, especially when compared to chemical synthesis methods.

Researchers have observed the importance of food security and the use of food security and the use of green synthesis products as reducing agents in iron nanoparticles. Therefore, more emphasis should be placed on using agro/biowaste and natural herbs to treat this issue. Additionally, in-depth research is required to comprehend the fate and transportation of NPs in the environment.

The process of biosynthesizing nanoparticles varies depending on the microorganisms involved, including bacteria, fungi, or yeast. Different interactions among metal ion precursors and biological substances lead to nanoparticles with diverse characteristics and morphology. Intracellular synthesis occurs when metallic ions are decreased on the inside of the cell wall, while extracellular production involves the transfer of electrons among enzymes and metal ions. Due to the complexity of these reactions, extensive research is necessary to compressively comprehend the mechanisms involved. This understanding can enable better control over the properties and morphology of the synthesized nanoparticles.

Due to their elevated oxidation state, iron oxide nanoparticles, particularly zerovalent irons, possess considerable reactivity. During their transportation between different environments, these nanoparticles can induce severe toxic effects on both soil and aqueous systems, thereby, posing a potential threat to the ecosystem. As a result, researchers must prioritize efforts in improving the stabilization of prepared nanoparticles using environmentally friendly methods and enhancing the management of nanoparticle waste.

The research focus should be directed towards employing agricultural waste for environmentally friendly synthesis of nanoparticles. Additionally, there should be a greater emphasis on the synthesis of metal-metal nanoparticles through eco-friendly processes. This approach has facilitated the removal of heavy metals for various application purposes.

Through this research, it has been discovered that several microorganisms can produce nanoparticles that possess distinct physical and chemical characteristics. To gain a comprehensive understanding of these synthesized nanoparticles, further research should be driven toward investigating the specific biomolecules responsible for capping and stabilizing them.

Additionally, there is a need to explore new microorganisms for the formation of FeNPs. Previous literature suggests that the use of microorganisms in the fabrication process leads to less stable nanoparticles with narrower morphological variations, leading to poorer yields. Therefore, to find NPs with consistent, crystallinity, and uniformly dispersed features, it is crucial to focus research efforts on discovering and studying different species of microorganisms.

To manage the risk associated with the toxic environment of produced FeNPs, it is crucial to establish a standardized synthesis method. Currently, research utilizes different synthesis methods, leading to variations in the properties and characteristics of nanoparticles. Consequently, the proper screening of nano waste before disposal is often neglected, resulting in inconsistencies in research outcomes and posing challenges for predicting human well-being and analyzing risks to the environment.

The exploration of bimetallic NPs is a captivating research domain that shows promise in enhancing the properties of individual Fe nanoparticles. However, this area remains relatively underrepresented in literature. Bimetallic nanoparticles offer the potential to improve catalytic properties, increase adsorption surface area, and enhance a variety of other chemical attributes. Therefore, study efforts must be directed more toward investigating these bi-metallic nanometals for diverse natural uses.

The major issue in using these nanoparticles as absorbents in column operations is the significant increase in pressure drops caused by the extremely reduced bed void volume. To address these challenges, it is possible to integrate nanoparticles onto a support material, such as adsorbent fragments, carbon composites, or carbon-activated composites. In this context, chemosorption, which involves a strong chemical interaction between the absorbate and adsorbent, is the preferred mode of reaction for the operation. Therefore, the main focus lies in preparing and selecting specific nanoparticles with improved surface functional groups.

16. Conclusion

The exploration of nanoparticles reveals significant potential, particularly in cancer treatment and environmental remediation. Recent advancements in the green synthesis of nanoparticles for wastewater treatment highlight innovative solutions to urgent environmental challenges. However, controlling particle size and morphology remains a critical challenge. Factors such as pH, temperature, and reaction time play crucial roles in determining these properties, which are essential for optimizing the effectiveness of nanoparticles in removing contaminants.

This review underlines the importance of sustainable practices in Fe nanoparticle production. A particularly promising area of research is the use of microbial bioflocculants for synthesizing iron nanoparticles. According to current knowledge, only three studies have reported the successful synthesis of FeNPs using bioflocculants. This limited research highlights a significant gap in the literature and calls for further investigation to improve production processes.

Additionally, the review offers a balanced perspective on the toxicological effects of iron nanoparticles, emphasizing the need for comprehensive assessments of their environmental impact. By examining both the challenges and opportunities associated with green synthesis, this paper serves as a valuable resource for researchers and practitioners in this dynamic field.

In conclusion, advancing research in nanoparticle synthesis is essential for promoting sustainable practices that benefit both human health and the environment. The integration of nanotechnology into therapeutic and industrial applications holds transformative potential, paving the way for innovative solutions to today's most pressing medical and ecological challenges. This paper might be useful to the readers for acquiring in-depth knowledge on biological synthesis of iron nanoparticles and its amazing success for the various environmental application.

CRediT authorship contribution statement

Nkanyiso C. Nkosi: Writing – original draft, Methodology, Investigation, Formal analysis. Albertus K. Basson: Supervision, Conceptualization. Zuzingcebo G. Ntombela: Writing – review & editing, Supervision. Nkosinathi G. Dlamini: Writing – review & editing, Supervision. Rajasekhar V.S.R. Pullabhotla: Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Funding

The research was funded by the National Research Foundation (NRF, South Africa) grant number (103,691) and the Research Development Grant Rated Research grant number (112,145).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.