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Home > Books > Green Chemistry for Environmental Sustainability - Prevention-Assurance-Sustainability (P-A-S) Approach

Green Synthesis of Nanoparticles: A Biological Approach

Submitted: 29 May 2023 Reviewed: 05 June 2023 Published: 11 August 2023

DOI: 10.5772/intechopen.1002203

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Green Chemistry for Environmental Sustainability - Prevention-Assurance-Sustainability (P-A-S) Approach

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Nanoparticles are often associated with their small size and numerous applications. However, the synthesis process is equally important as it determines the size and properties of the nanoparticles. While traditional nanoparticle synthesis methods require the use of hazardous chemicals and high-energy consumption, green synthesis offers a sustainable, cost-effective, and environmentally friendly alternative. This approach utilizes natural resources and biologically active compounds that can act as reducing, stabilizing, or capping agents in the one-step synthesis of nanoparticles. Green synthesis offers numerous advantages, including the development of processes with minimal environmental impact and improved safety for nanoparticle synthesis. Overall, the synthesis of nanoparticles using green chemistry is a promising approach for sustainable and efficient production. This chapter provides a general overview of nanoparticles, their applications, and green synthesis, and highlights the various biological resources used in these processes and the factors affecting their synthesis.

  • green synthesis
  • nanoparticles
  • plant extract
  • microorganisms
  • phytochemicals

Author Information

Rafael álvarez-chimal *.

  • Laboratory 113 Synthesis of Magnetic Nanomaterials, Condensed Matter Department, Institute of Physic, National Autonomous University of Mexico, Ciudad Universitaria, Mexico City, Coyoacán, Mexico

Jesús Ángel Arenas-Alatorre

*Address all correspondence to: [email protected]

1. Introduction

Nanoparticles are small particles with sizes ranging from 1 to 100 nanometers. These materials have gained importance and interest in recent years owing to their large number of applications, because the matter at this scale presents a more compact arrangement of atoms and molecules, generating phenomena and acquiring or enhancing mechanical [ 1 ], electrical [ 2 ], magnetic [ 3 ], optical [ 4 ], catalytic [ 5 ], and antibacterial [ 6 , 7 ] properties that are completely different from those of their macroscopic counterparts [ 8 ]. They can be classified based on their composition, shape, and size. The most common types of nanoparticles are metals, metal oxides, carbon-based, and quantum dots. Owing to their unique sizes and properties, nanoparticles have attracted significant attention in various fields including medicine, electronics, energy, and environmental science [ 9 , 10 ]. By reducing their size, nanoparticles can have a higher surface-to-volume ratio, enabling a greater number of atoms or molecules per volume, which means that less material is needed to obtain the same activity and exhibit other properties ( Figure 1 ) [ 11 ].

green synthesis method of nanoparticles

Surface-to-volume ratio of nanoparticles compared with that of bulk materials.

Nanoparticles have many potential benefits for the environment. For example, nanoparticles can be used to improve the efficiency of water treatment, air filtration, and soil remediation; reduce pollution, and develop new types of renewable energy technologies [ 12 ]. In medicine, nanoparticles have shown potential for drug delivery, imaging, and cancer therapy. They can be functionalized with targeting moieties, making them capable of selectively targeting cancer cells, while sparing normal cells. Additionally, nanoparticles can enhance the efficacy of chemotherapy by improving drug delivery to the tumor site and reducing systemic toxicity [ 13 ]. In electronics, nanoparticles are used to fabricate high-performance devices such as sensors, transistors, and solar cells [ 14 ]. Nanoparticles have potential applications in fuel cells, hydrogen storage, and catalysis [ 15 ].

However, it is also important to address the environmental impact of the nanoparticles. Some studies have shown that nanoparticles can harm plants, animals, and humans, but it depends on many factors, such as concentration, size, and time of exposure [ 16 , 17 ]. Nanoparticles can easily be released into the environment through various sources, such as industrial emissions, consumer products, and medical procedures. Once released into the environment, nanoparticles can be difficult to control and monitor. There is potential for long-term accumulation. Nanoparticles can accumulate in the environment, and they may be able to persist for long periods. This raises concerns about the potential for nanoparticles to cause long-term harm to the environment and human health [ 17 , 18 ]. However, one of the alternatives for reducing their environmental impact is to control the synthesis process.

There are many methods for synthesizing nanoparticles, including physical, chemical, and biological processes [ 19 ]. Green synthesis, which refers to the eco-friendly and sustainable production of nanoparticles without the use of hazardous chemicals or toxic solvents, has gained attention in recent years within biological processes. Natural sources, such as plants and microorganisms, are popular green synthesis approaches [ 20 ]. This method has several advantages over traditional synthesis methods, including low cost, scalability, and reduction of hazardous waste. Moreover, green synthesis can produce nanoparticles with unique shapes, sizes, and surface properties tailored for specific applications [ 21 ]. The biological sources used for the green synthesis of nanoparticles contain biologically active compounds, such as enzymes, proteins, polyphenols, flavonoids, and terpenoids, which can act as catalyzing, reducing, stabilizing, or capping agents for one-step synthesis [ 20 , 21 ].

In summary, this chapter provides a general overview of nanoparticles, their properties and applications, and how green synthesis is used to synthesize them. This chapter also discusses the different biological resources used for green synthesis, the factors that participate, and the mechanisms involved in their production.

2. Traditional nanoparticle synthesis methods

Chemical reduction: This method involves the reduction of metal ions in solution using chemical reagents such as sodium borohydride or sodium hydroxide to form nanoparticles [ 22 ].

Coprecipitation: Synthesis involves mixing two or more solutions containing metal ions. When the solutions are mixed, metal ions precipitate out of the solution and form nanoparticles [ 23 ].

Sol-gel: The process requires mixing a metal salt with a solvent and gelling agent. The solvent is evaporated leaving behind the gel. The gel is then heated, causing it to solidify and form nanoparticles [ 24 ].

Microemulsion: This method needs surfactants, water-soluble compounds, and oil-soluble compounds. The mixture forms small droplets that contain the metal ions. When droplets are heated, metal ions precipitate out of the solution and form nanoparticles [ 25 ].

Solvothermal/hydrothermal synthesis: This reaction involves heating a solution of metal ions in water or an organic solvent under high pressure. High pressure and temperature cause metal ions to precipitate out of the solution and form nanoparticles [ 26 ].

Sonochemical/electrochemical synthesis: This process uses ultrasound or an electrical current to break down metal salts into nanoparticles [ 27 ].

green synthesis method of nanoparticles

Nanometric scale and different approaches to nanoparticle synthesis.

In addition, there are physical processes, such as laser ablation, milling, and sputtering, where the material is reduced to nanoparticles by the mechanical action of the equipment used [ 28 ].

The choice of method depends on the type of nanoparticles being synthesized, the desired size and shape, and the availability of equipment and reagents.

2.1 Environmental limitations in nanoparticle synthesis

Traditional methods for synthesizing nanoparticles have several limitations.

Using organic reagents can harm the environment, humans, and animals, causing illnesses, such as liver damage [ 18 ]. In addition, wastewater generated from nanoparticle synthesis can contain harmful chemicals [ 29 ].

The low yield is another disadvantage: only a small percentage of the starting materials is converted into nanoparticles, generating raw material waste. The high cost of the starting materials, equipment, labor required, long-time synthesis, and the inability to control the size and shape can limit their applications [ 30 , 31 ].

2.2 Strategies to overcome barriers to nanoparticle synthesis

Several strategies can be used to overcome the disadvantages of nanoparticle synthesis, such as the use of environmentally friendly solvents, reagents, and processes. Using water, ionic liquids, and supercritical fluids are examples of eco-friendly solvents [ 21 , 32 ] or we can even perform solvent-free synthesis, eliminating the need for hazardous chemicals and reducing the environmental impact of nanoparticle synthesis [ 33 ].

Many nanoparticle synthesis methods are not scalable, which limits their application. Therefore, it is necessary to develop cost-effective and efficient processes to obtain large quantities of nanoparticles [ 8 ].

Multipurpose nanoparticles can be used to improve their performance in a variety of applications and fields. For example, biocompatible nanoparticles are used in biomedicine or as stable nanoparticles for long-term applications [ 34 ].

The characterization of nanoparticles is important for understanding their size, shape, surface properties, and chemical composition. This information can be used to understand how nanoparticles interact with their environment and ensure they are safe [ 35 ].

Strategies to overcome these barriers in nanoparticle synthesis are still under study to develop more innovative, efficient, cost-effective, and environmentally friendly methods.

3. Green synthesis of nanoparticles: an overview

Green synthesis aims to promote innovative chemical technologies to reduce or eliminate the use and production of hazardous substances in the design, manufacture, and use of chemical products. This involves minimizing or, if possible, eliminating the pollution produced in the synthesis processes, avoiding the consumption and wastage of nonrenewable raw materials, using hazardous or polluting materials in product manufacturing, and reducing the synthesis time. Paul J. Anastas, considered the father of green chemistry, defined it as “a work philosophy that involves the use of alternative tools and pathways to prevent pollution,” referring to both the design of the synthetic strategy and the treatment of possible secondary products originating from that route [ 36 , 37 ].

Two approaches can be used to generate nanoparticles [ 37 , 38 ] ( Figure 2 ).

“Top-down” approach: In which nanoparticles are produced using physical techniques such as grinding or abrasion of a material.

Chemical synthesis: The method of producing molecules or particles by the reaction of substances used as raw materials.

Self-assembly: A technique in which atoms or molecules self-order through physical and/or chemical interactions.

Positional assembly: The atoms, molecules, and aggregates are deliberately manipulated and positioned individually. However, this method is extremely laborious and unsuitable for industrial applications.

The “bottom-up” approach is preferred over the “top-down” approach because specialized equipment is not required and the time to obtain nanoparticles is shorter. Green synthesis is gaining relevance in producing nanoparticles within the “bottom-up” approach [ 37 ].

The use of plant species, algae, or microorganisms such as bacteria or fungi is one of the most commonly used resources for this procedure. Various compounds from plants or microorganisms, including terpenes, polyphenols, alkaloids, carbohydrates, proteins, and genetic materials, play an important role in the synthesis of nanoparticles by acting together [ 39 , 40 ].

In addition to the biological resources used to perform the synthesis (plants, algae, or microorganisms), other factors influence the shape and size of nanoparticles, such as the concentration of the metal ion, pH, reaction time, and temperature [ 39 , 41 ].

Initial phase: Obtaining the reaction medium, which is the aqueous extract of one or several parts of the plant species or the culture media for the growth of microorganisms, in addition to the precursor salt, which is the source of metal ions.

Activation phase: Chemical reduction of metal ions and generation of nucleation centers occur where nanoparticles emerge and grow.

Growth phase: Small adjacent nanoparticles spontaneously fuse into larger particles, forming aggregates, which are influenced by factors such as temperature, concentration, and type of compounds, pH, and reaction time.

Termination phase: The final shape of the nanoparticles is determined, and the compounds that participate in the reaction help stabilize and enhance their properties.

green synthesis method of nanoparticles

Phases involved in the green synthesis of nanoparticles.

3.1 Biological resources for the green synthesis of nanoparticles

As stated previously, nanoparticles have attracted attention in the fields of biology, medicine, and electronics in recent years, owing to their remarkable applications ( Figure 4 ). Numerous nanoparticle synthesis techniques have been developed; however, these may involve the use of toxic compounds and high-energy physical processes. An alternative is the use of biological methods to circumvent these obstacles. Bacteria, fungi, algae, and plant species are some of the most commonly used biological resources for the green synthesis of nanoparticles ( Figure 4 ). This biological approach has provided a method that is reliable, straightforward, benign, and environmentally beneficial [ 40 , 42 ].

green synthesis method of nanoparticles

Biological resources and compounds used for the green synthesis of nanoparticles and some of their applications [ 9 ].

3.1.1 Bacteria

Nanoparticle synthesis using bacteria is performed both extracellularly and intracellularly [ 38 ].

Intracellular: The synthesis is carried out inside the living microorganism, using its growth conditions to favor synthesis, known as “nanoparticle micro-factories.” To recover nanoparticles, bacteria must be destroyed [ 43 ].

Extracellular: The components released by the bacteria after lysis are used. The synthesis is performed by adding a metal salt precursor to the medium in which these components are located. Extracellular synthesis has the advantage of being faster because it does not require additional steps to recover nanoparticles from microorganisms [ 43 , 44 ].

Enzymes, such as reductases, which catalyze the reduction of metal ions into nanoparticles, participate in the synthesis. Even components of the genetic material participate in this process [ 45 , 46 ].

3.1.2 Fungi

Fungi contain active biomolecules, such as proteins or enzymes, that participate in nanoparticle synthesis, improving their yields and stability [ 47 ].

Some fungal species can synthesize nanoparticles using extracellular amino acids. For example, glutamic and aspartic acids on the surface of yeast or the reductase enzyme in the cytosol of fungi reduce metal ions to form nanoparticles. This is facilitated by the presence of hydroxyl groups in the mycelium, which donate electrons to the metal ion and reduce it to form nanoparticles. Aliphatic and aromatic amines or some proteins act as coating agents to stabilize them [ 48 , 49 ].

3.1.3 Algae

Algae are used in nanotechnology because of their low toxicity and their ability to bioaccumulate and reduce metals [ 50 ].

Nanoparticle synthesis can be intracellular, with the metal ion entering the alga, or extracellular, and involves compounds such as polysaccharides, proteins, and pigments that direct the reduction of metal ions and coat the newly formed nanoparticles. These particles are subsequently released from the cell in the form of colloids [ 51 ].

3.1.4 Plant species

The use of plants in nanoparticle synthesis is one of the most widely used methods because of its environmentally friendly nature, as it avoids the use of toxic or harmful substances. It is also one of the fastest and most economical methods because it involves fewer steps [ 39 , 40 ]. This makes it highly efficient in the nanoparticle production process compared to synthesis using microorganisms.

Plants contain several compounds (terpenes, flavonoids, polyphenols, alkaloids, proteins, etc.) that reduce metal ions and stabilize the resulting nanoparticles [ 52 ].

This type of synthesis can be performed using intracellular, extracellular, and phytochemical-mediated methods [ 53 ].

Intracellular: The synthesis is carried out inside the plant cell, and the nanoparticles are recovered by breaking down the structure, which is very similar to the intracellular method using microorganisms. Control of the growth factors of plant species is required so that they do not interfere with synthesis [ 54 ].

Extracellular: This method is the most commonly used because of its ease and speed. The process begins by obtaining a plant extract, which is generally water-based, to which a metal salt precursor is added. Owing to the action of the different compounds present in the extract, nanoparticles are generated and stabilized in a single step [ 54 , 55 ].

Phytochemically mediated: This is based on the extracellular method, but with the difference that isolated phytochemical compounds are used and other substances are added to stabilize the nanoparticles. There is greater control over the synthesis, but more components and steps are involved [ 53 ].

3.2 Factors involved in the green synthesis of nanoparticles

As in any synthesis process, reaction conditions, such as temperature, pH, and reaction time, play an important role in the shape, size, and yield of the synthesized nanoparticles [ 39 , 40 , 41 ] ( Figure 3 ).

Temperature: This is one of the most influential factors, as the shape (spherical, prismatic, flakes, triangular, octahedral, etc.), size, and synthesis depend on temperature. As the temperature increases, the reaction rate and the formation of nucleation centers increase, resulting in higher yields. Different temperatures promote different interactions between the reactants, giving rise to various shapes; the larger the temperature increase, the larger the size of the nanoparticles [ 56 , 57 ].

pH: This influences the nucleation centers, generating more centers at higher pH values. Another important influence of pH is that some nanoparticles can only be synthesized in acidic or alkaline media. For example, magnetic nanoparticles are synthesized at an alkaline pH, and metal oxide nanoparticles are generally synthesized at an acidic or neutral pH [ 58 ].

Time: This parameter plays an important role in defining the size of the nanoparticles. It has been observed that longer reaction times favor an increase in the size of the nanoparticles and higher yields, owing to the prolonged interaction time between reactants [ 59 ].

3.3 The mechanism involved in the green synthesis of nanoparticles

The plant extract or organism used for the synthesis is an important factor that influences the morphology and size of nanoparticles because different concentrations of metabolites or cellular components give rise to differences in the nanoparticles [ 40 , 60 ] ( Figure 5 ).

green synthesis method of nanoparticles

Green-synthesized nanoparticles. (a) Spherical ZnO nanoparticles using the leaves of Dysphania ambrosioides (plant). (b) Prismatic ZnO nanoparticles using the stems and leaves of Dysphania ambrosioides (plant). (c) Quasi-spherical Fe 3 O 4 nanoparticles using the leaves of Datura innoxia (plant). (d) Quasi-spherical Ag nanoparticles using stems of Aloe vera (plant) [ 61 ]. (e) Spherical and triangular Au nanoparticles using Lentinula edodes (fungus) [ 43 ]. (f) Irregular Ag and triangular Au nanoparticles using Ganoderma lucidum (fungus) [ 43 ]. (g) Hexagonal MgO nanoparticles using the flowers of Saussurea costus (plant) [ 62 ]. (h) Irregular Cu nanoparticles using Salmonella typhimurium (bacterium) [ 63 ]. (i) Quasi-spherical Ag nanoparticles using Dunaliella salina (alga) [ 64 ].

Proteins and enzymes facilitate the formation of nanoparticles from metal ions. Because of their high reducing activity, proteins and enzymes can attract metal ions to specific regions of a molecule responsible for reduction, facilitating the formation of nanoparticles; however, their chelating activity is not excessive. The amino acids of a protein can greatly influence the size, morphology, and quantity of nanoparticles generated, thus playing a very important role in determining their shape and yield. Removing a proton from amino acids or other molecules results in the formation of resonant structures capable of further oxidation. This process is accompanied by the active reduction of metal ions followed by the formation of nanoparticles [ 39 ].

Flavonoids are a large group of polyphenolic compounds that can actively chelate and reduce metal ions because they contain multiple functional groups capable of forming these structures. Structural transformations of flavonoids also generate protons that reduce metal ions to form nanoparticles; therefore, they are involved in the nucleation stage, their formation, and further aggregation. Saccharides can also play a role in nanoparticle formation. Monosaccharides, such as glucose, can act as reducing agents, as the aldehyde group of the sugar is oxidized to a carboxyl group through the addition of hydroxyl groups, which in turn leads to the reduction of metal ions and the synthesis of nanoparticles [ 39 ].

The mechanism of green synthesis of nanoparticles has been associated with the action of polyphenols, which act as ligands. Metal ions form coordination compounds, in which the fundamental structural unit is the central metal ion surrounded by coordinated groups arranged spatially at the corners of a regular tetrahedron. The aromatic hydroxyl groups in polyphenols bind to metal ions and form stable coordinated complexes. This system undergoes direct decomposition at high temperatures, releasing nanoparticles from the complex system [ 65 ].

Flavonoids, amino acids, proteins, terpenoids, tannins, and reducing sugars have hydroxyl groups that surround the metal ions to form complexes. After this process, the hydroxyl ions are oxidized to carbonyl groups, which stabilize the nanoparticles. Synthesis is favored if the participating molecules have at least two hydroxyl groups at the ortho- and para-positions [ 52 , 65 ].

Amino acids influence the size, morphology, and yield of nanoparticles generated [ 23 ], depending on the specific amino acids present in the extract and their concentration, along with the reaction conditions that give rise to nanoparticles with different shapes [ 65 ].

4. Confirming that the biological approach of nanoparticle synthesis is a green chemistry method

To corroborate that the processes of nanoparticle synthesis using biological resources are “green synthesis methods,” the 12 principles mentioned above are revisited [ 66 , 67 , 68 ] ( Table 1 ).

The plant extract or culture medium used in the synthesis can be easily disposed of, either by using it for composting in the case of plant extract or by sterilizing the culture medium and similarly disposed of without causing environmental harm. The synthesis yields are high, which supports the great incorporation of the raw material into the final product.
Few or no toxic wastes are generated because aqueous extracts or culture media are used and treated after use. Nanoparticles have the same or better properties than those generated using other methods.
Water is preferably used as the solvent. Syntheses are carried out at room temperature; although the temperature is a factor that influences synthesis, depending on the nanoparticles to be synthesized, it may be necessary to vary the synthesis temperature.
Vegetal extracts and microbial culture media can be reused more than once for several syntheses. The method focuses on nanoparticle synthesis; there are no subproducts, or those generated are the metabolites that participate in the reaction and can be reused in the synthesis.
Synthesis is catalyzed by biological compounds found in organisms. Making a faster or one-step synthesis. Nanoparticles should be handled with the necessary precautions, regardless of the process used in their synthesis.
There is considerable control over the synthesis process; it can be stopped at any time if there is a problem and resumed without issue. The reactants and raw materials are handled with the necessary care, and the generated products are easily treated. The risk of accidents is minimized because the synthesis is performed at ambient temperature and pressure.

The 12 principles of green synthesis are fulfilled with the biological approach to produce nanoparticles.

Considering the above, the 12 principles of green synthesis are fulfilled using biological resources, such as plants, bacteria, fungi, and algae, to synthesize nanoparticles [ 69 , 70 , 71 ].

Finally, green synthesis of nanoparticles is a sustainable and environmentally friendly alternative to traditional methods of nanoparticle synthesis. Traditional methods often take long periods of time, use toxic chemicals and solvents, or generate waste products that can pollute the environment and pose health risks to humans and animals. In contrast, the green synthesis method uses renewable natural resources, such as plant extracts and microorganisms, which are less damaging and can be replenished over time. In addition, these methods are often more cost-effective and faster than traditional procedures because they do not require expensive chemicals or equipment and are considered one-step syntheses, which contribute to energy savings [ 72 ].

In furtherance of these advantages, green synthesis methods are still being developed to improve their efficiency and scalability, leading to the potential benefits of green synthesis of nanoparticles or even their application to the synthesis of other molecules as drugs or nutraceuticals.

5. Conclusion

Nanoparticles have emerged as a versatile and promising class of materials with unique properties that can be harnessed for various applications. The use of green synthesis utilizing natural resources and biologically active compounds to produce nanoparticles is an area of continuous research to improve processes, reduce environmental damage, and meet the increasing demand for the application of these nanostructures. Utilizing biological resources, the synthesis of nanoparticles is inexpensive, faster, and considered a one-step synthesis while preserving or even improving the physical and chemical properties of the nanoparticles. With the great potential of this method and the sustainable and efficient production of nanoparticles, different sizes and shapes can be obtained, which makes it a very attractive option not only for the synthesis of nanostructures, but also for the application of this technique in the synthesis of other compounds.


The authors acknowledge Dr. Samuel Tehuacanero Cuapa, Physicist. Roberto Hernández Reyes, and Arq. Diego Quiterio Vargas for their technical support.

Thanks to the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) for the scholarship granted to Rafael Álvarez-Chimal with the CVU number: 579637.

Funding was provided by the UNAM-DGAPA- PAPIIT project IN112422.

Conflict of interest

The authors declare no conflicts of interest.

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Green synthesis of nanoparticles and their energy storage, environmental, and biomedical applications.

green synthesis method of nanoparticles

1. Introduction

2. green synthesis, 2.1. green synthesis of nps from biogenic wastes, 2.2. green synthesis of nps from plant extracts, 3. metal and metal oxide-based nanoparticles, 4. mn oxide nps, 4.1. mno 2 nps, 4.2. crystal structure of mno 2 nps, 5. synthesis of nanostructured mno 2, 5.1. traditional synthesis of mno 2 nps, 5.2. green synthesis of mno 2 nps, 6. plant extracts for green-synthesized mno 2 nps and recent applications, 6.1. lemon juice and lemon peel extracts, 6.2. black and green tea extracts, 6.3. broccoli vegetable extract, 6.4. orange juice and orange peel extracts, 6.5. moringa and cinnamon herb extracts, 7. iron oxide nanoparticles, 7.1. iron oxide np applications, 7.1.1. antioxidant activity, 7.1.2. anti-inflammatory activity, 7.1.3. anti-diabetic activity, 8. silver nanoparticles, 8.1. ag np applications, 8.2. antiviral activity, 9. gold nanoparticles, 9.1. au np synthesis, 9.2. green au np applications in cancer therapy and diagnosis, 10. future prespectives, 11. concluding remarks, author contributions, conflicts of interest, abbreviations.

AFMatomic force microscopy
BETBrunauer, Emmett and Teller
GCDgalvanostatic charge and discharge
CR dyeCongo red dye
EDXenergy dispersive X-ray
E energy bandgap
HAuCl chloroauric acid
HCT-116colon cancer cell line
HepG-2human liver cancer cell line
HIVhuman immunodeficiency virus
photon energy
HSV-1 and 2herpes simplex virus
KMnO potassium permanganate
LDHAlactate dehydrogenase analysis
LIBslithium-ion batteries
MB dyemethylene blue dye
MCF-7breast cancer cell line
MDOmanganese dioxide
NIH 3T3non-cancerous cell
SCspecific capacitance
SEMscanning electron microscopy
SPRsurface plasmon resonance
TEMtransmission electron microscopy
XRD X-ray diffraction
ZnOzinc oxide
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Crystal Structureα-MnO β-MnO γ-MnO δ-MnO λ-MnO
Chemical name
Crystal structure
Lattice parameter (Å)

Tunnel shape
Tunnel size (Å)
a = 9.96
c = 2.85
(2 × 2)
a = 4.39
c = 2.87
(1 × 1)
a = 9.65
c = 4.43
(1 × 1), (1 × 2)
1.82, 2.3
a = 2.94
c = 21.86
interlayer distance
a = 8.04

MaterialBiological AgentParticle Size (nm)Application
Fe O
Peel and juice of lemon
Black and green tea
Broccoli extract
Peel and juice of orange
Moringa oleifera
Echinochloa frumentacea
Ginger rhizome
Plumeria alba flower
Cassiem auriculata leaf
7.2 (pore size)
Cathode in Li-ion batteries
Cathode in Li-ion batteries
Cathode in Li-ion batteries
Electrode of supercapacitor
Photocatalysts (dye degradation)
Antioxidant, anti-inflammatory, and anti-diabetic activities
Degradation of textile dye
Antiviral (HIV, SARS)
Cancer therapy
Cancer therapy
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Abuzeid, H.M.; Julien, C.M.; Zhu, L.; Hashem, A.M. Green Synthesis of Nanoparticles and Their Energy Storage, Environmental, and Biomedical Applications. Crystals 2023 , 13 , 1576.

Abuzeid HM, Julien CM, Zhu L, Hashem AM. Green Synthesis of Nanoparticles and Their Energy Storage, Environmental, and Biomedical Applications. Crystals . 2023; 13(11):1576.

Abuzeid, Hanaa M., Christian M. Julien, Likun Zhu, and Ahmed M. Hashem. 2023. "Green Synthesis of Nanoparticles and Their Energy Storage, Environmental, and Biomedical Applications" Crystals 13, no. 11: 1576.

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  • Published: 18 May 2024

Green synthesis of metal nanoparticles and study their anti-pathogenic properties against pathogens effect on plants and animals

  • Osama Usman 1 ,
  • Mirza Muhammad Mohsin Baig 2 ,
  • Mujtaba Ikram 3 ,
  • Tehreem Iqbal 1 ,
  • Saharin Islam 4 ,
  • Wajid Syed 5 ,
  • Mahmood Basil A. Al-Rawi 6 &
  • Misbah Naseem 7  

Scientific Reports volume  14 , Article number:  11354 ( 2024 ) Cite this article

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  • Biotechnology
  • Engineering
  • Materials science
  • Microbiology
  • Nanoscience and technology

An Author Correction to this article was published on 29 May 2024

This article has been updated

According to an estimate, 30% to 40%, of global fruit are wasted, leading to post harvest losses and contributing to economic losses ranging from $10 to $100 billion worldwide. Among, all fruits the discarded portion of oranges is around 20%. A novel and value addition approach to utilize the orange peels is in nanoscience. In the present study, a synthesis approach was conducted to prepare the metallic nanoparticles (copper and silver); by utilizing food waste (Citrus plant peels) as bioactive reductants. In addition, the Citrus sinensis extracts showed the reducing activity against metallic salts copper chloride and silver nitrate to form Cu-NPs (copper nanoparticles) and Ag-NPs (Silver nanoparticles). The in vitro potential of both types of prepared nanoparticles was examined against plant pathogenic bacteria Erwinia carotovora ( Pectobacterium carotovorum ) and pathogens effect on human health Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) . Moreover, the in vivo antagonistic potential of both types of prepared nanoparticles was examined by their interaction with against plant (potato slices). Furthermore, additional antipathogenic (antiviral and antifungal) properties were also examined . The statistical analysis was done to explain the level of significance and antipathogenic effectiveness among synthesized Ag-NPs and Cu-NPs. The surface morphology, elemental description and size of particles were analyzed by scanning electron microscopy, transmission electron microscopy, energy-dispersive spectroscopy and zeta sizer (in addition polydispersity index and zeta potential). The justification for the preparation of particles was done by UV–Vis Spectroscopy (excitation peaks at 339 nm for copper and 415 nm for silver) and crystalline nature was observed by X-ray diffraction. Hence, the prepared particles are quite effective against soft rot pathogens in plants and can also be used effectively in some other multifunctional applications such as bioactive sport wear, surgical gowns, bioactive bandages and wrist or knee compression bandages, etc.

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Fruit and vegetable wastes (FVW) are produced in large quantities in markets and constitute a source of nuisance in municipal landfills because of their high biodegradability 1 . It has been documented that the wastage of fruit contains a major portion of citrus peels, with a world production estimation of 15 × 10 6 tons/year 2 . The disposal of citrus peels may cause bad environmental impact due to their high chemical oxygen demand (COD) and high biological oxygen demand (BOD). Therefore, the utilization of citrus peels in the area of nanotechnology is expected to minimize problems concerned with their long-term sustainability in the environment and cause pollution 3 . Several studies reported the presence of bioactive compounds, such as phenolic compounds, ascorbic acid and carotenoids in the citrus peel extract. These compounds may act as bioreductants, biodispersents and stabilizing agents during the green synthesis of metal nanoparticles 4 . Among all extracted phytochemicals, the phenolic contents (key compound) play a vital role during the reduction of metallic ions and in converting them into stabilized metallic particles 5 . According to information obtained from recent research, the average total phenolic contents (TPC) were reported between 4.9 and 6.9 mg gallic acid equivalent (GAE)/g fresh weight (FW) citrus peels. Moreover, they also described the yield recovery of phytochemicals as about 73%, obtained in comparison to total solid mass 6 . In fact, the synthesis of metal nanoparticles by synthetic reducing agents has toxic effects not only on human health but also on crop growth and yield (by accumulating in plant tissues, including edible parts). Hence, among all the famous techniques to prepare the metal nanoparticles such as mechanical milling, sputtering, physical method, chemical method and laser ablation etc. 7 . The green synthesis technique is more ecologically satisfactory, nontoxic, perfect, modest, dependable and one stage process. The effects of green synthesized metallic nanoparticles on plant species have been the subject of few studies 8 . A broad exploration has been led to limit the weighty reliance on engineered fungicides for controlling postharvest infections 9 . However, this growing problem necessitates eco-friendly and safe solutions for perishable crops like sweet potato quality 10 . Pectobacterium carotovorum ( P. carotovorum ) ubiquitous plant pathogen has been reported frequently with an adverse effect on vegetable host potatoes 11 . A recent study showed the strongest antibacterial effect of copper nanoparticles during the In-vivo and In-vitro analysis of ( P. carotovorum ) effect of copper nanoparticles. On the other hand, the current interest of researchers has also focused on human-infected viruses and bacteria. These viruses and bacteria have a broad effect on hospital-acquired infections. After the outbreak of deadliest pandemic COVID-19, a number of trust worthy sources has declared the use of metallic nanoparticles against SARS-CoV-2 12 . Shahid et al. studied the effect of cuprous oxide nanoparticles coated cotton fabrics against various types of pathogens to deal with hospital-acquired infections. The Cu 2 O coated fabrics showed excellent antibacterial effects against, E. coli and S. Aureus . Moreover, various studies demonstrated the In-vivo and In-vitro effect of copper and silver nanoparticles against plant and human pathogenic fungus Aspergillus niger ( A. niger ) 13 , 14 . Usha et al. analysed effectiveness of copper oxide nanoparticles coated fabrics against A. niger and exhibited about 100% reduction after 48 h of incubation 15 . So, the researcher has been using different types of antimicrobial agents on hospital textiles to reduce the risk factor against infections. However, the use of antimicrobial agents is limited because of their toxicity 16 . So, as an alternative to all aforementioned techniques the use of green synthesis metal nanoparticles is quite well and suitable against several types of pathogens (human and plant infected) 17 , 18 . Manal et al. 19 attempted to synthesize Ag-NPs using biological waste material from citrus limon peels. The synthesized Ag-NPs had an average size of 59.74 nm according to DLS measurements and showed strongest antipathogenic effect.

The anti-microbiological activity of copper and silver nanoparticles (prepared from synthetic source) used in plant soft roots has been widely reported. However, the antimicrobial activity of Cu-NPs and Ag-NPs and their role against the pathogens on potato soft roots (from citrus plants waste source has not been reported). Moreover, the same particles were applied on cotton fabric to fabricate the hygienic textiles for human use. The current study was conducted to study the in-vivo and in-vitro potential of green synthesized nanoparticles against bacterial infection in plants and human. To address the aforementioned issues, the waste of citrus fruit Citrus sinensis was used as a reducing and stabilizing agent for silver and copper salts. Thus, the coating was done over plant source (potato slices) and cotton bandages. The end applications of the developed textiles are their use as an active agent against soft roots plants and also for the benefit of humans in the fabrication of antimicrobial compression bandages, surgical drapes, panels, covers, shoe mats, scrub suits, table coverings, chair coverings, socks for doctors and patients etc.

Materials and methods

The peels of fruit ( Citrus sinensis ) were collected from local juice points, a market of Lahore, Pakistan. Copper (II) chloride (CuCl 2 .2H 2 O) and silver nitrate (AgNO 3 ) with 99% purity were acquired by Germany’s Riedel–de Haen. While, Merck (Germany) provided 98% pure L-ascorbic acid (used as a capping and reducing agent). None of the compounds were further chemically treated or purified, as they were all of analytical reagent grade. Merck provided LB (Luria–Bertani) agar-based broth and Lennox broth for antibacterial testing. For usage in all of the chemical reactions, every chemical solution was newly synthesized.

Preparation of plant extract and phytochemical analysis

Experimental research and field studies on plants involving the collection of plant material, were conducted in accordance with relevant institutional, national, and international guidelines and legislation.

The peels of Citrus sinensis were properly washed and let to air dry as shown in Fig.  1 a. Then, the dry peels were cut into flakes and chopped by using lab scale pestle and mortar. Subsequently, 40 g of chopped peels were added in round bottom flask followed by the addition of 100 mL of distilled water and refluxed at 80 °C for 60 min. Then obtained extraction was heated at 80 °C for 2 h and was left at room temperature to cool down. Afterward, the whole solution was filtered through Whatman filter paper to obtain the fine extract. The obtained extract (Fig.  1 b) was then stored in refrigerator at 4 °C for further use. The phytochemical analysis of obtained extract was also analyzed by using standard procedures as described previously 20 , 21 .

figure 1

( a ) Citrus sinensis peel flakes, ( b ) extraction through reflux, ( c ) CuCl 2 ·2H 2 O solution, ( d ) greenish black solution of Cu-NPs, ( e ) calcinated obtained Cu-NPs, ( f ) AgNO 3 solution, ( g ) greenish grey solution of silver nanoparticles, ( h ) calcinated obtained Ag-NPs.

Green synthesis of Cu-NPs from Citrus sinensis fruit peel extract

The hydrated copper chloride (CuCl 2 ·2H 2 O) was used as a precursor salt for green synthesis of Cu-NPs. About 30 g/L of CuCl 2 ·2H 2 O was mixed by using magnetic stirrer in 100 mL of deionized water (Fig.  1 c). The solution was stirred at 60 °C for 15 min followed by the slow addition of prepared extract into the solution. The color of the solution changed from blue to bluish orange after the addition of extract. Then it was stirred further for 30 min. The color of solution turned to greenish black, which justified the synthesis of Cu-NPs (Fig.  1 d). Afterward the solution was centrifuged at 8000 rpm for 10 min and placed in furnace for calcination. The blackish green colored copper particles were obtained after the procedure of calcination (Fig.  1 e) .

Green synthesis of Ag-NPs from Citrus sinensis fruit peel extract

The silver nitrate (AgNO 3 ) was used as a precursor salt for green synthesis of Ag-NPs. About 40 g/L of AgNO 3 was mixed by using magnetic stirrer in 100 mL of deionized water (Fig.  1 f). The solution was stirred at 60 °C for 30 min followed by the slow addition of prepared extract into the solution. The color of solution changes to brownish grey, which justified the synthesis of Ag-NPs (Fig.  1 g). Then, the solution was centrifuged at 8000 rpm for 10 min. The obtained particles were then placed in furnace for calcination. Greenish grey colored Ag-NPs were obtained after calcination (Fig.  1 h).

Application of prepared particles on substrate (cotton fabric and potato slices)

At first, three different concentrations of each particles were decided (0.25 g, 0.5 g, and 1 g) and dissolved 20 ml of de-ionized water.

Application on potato slices Fresh potatoes having almost same size with (no buds or eyes) were selected to make the slices. All slices were cut in same size and same width (1.5 mm). Then, the slices were transferred into beakers, containing the solutions of different nanoparticles with different concentration (see Table 1 ). The slices were remained in solutions for whole night to soak the maximum solution contains particles. Then, dried at 50 °C for 20 min in an oven. The schematic for applying the particles over the potato slices is shown in Fig.  2 a.

figure 2

The process of coating the nanoparticles over the ( a ) potato slices, ( b ) fabric structure.

Application on cotton bandages Now, within every solution, 0.5 g of binder was dissolved. Citric acid was used to kept the pH between 5 and 6. After that cotton fabrics with 10 × 10 cm pieces were dipped in the solutions containing different nanoparticles with different concentrations (see Table 1 ). Subsequently, the cotton cloth was pad in solution and dried for 20 min at 90 °C. The following procedure illustrates the application of particles over the structure of cotton fabric (Fig.  2 b). The experimental design for all the developed samples is given in Table 1 .

Testing and characterizations

Surface characterization of the synthesized nanoparticles.

Surface characterizations involved UV–Vis spectroscopy, XRD, and SEM analysis. UV spectroscopy was performed to analyze the absorbance spectra in a wavelength range of 200–1000 nm. The crystalline nature of the nanoparticles was observed by XRD analysis by using Japan Made Model JEOL JDX 3532. Approximately 1 mg of particles powder was used for XRD analysis. The scanning electron microscopy (SEM) was done to observe the surface morphology of the synthesized nanoparticles. The element composition of the biosynthesized nanoparticles was examined by EDX Oxford Company Model INCA 200. Nicolet Nexus 470 spectrometer was used to measure the infrared spectra. The instrument was equipped with an Attenuated Total Reflection (ATR) Pike-Miracle accessory.

In-vitro and in-vivo testing of synthesized nanoparticles against plant pathogens

The in-vitro antibacterial effectiveness was evaluated by using Disc diffusion method. Plant pathogens bacterial strains ( P. carotovorum ) were used for this purpose. Nutrient agar was used as a culture media. The zone of inhibition against different concentrations of Ag-NPs (0.25, 0.50 and 1.00 g) and different concentrations of Cu-NPs (0.25, 0.50 and 1.00 g) was measured. For in-vivo study, the bacterial strains of ( P. carotovorum ) with constant concentration 50 µl were applied over each sample (copper particle coated potato slice, silver particles coated potato slice and uncoated potato slice). Then, the slices were placed in petri dishes and sealed tightly with parafilm, and incubated for 24–48 h at 35 °C 22 .

In-vitro testing of synthesized nanoparticles against human pathogens

The in-vitro testing of synthesized nanoparticles was involved antibacterial Qualitative and Quantitative test.

Qualitative analysis (zone of inhibition measurement)

Bacterial strain preparation.

To perform the qualitative analysis, two types of bacterial strains gram positive S. aureus (CCM-3953) and gram-negative E. coli (CCM-3954) were selected. Each time fresh bacterial suspensions were prepared for cultivating even a single colony in a nutrient bath. The bacterial suspensions were remained in nutrient bath for the duration of the whole night at 37 °C. Prior to start the antibacterial tests the agar plates were prepared carefully by adjusting the sample turbidity at 0.1 with optical density of (OD 600). Subsequently, the Cells were evenly distributed on the agar plates with the help of cotton swab (soaked in the culture media). These plates were used for qualitative analysis of antibacterial testing.

Determining zone of inhibition

The qualitative analysis Zone of Inhibition was found against the Ag-NPs and Cu-NPs coated textile samples. The particles coated textile samples, and controlled fabric (not coated with particles) having (6 × 6 mm sq.) dimensions were placed directly on inoculated agar plates. Then whole assembly (samples and inoculated agar plates) were placed at 37 °C for 24 h. Zone of inhibition was analysed around the entire diameter (mm) of the particles coated textile. The calculation was made to measure the area where bacterial growth was inhibited.

Quantitative test (reduction factor)

The standard test according to AATCC (100-2004 procedure was used to conduct quantitative measurements). This approach describes the reduction in inoculation bacterial concentration caused by the sample effect by using the reduction factor. The inhabitation in reduction normally calculated by considering the number of surviving bacterial colonies (CFU). Consequently, a comparison of both treated and untreated samples is required (standardized). First, a sample that had been cut into 18 × 18 mm squares was placed in a sterile container for thirty minutes. Next, for each test, a specific bacterial strain (100l) containing 105 CFU/ml was used. After 24 h of incubation at 37 °C in a thermoset, physiological solution was added.

Antifungal activity

To assess the antifungal properties of the treated samples, the standardized test method AATCC 100-2004 was consistently employed. The specific fungus used in this experiment was Aspergillus niger . The antifungal activity was estimated as a percentage change using Eq. ( 1 ):

Here, A represents the fungal spore counts for the control fabric, and B represents the spore counts for the treated dyed fabric specimens.

Antiviral activity

The infectious viral titer was determined using Behrens and Karber method. Briefly, the Vero-E6 cell line cultures were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 9% foetal bovine serum (FBS) and 2% penicillin–streptomycin (PSA). The cultures were infected with the coronavirus and were grown under standard test conditions (24 h at 37 °C in 6% CO 2 ) and stored in 96-well plates at a concentration of 2 × 10 5 . To evaluate the viral titer, the supernatant was carefully centrifuged for 30 min at 3700 rpm and temperature of 5–7 °C. The viral titer in the developed cell lines was calculated using the Behrens and Karber’s method. For Next, vials containing the fabric samples were filled with a 20 mm × 20 mm section of fabric. The filter was used to eliminate any extractable viral loads in the channels after passing a 100 µl infection rate through the test sample. The infectious coronavirus was diluted from 101 to 108 repetitions. Vero-E6 cell cultures were grown for three days under optimal conditions at 37 °C and 6% CO 2 after injecting each serial dilution. The titers of the corona virus in the cultured cell lines were determined the same method.

Statistical analysis

The extent of relationships between dependent and independent variables was determined using the ANOVA tool by MINTAB software.

Results and discussion

The nanoparticles synthesized by green method were characterized by various characterization techniques.

Phytochemicals screening analysis

At first, the screening of extracted phytochemicals was done by using the standard methods obtained from various studies. The screening showed the presence of all active compounds necessary for the reduction, dispersion and stabilization of metal ions. The obtained extract from the citrus leaves were contained the phytochemicals such as flavonoids, phenols, steroids, glycosides and terpenoids. Several studies have already been reported about the presence of alkaloids, saponins, tannins, sterols and flavonoid in citrus extract. Flavonoids are most desiring and key component among all phytochemicals. In fact they played a dual role, as a reducing agent and as an antibacterial agent due to their inherent antipathogenic characteristics. Flavonoids also have antioxidative, cytotoxic, chemopreventive, and antiprnoliferative properties 23 . The list of extracted phytochemicals in accordance with the relevant studies is given in Table 2 .

Distribution of particle sizes, polydispersity index and zeta potential

The molecule size was calculated by using DLS methods, based on the Brownian motions of the particles. Figure  3 a,b respectively demonstrate the average particle size distribution for Cu-NPs and Ag-NPs. The sizes of particles were varying in size from nano meter to milli meter range in a multi-modal manner. The average size of copper and silver particles were notices about 510 nm and 470 nm respectively, at a zeta potential of − 41.6 mV and − 30.6 mV respectively. The Zeta potential values showed the colloid stability of both types of nanoparticles, as these values were > ± 30 mV 22 .This ensured that the particles were evenly distributed throughout the suspension and had a high negative potential from the Nano meter to the micro range. The stability of particles is well analyzed by zeta potential, while the particle size distribution in nano sciences is more articulate with polydispersity index (PDI) values. The polydispersity Index (PDI) of Ag-NPs and Cu-NPs was noted about 0.312 and 0.258 respectively. The values show that the synthesised particles are highly polydisperse 26 .

figure 3

The particle size distribution of ( a ) Cu-NPs and ( b ) Ag-NPs.

Surface characterizations

The surface morphology of synthesized copper and silver nanoparticles was examined using scanning electron microscopy (SEM). The external morphological investigation through SEM revealed the formation of Ag-NPs, and Cu-NPs at the nano to micrometric scale. The rough surface and random clusters with cylindrical form for both types of particles was observed. The tiny agglomerations with constant repetition and even deposition was also noticed. SEM images also revealed the irregular spherical morphological features of the biosynthesized nanoparticles as shown in Fig.  4 b,e. However, the sizes of all particles were noticed within the nanometric sale by zeta size analysis as described in the previous Section “ Distribution of particle sizes, polydispersity index and zeta potential ”. More SEM images at higher magnification in their respective box are added to deeply analyze the connectivity of particles. Which showed the dense coating and homogeneous connectivity between the particles over the fabric structure. However, they were observed within the range of nanometric scale. Moreover, the TEM analysis were also performed to better clarify the sizes and morphologies of nanoparticles as shown in Fig.  4 a,d. The TEM analysis estimates the sizes of copper and silver particles between 400 and 500 nm and report the morphologies nearly spherical. As aforementioned, SEM analysis shows the nanoclusters of particles, ranging in 500 nm. While during TEM analysis, it seems that the separate particles of copper and silver are broken parts from their respective clusters 27 .

figure 4

( a – c ) TEM analysis, SEM analysis and EDX spectra of silver particles ( d – f ) TEM analysis, SEM analysis and EDX spectra of copper particles.

Additionally, the elemental compositions of Cu-NPs and Ag-NPs were also estimated to found the amount of metal in percentage. The elemental composition of EDX was estimated using spectrum analysis also uncover additional information about the makeup and components of particles as shown in Fig.  4 c,f. Except of oxygen and carbon, some other peaks of impurities in least amount were also noticed such as Ca, Mg, and Cl. The existence of trace elements with low quantities is normal behavior during elemental analysis 28 .

Justification for the formation of copper and silver particles

The UV–Visible spectroscopy was conducted to justify the synthesis of Cu-NPs and Ag-NPs.

The aqueous solution of nanoparticles was mixed with constant ratio (1:2) in distilled water. Subsequently, were mixed well and prepared for UV analysis. The UV spectrum obtained from the synthesized nanoparticles were noticed at 339 nm and 415 nm for copper and silver respectively. While the UV–Vis absorbance spectrum of orange peels extract was notes at λ max was noted 320 nm due to the respective signal of phenolics groups Fig.  5 a 29 . In fact, the significant shifts in values and peaks of metal particles as compared to orange extract values is due to the changes in the morphology, size or surface microstructures of silver and copper nanostructures.

figure 5

( a ) UV–Vis Spectrum of the orange peels extract, synthesized copper and silver nanoparticles, ( b ) XRD peaks of silver nanoparticles and ( c ) XRD pattern of Cu-NPs.

Moreover, the XRD analysis was done to justify the formation of crystalline nature of coper and silver particles. The phase purity of manufactured Ag particles was confirmed by exact indexing of all the peak intensity to the silver structure, as shown in Fig.  5 b. The four peaks for Ag-NPs appeared at 2 values of 77.5, 64.5, 44.3, and 38.1, respectively. These peaks were attributed to cubic-shaped diffraction planes (3 1 1), (2 2 0), (2 0 0), and (1 1 1), according to data from the International Diffraction Centre (data number JCPDS 04-0783 card) 30 . No significant peaks were observed for other impurities, such as silver oxide. Figure  5 c represents the XRD spectrum of Cu-NPs. A precise identification of every diffraction peak to the copper structure reveals the elemental composition of Cu particles. Cu diffraction planes (2 2 0), (2 0 0), and (1 1 1) are characterized by the occurrence of Cu diffraction pattern (2θ) at 74.2, 59.5, and 43.3°. From either the presence of peak position, the copper particles crystalline structure was investigated. Because no distinct impurity peaks were found, other than the development of the Cu 2 O peak (2θ) at 38°, the widening of the peaks instead indicated the synthesis of Cu particles at the nano range, respectively 31 .

The extract of orange peels was used as a bio-reductant to synthesized the nanoparticles of copper and silver. Therefore, FT-IR spectroscopy was employed to confirm this reduction process. An analysis of the FT-IR spectroscopy analyzed the presence of functional groups on green synthesized silver and copper particles. The Fig.  6 is illustrating the respective FT-IR spectra of orange peels extract coated cotton fabric, copper particles coated and silver particles coated fabrics. The absorption peaks around 2950, 3331, 2115, 1636, and 597 cm −1 . On 2950 are C–H stretching vibration absorption peaks in cellulose. While, the broad absorption band on 3670 cm −1 corresponds to the O–H stretching frequency, while at 1636 cm −1 depicts the C=O stretching of the carbonyl group. The peak 1059 cm −1 was noted due to the to the link of alcohols or esters (C–O–H or C–O–R) 32 .

figure 6

The FT-IR spectra of orange peels extract, copper particles and silver particles coated fabrics.

The in-vitro analysis of synthesized nanoparticles against plants pathogens

The In-vitro analysis of the biosynthesized silver and copper nanoparticles was carried out by using disc diffusion method. The bacterial strains P. carotovorum, was used to check the antibacterial potential of biosynthesized Ag-NPs and Cu-NPs. Nutrient agar was used as a culture media. Different dilutions of the synthesized nanoparticles were used to analyze zones of inhibitions against the bacterial strains. The zone of inhibition against different concentrations of silver particles coated potato slices samples S1 (0.25), S2 (0.50) and S3 (1.00 g) and different concentrations of copper particles coated potato samples S7 (0.25), S8 (0.50) and S9 (1.00 g) are shown in Fig.  7 a,b.

figure 7

Growth inhibitions in response to ( a ) Cu-NPs and ( b ) Ag-NPs against plant pathogens ( P. carotovorum ) and ( c ) graphical representation of zone of inhibition values for samples coated with silver and copper particles.

The silver particles coated potato sample S3 (1.00 g) showed the maximum zone about 16 mm. In a similar study, the antibacterial activity was noted against P. carotovorum by silver nanoparticles. Their results revealed that the Ag-NPs showed largest inhibition zone of about with the 14.33 mm 33 . While the copper particles coated sample S9 (1.00 g) showed the zone about 14 mm. It means the silver particles are little bit more effective as compared to copper particles. Azam et al., conducted a comparative analysis of copper and silver particles against different pathogens. Where, silver particles coated substrate showed better performance as compared to copper particles 34 . However, the overall efficiency of both particles is quite effective against bacterial strains P. carotovorum. Figure  7 c is showing the bar graphs with standard errors against inhibition zone of all silver and copper particle coated potato samples. The group S3 and S9 contains the zone of inhibition values against Ag-NPs and Cu-NPs coated potato samples. During the observation the tail of error bar of silver particles coated sample S3 (1.00 g) group is not coinciding with the head of the error bar of copper coated S9 sample. It means there is a insignificant difference between two groups, which implies that silver and copper particles coated samples have different zone of inhibition range. The ANOVA analysis at 95% confidence interval was applied. The P value for group control sample and copper coated sample was calculated as 0.047 which is P  < 0.05. Hence the P value was less than 0.05 which means null hypothesis is insignificant and there is significant difference between the values of sample group S3 and S9. So, there is significant difference between the inhibition Zone against Ag-NPs and Cu-NPs.

The in-vivo antagonistic potential of nanoparticles against plant pathogens

The potato slices coated with different concentrations of silver particles coated potato slices samples S1 (0.25), S2 (0.50) and S3 (1.00 g) and different concentrations of copper particles coated potato samples S7 (0.25), S8 (0.50) and S9 (1.00 g) were also subjected to In-vivo Antagonistic potential. The bacterial strains of ( P. carotovorum ) with constant concentration 50 µl were applied over each sample (silver particle coated potato slice S3 (1.00 g), copper particles coated potato slice S9 (1.00 g) and uncoated potato slice). Afterwards, the slices were put in petri plates and covered with para-film and incubated for 24–48 h at 35 °C. The diameter of the infectious zones with measured values are shown in Fig.  8 a–c and their values of infection in graphical representation are shown in Fig.  8 d respectively. No or almost zero zone of infection was observed on potato slice coated with silver particles S3, while the potato slice coated with copper particles S9 showed slight zone of infection. The reason we have already described in previous section (see section In-Vitro antagonistic), where silver particles proved more effective against pathogens as compared to copper particles. Furthermore, the clear and large zone of infection was seen on uncoated potato slice. It means nanoparticles are quite effective against the bacterial strains of ( P. carotovorum ) . The tail of error bar on control sample group is not coinciding with the head of the error bar of copper coated error bar. It means there is significant difference between two groups, which implies that the coating of copper over the potato sample significantly reduce the infection against the bacterial strains of ( P. carotovorum ) . Also, there is no group in the place of silver coated sample (as there was no zone of infection). So, A huge difference between each group is present and promotes to the significant difference. The ANOVA analysis at 95% confidence interval was applied. The P value for group control sample and copper coated sample was calculated as 0.029 which is P  < 0.05. Hence the P value was less than 0.05 which means null hypothesis is insignificant and there is significant difference between the control and copper coated sample. So, there is significant difference between the infection zones.

figure 8

Potato slices with zone of infection caused by P. carotovorum ( a ) uncoated sample, ( b ) coated with copper particles and ( c ) coated with silver particles and ( d ) bar graphs showing the values of their respective zone of infections.

Moreover, the area of slice without infection in percentage (area free from bacterial attack) was also calculated by using the following Eq. ( 2 ).

The calculated values of percentage of clear area from bacterial strains are given in Table 3 . The silver particles coated samples showed almost 100 percent clear area (i.e. not a single spot or colony of bacterial strain). While there was 87 percent bacterial free area was calculated for potato slice coated with copper particles. In case of potato slice having no coating of particles showed less bacterial free area, which is only 54.5 percent.

In-vitro potential of synthesized nanoparticles against human pathogens

To evaluate the effectiveness of the coated textiles for antibacterial properties, both qualitative and quantitative test were conducted.

Reduction factor (quantitative test)

The quantitative technique according to AATCC-100 method was used to measure bacterial resistance against S. aureus and E. coli strains. Figure  9 presents the reduction percentage of the bacterial cultures on the treated and untreated textile samples. The effectiveness was checked against different concentrations of silver particles coated fabric samples S4 (0.25 g), S5 (0.50 g) and S6 (1.00 g) and different concentrations of copper particles coated fabric samples S10 (0.25 g), S11 (0.50 g) and S12 (1.00 g). The control sample was ineffective against the tested microorganisms. All of the treated samples showed higher reduction in percentage against both type of bacteria, as the amount of Cu-NPs (from 0.25 to 1 g) and Ag-NPs (from 0.25 to 1 g) on the fabric increased. The maximum reduction about 99.99% was found in case of both types S. aureus and E. coli bacterial colonies. It was noteworthy that all fabric samples coated with silver (S4 to S6) and copper particles (S10 to S12) showed about 99.99% reduction against E. coli as the compared to S. aureus . It means that E. coli is more susceptible to metal particles than to S. aureus . The reason is E. coli can survive less in open environment (cause less infections), easily vulnerable to antibiotics due to its interactive membrane. While the S. aureus can stay longer and resist a range of antibiotics and cause serious infections and leads to different physical rheological responses 35 , 36 .

figure 9

Antibacterial activity in terms of log CFU/ml (left) and percentage reduction (right) of fabrics treated with silver and copper nanoparticles and untreated cotton fabric.

Figure  10 provides additional evidence for the aforementioned trend by displaying the development of bacteria concentrations for silver coated fabric sample S6 (1 g of particles) and copper coated fabric sample S12 (1 g of particles). The untreated cloth was shown to be inefficient against bacterial growth when compared to textiles coated with copper and silver particles. The copper and silver particles coated samples showed maximum reduction in bacterial colonies against both type of pathogens ( E. coli and S. aureus ). At greater concentrations, colony reductions showed a substantial increase, with more than 99 percent efficiency for both species of bacteria 37 .

figure 10

Images of concentration of bacterial growth for the ( a , b ) copper particles, ( c , d ) for silver particles and ( e , f ) for untreated fabrics.

The silver particles coating on cotton fabric in present study showed better performances compared to previously reported study, where the incorporation of Ag NPs into cotton fabrics using UV photo-reduction was performed 38 . Their results also support the declaration about increase in concentration has direct relation on the reduction of antimicrobial activity of E. coli . Several researches have been conducted for the analysis of antimicrobial activities of Ag-NPs coated bandages, and their impact on bacterial strains. The exact mechanism of reduction or inhibition of bacteria growth is still partially understood. In fact, some vibrant concepts involve the release of Ag + and interaction with cell walls. Moreover, these silver ions can also interact with released -SH groups from cellular excretions; and leads to further inactivation of proteins. Hence, the released Ag + ions may again combine another protein when the current protein is decomposed. The silver ions also expediate the production of oxidized radicals; which can penetrate easily into cell wall structure 39 .

Zone of inhibition test (qualitative measurements)

Zone of inhibition test was also used to assess the samples antibacterial abilities. Both Gram-positive ( S. aureus ) and Gram-negative ( E. coli ) bacteria were incubated for 24 h at 37 °C in the dark, all fabric samples had distinct inhibitory zones, as seen in Fig.  11 a,b. The effectiveness was checked against different concentrations of silver particles coated fabric samples S4 (0.25 g), S5 (0.50 g) and S6 (1.00 g) and different concentrations of copper particles coated fabric samples S10 (0.25 g), S11 (0.50 g) and S12 (1.00 g). The textiles treated with silver nanoparticles (S4 to S6) had the greatest antibacterial zones against the strains of S. aureus and E. coli , whereas the fabrics treated with Cu-NPs (S10-S12) exhibited a smaller zone of inhibition. The average values were computed by conducting three readings of each sample. The outcomes showed that the free-standing nature of the copper and silver particles led to considerable disinfection of both bacterial strains, where S. aureus demonstrating more sensitivity than E. coli. As an illustration, using copper and silver particles raised the area of inhibition for E. coli from 4.5 to 10.7 mm, while increasing the area of inhibition for S. aureus from 6.5 to 14 mm as shown in Fig.  11 c. It should be noted that the increase in inhibition zone with the increase in the concentration of nanoparticles had already been discussed in some previously published research works 30 . The combination of physical and chemical action of bacteria with particles is assumed to be the cause of coated textiles antibacterial properties. Through endocytosis procedures, the nanoparticles are absorbed by the cells. Ionic species are produced inside the cells during the nanoparticles degradation, increasing the cells ability to absorb ions 40 . Silver is showing good antimicrobial ability. In fact, the less antipathogenic effect of copper coating over the substrate as compared to silver was due to the less stability of copper. The similar effect of antimicrobial effectiveness was observed in some relevant studies. Where the in-situ deposition of copper and silver particles was performed to achieve the electrical conductivity and antimicrobial effectiveness. The reason for low electrical performance and bioactive performance was due to the susceptibility of copper particles to oxidation and carbonization 41 .

figure 11

Inhibition zones ( a ) against S. aureus , ( b ) E. coli. , ( c ) graphical representation of Zone of inhibition around all samples.

The group S6 contains the Ag-NPs coated fabric samples showing the zone of inhibition values against S. aureus, E. coli, whereas group S12 contains the Cu-NPs coated fabric samples showing the zone of inhibition values against S. aureus, E. coli. While comparing the zone of inhibition, values against S. aureus were between S6 and S12. The tail of error bar of silver coated S6 sample group (zone of inhibition around S. aureus black bar) is not coinciding with the head of the error bar copper coated S9 sample of (zone of inhibition around S. aureus black bar). It means there is significant difference between two groups, which implies that silver and copper particles coated samples have different zone of inhibition range. The P value between these two groups was observed as 0.037 which is P  < 0.05. Hence the P value was less than 0.05 which means there is significant difference between silver coated S6 sample group (zone of inhibition around S. aureus black bar) and copper coated S9 sample of (zone of inhibition around S. aureus black bar).

In the same way, while comparing the zone of inhibition values against E. coli between S6 and S12. The tail of error bar of silver coated S6 sample group (zone of inhibition around E. coli green bar) is not coinciding with the head of the error bar copper coated S9 sample of (zone of inhibition around E. coli green bar). It means there is significant difference between two groups, which implies that silver and copper particles coated samples have different zone of inhibition range. The P value between these two groups was observed as 0.029 which is P  < 0.05. Hence the P value was less than 0.05 which means there is significant difference between silver coated S6 sample group (zone of inhibition around E. coli green bar) and copper coated S9 sample (zone of inhibition around E. coli green bar).

Antifungal activity of treated samples

In order to assess the effectiveness of various fabric samples against the A. niger fungus, the AATCC-100 method was utilized in this study. Figure  12 a–d showed the results related to fungus growth against each particle coated sample and percentage reduction in fungal spore germination for each fabric specimen. However, it was observed that fabrics with particle coatings were better in combating fungi when compared to untreated samples . Silver coated fabrics had the greatest inhibition of fungal growth among the particles-coated samples with antifungal effectiveness of approximately 77%. In fact, the present study showed better antipathogenic properties of silver particles overall. The statement can be further justified from a related study; where green synthesized silver particles showed almost the same reduction in percentage of fungus 36 . The group S6 contains the Ag-NPs coated fabric samples showing the reduction percentage of fungal activity values against A. niger, whereas group S12 contains the Cu-NPs coated fabric samples showing the reduction percentage of fungal activity values. While comparing the reduction percentage of fungal activity values against A. niger between S6 and S12. The tail of error bar of silver coated S6 sample group is not coinciding with the head of the error bar copper coated S9 sample. It means there is significant difference between two groups, which implies that silver and copper particles coated samples have reduction percentage of fungal activity values. The P value between these two groups was observed as 0.013 which is P  < 0.05. Hence the P value was less than 0.05 which means there is significant difference between silver coated S6 sample group (percentage of fungal activity values against A. niger ) and copper coated sample S12.

figure 12

Images shows fungus growth against ( a ) silver particles coated samples, ( b ) against copper particles coated samples, ( c ) raw cotton and ( d ) percentage reduction in fungal spore germination for each fabric specimen.

Antiviral effectiveness

The Behrens and Karber method was used to measure the antiviral effectiveness. Starting with initial viral titer of infectivity to determine the decrease in viral titer for coronavirus. The viral infectivity titer log is shown in Fig.  13 for both 0 h and 6 min. Overall, it showed that all treated samples (S4-S6 and S10-S12) with nanoparticles had sharply reduced viral infectious titer more than double as compared to untreated samples. However, there was no considerable difference of the titer amount in samples treated with either Ag-NPs or Cu-NPs. It indicates both silver and copper nanoparticles are almost equally effective in reducing viral infection in tested cell lines.

figure 13

Reduction in viral infectivity titer ( a ) and percentage adsorption ( b ) calculated from viral infectivity at a contact time of 0 and 60 min.

One possible mechanism for the suppression of viruses and the antiviral effects seen involves the interaction between particles and glycoproteins on the viral surface. In a recent research silver particle coated fabric were fabricated by photo deposition method. The couple effect of Ag 0 /Ag + redox active agent exhibits 97% viral reduction specific to SARS-CoV-2 42 . The group S6 contains the Ag-NPs coated fabric samples showing the virus adsorption in percentage by nanoparticles, whereas group S12 contains the Cu-NPs coated fabric samples showing the virus adsorption in percentage by nanoparticles. While comparing the virus adsorption in percentage between S6 and S12. The tail of error bar of silver coated S6 sample group is not coinciding with the head of the error bar copper coated S9 sample. It means there is significant difference between two groups, which implies that silver and copper particles coated samples have significant difference in virus adsorption in percentage. The P value between these two groups was observed as 0.03 which is P  < 0.05. Hence the P value was less than 0.05 which means there is significant difference between silver coated S6 sample group and copper coated sample S12.

The current research employed sustainable, inexpensive and eco-friendly method to synthesized two different types of nanoparticles. In present study the phytochemical analysis of the peels of Citrus sinensis revealed the phenolic contents (rich in phenols and flavonoids), served as reducing and as a dispersing agent during the green synthesis of metal nanoparticles. The study was conducted to reveal the antagonistic (in vivo and in vitro) potential of synthesized nanoparticles against plant pathogenic bacteria ( Pectobacterium carotovorum ) and pathogens effective against humans ( E. coli , S. aureus ) was studied.

A total of 12 samples were prepared, (S1 to S6 with silver particles and S7 to S12 with copper particles) with different concentration. The prepared particles were coated on potato slices and cotton bandages. It was observed that the silver-coated samples S3 (1 g of particles on potato slices) and S6 (1 g of particles on fabric) had a higher ZOI than copper particles coated samples S9 (1 g on potato slices) and S12 (1 g of particles on fabric) samples. The results were also justified statistically, where the significant difference (as P  < 0.05) between the two groups (of silver and copper coated potato samples) S3 and S9, and between the groups (of silver and copper coated fabric samples) S6 and S12 was found.

During the In-vivo analysis of particles over the potato slices. The bacterial strains of ( P. carotovorum ) showed almost zero infection against silver particles coated potato sample S3, while the potato slice coated with copper particles S9 showed very slight zone of infection, However, the clear and large zone of infection was seen on uncoated potato slice. Moreover, during in vitro analysis (antibacterial, antiviral, and antifungal) of prepared bandages the silver particles coated fabrics S6 with higher concentration (1 g) showed the 78% and 84% of antifungal and antiviral activity respectively. It means, the waste of peels contains quite effective bioactive agents that can be used against diverse types of pathogens. The surface morphology and existence of metals were analyzed by SEM, dynamic light scattering, EDS and XRD. The durability and retention of particles over the fabric surface was also analyzed by sever washing cycles. The developed fabrics can be effectively used to fabricate bioactive sportswear or active wears, bioactive compression garments as well as winter gloves, and compression bandages.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Change history

29 may 2024.

A Correction to this paper has been published:

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The authors of this study extend their appreciation to the Research Supporting Project, King Saud University, Riyadh, Saudi Arabia, for supporting this study (RSP2024R378) and for funding this work.

The authors of this study extend their appreciation to the Research Supporting Project, King Saud University, Riyadh, Saudi Arabia, for supporting this study with the following Grant Number (RSP2024R378).

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Mujtaba Ikram

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Saharin Islam

Department of Clinical Pharmacy, College of Pharmacy, King Saud University, 11451, Riyadh, Saudi Arabia

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Conceptualization: O.U., S.T.I.; Methodology: M.M.M.B. and W.S.; Software: M.B.A.A.; Investigation: M.B.A.A.; Validation: M.I., S.I.; Writing—original draft: O.U.; Visualization: M.I.; Data curation: O.U.; Supervision: S.T.I.; Writing—review and editing: S.T.I., M.N. and O.U.; Funding acquisition: W.S. and M.B.A.A.

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green synthesis method of nanoparticles

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Green synthesis of metal nanoparticles using microorganisms and their application in the agrifood sector

  • Howra Bahrulolum 1   na1 ,
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  • Hossein Tarrahimofrad 2 ,
  • Vasighe Sadat Mirbagheri 1 , 3 ,
  • Andrew J. Easton 4 &
  • Gholamreza Ahmadian 1  

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The agricultural sector is currently facing many global challenges, such as climate change, and environmental problems such as the release of pesticides and fertilizers, which will be exacerbated in the face of population growth and food shortages. Therefore, the need to change traditional farming methods and replace them with new technologies is essential, and the application of nanotechnology, especially green technology offers considerable promise in alleviating these problems. Nanotechnology has led to changes and advances in many technologies and has the potential to transform various fields of the agricultural sector, including biosensors, pesticides, fertilizers, food packaging and other areas of the agricultural industry. Due to their unique properties, nanomaterials are considered as suitable carriers for stabilizing fertilizers and pesticides, as well as facilitating controlled nutrient transfer and increasing crop protection. The production of nanoparticles by physical and chemical methods requires the use of hazardous materials, advanced equipment, and has a negative impact on the environment. Thus, over the last decade, research activities in the context of nanotechnology have shifted towards environmentally friendly and economically viable ‘green’ synthesis to support the increasing use of nanoparticles in various industries. Green synthesis, as part of bio-inspired protocols, provides reliable and sustainable methods for the biosynthesis of nanoparticles by a wide range of microorganisms rather than current synthetic processes. Therefore, this field is developing rapidly and new methods in this field are constantly being invented to improve the properties of nanoparticles. In this review, we consider the latest advances and innovations in the production of metal nanoparticles using green synthesis by different groups of microorganisms and the application of these nanoparticles in various agricultural sectors to achieve food security, improve crop production and reduce the use of pesticides. In addition, the mechanism of synthesis of metal nanoparticles by different microorganisms and their advantages and disadvantages compared to other common methods are presented.

green synthesis method of nanoparticles

Nanoparticles now play a key role in most technologies, including medicine, cosmetics, agriculture and the food sciences [ 1 ]. Recently, the synthesis of metal nanoparticles (MtNPs) using microorganisms and plants has been recognized as an efficient and green method for further exploitation of microorganisms as nanofactories [ 2 ]. Given the challenges facing the international community, especially in terms of population growth and climate change, nanotechnology can have positive effects on improving the quality of agricultural products, minimizing the adverse effects of agricultural pesticides on the environment and human health, and increasing productivity and food security. Unique properties of nanoscale materials make them an excellent candidate for using in the design and development of new tools for supporting agriculture and related industries. Nanotechnology can improve agricultural processes such as soil quality and the quality of agricultural products by using nanoparticle-based fertilizers or by stimulating plant growth. In addition, the use of fertilizers and pesticides using nanoparticle-based carriers and compounds is reduced without reducing productivity [ 3 ]. Nanotechnology can also minimize waste by fabricating products that are more efficient. Applications of nanosensor technology can lead to the development of precision agriculture and efficient management of resources, including energy and materials used [ 4 ]. In particular, the goal of developing green nanotechnology, which utilizes biological pathways for the synthesis of nanomaterials is minimizing the production of hazardous substances. Meanwhile, the amount of energy input in green nanotechnology is much lower than in other technologies; almost no toxic chemicals are produced during synthesis, and their environmental compatibility is very high. Therefore, green nanomaterials produced can be widely used in various industries [ 5 ]. Depending on the application required, different types of nanomaterials are used in agriculture. For example, for use in pesticides, nanoparticles are used as carriers, which gradually release the active ingredient(s) to reduce their overall consumption. When the goal is to improve the packaging of agricultural products, the nanomaterials used are selected to be biocompatible and do not have negative effects on human health while increasing the shelf life of food. Alternatively, high-sensitivity nanosensors with plasmonic properties such as silver or gold nanoparticles can be used to measure environmental conditions, report changes in a timely way, and intelligently control plant needs in greenhouses. In all cases, the small size and unique physical and chemical properties of the MtNPs make them attractive for use in various agricultural sector [ 1 ]. To date, a broad range of nanotechnology applications have emerged in the agrifood sector, such as nanosensors, tracking devices, targeted delivery of required components, food safety and intelligent packaging which can affect different aspects of our lives [ 6 , 7 , 8 ].

Several advanced techniques are available to improve precision breeding methods and enable precise control of the green synthesis process at the nanometer scale. Nanotechnology can also be an alternative source for generating fertilizer, as MtNPs have been shown to be able to increase germination in agricultural seeds. Other applications include the use of nanoscale carriers for effective delivery of fertilizers, pesticides, plant growth regulators, and other similar compounds. These processes improve the stability of these materials to environmental degradation and ultimately reduce their amount used, which in turn leads to reductions in chemical runoff and associated environmental problems. Carriers can also be designed to increase the communication between plant roots and the surrounding soil structure [ 9 ]. Modified nanoparticles can be added to conventional fertilizers for improving nitrogen storage capacity which leads to reduced nitrogen loss and better nutrition for agricultural products. Several nanoemulsions have also been formulated to increase the biological compatibility of herbicides and pesticides [ 10 ].

Microorganisms are important nanofactories that are able to accumulate and detoxify heavy metals due to the presence of various reductase enzymes that are able to reduce metal salts to MtNP [ 2 ]. In recent research, bacteria such as Pseudomonas deptenis [ 11 ], Visella oriza [ 12 ] Bacillus methylotrophicus [ 13 ], Bhargavaea indica and Brevibacterium frigoritolerans have been shown to be able to synthesize silver (Ag) and gold (Au) nanoparticles. MtNPs have also been synthesized by various genera of microorganisms such as Lactobacillus, Bacillus , Pseudomonas , Streptomyces, Klebsiella , Enterobacter , Escherichia , Aeromonas , Corynebacterium , Weissella , Rhodobacter , Rhodococcus , Brevibacterium , Trichoderma , Desulfovibrio , Sargassum , Shewanella , Plectonemaboryanum , Pyrobacul um and Rhodopseudomonas [ 2 ]. The synthesis of nanoparticles by actinomycetes has not yet been well studied, although studies to date have shown that nanoparticles produced by actinomycetes have very good dispersion and stability and have significant lethal activity against various pathogens [ 14 ]. In particular, various microorganisms, such as bacteria, fungi, yeasts and microalgae have been shown to produce MtNPs either intra- or extracellularly. These microorganisms are able to produce organic matter inside, and to transport it to the outside of their cells [ 15 ]. Microorganisms as nanofactories have great potential as environmentally friendly, inexpensive, and non-toxic tools that do not require much energy for MtNPs synthesis compared to physicochemical methods. Among the various mechanisms for the green synthesis of MtNPs, those that perform extracellular synthesis are of great interest because the extracellular location of the material eliminates the need for costly and complex downstream processing steps to recover intracellular nanoparticles [ 2 ]. Green synthesis of MtNPs using microorganisms has several advantages compared to conventional physicochemical methods. In particular it offers a rapid, cost-effective, clean, non-toxic and environmentally friendly method for the synthesis of MtNPs with a wide range of sizes, shapes, compositions and physicochemical properties [ 16 , 17 ]. However, the main drawbacks of microorganism-based synthesis of MtNPs includes complicated steps such as microbial sampling, isolation, culturing and storage. In addition, the recovery of MtNPs produced by this method requires downstream processing [ 2 ].

In this review, we explore the various potential applications of green synthesized MtNPs with an emphasis on agriculture. This includes consideration of advantages of green synthesis of MtNPs using different microorganisms.

Green synthesis of MtNPs by microorganisms and their characterization

Various approaches have been used for MtNP synthesis, such as physical, chemical, and biological methods. The physical and chemical methods for MtNP synthesis have many disadvantages including the use of expensive equipment, high heat generation, high energy consumption and low production yield [ 18 , 19 ]. The main drawback of these methods is the use of toxic chemicals, which present several environmental problems [ 19 , 20 ]. This has generated a need for an environmentally friendly option for the synthesis of MtNPs, the current focus of which is the green synthesis of MtNPs from biological routes such as microorganisms, plants, microbial enzymes, polysaccharides and degradable polymers [ 21 ]. Green synthesis methods are more beneficial than traditional physical and chemical methods because they are simple, cost-effective, free of toxic and environmentally unfriendly chemicals, and as a result they have gained considerable importance in recent years [ 20 ].

The innovative and diverse applications of MtNPs in various fields including medical sciences, environmental sciences and agriculture, research on MtNPs and different approaches of their synthesis has increased rapidly over recent years [ 18 , 22 ]. The synthesis of MtNPs is generally performed using one of two different approaches, broadly considered as top-down and bottom-up approaches. In top-down approaches, bulk materials are broken down into nano-sized particles to form MtNPs, based on their reduction in size, using various physical and chemical techniques [ 18 , 23 ]. The main drawback of this method is the production of nanoparticles with imperfect surface structures. Also, it is an expensive and time consuming approach so it is not appropriate for large-scale production [ 23 ]. In bottom-up approaches, nanoparticles are produced by self-assembly of structures at the atomic and molecular scales, resulting in a more precise size, shape and molecular composition [ 24 ]. This method includes chemical and biological methods of production [ 18 ].

Among the various biological sources for the green synthesis of MtNPs, green synthesis mediated by microorganisms has acquired a special place due to their high growth rate, ease of cultivation and ability to grow in ambient conditions of temperature, pH and pressure [ 25 ]. Different microorganisms can serve as potential biofactories for the eco-friendly and inexpensive synthesis of various MtNPs containing metals such as silver, gold, copper, zinc, titanium, palladium and nickel. This can be achieved to generate MtNPs with a defined shape, size, composition and monodispersity of particles [ 18 , 22 , 26 ]. The biosynthetic mechanism of MtNPs in microorganisms can be carried out by trapping target metal ions from the surrounding environment and enzymatically converting them into elemental form, following a reduction mechanism [ 26 ]. Not all microorganisms are able to produce MtNPs because they are produced through metabolic pathways and through cellular enzymes that may not be present in some organisms. The synthesis of MtNPs also is dependent on the capacity of microorganisms for tolerating heavy metals. High metal stresses can affect various microbial activities and some microorganisms are able to reduce metal ions to the respective metals under stress condition. In general, microorganisms that live in metal-rich habitats are highly resistant to those metals due to their uptake and chelation of by intracellular and extracellular proteins. Consequently, this method, which mimics the natural bio-mineralization process, could be a favorable approach for the MtNPs synthesis [ 27 ]. Figure  1 shows a schematic illustration of intracellular and extracellular mechanisms of MtNPs biosynthesis. Intracellular biosynthesis involves unique transport systems in microorganisms in which the cell wall plays an important role due to its negative charge: positively charged metal ions are deposited in negatively charged cell walls through electrostatic interactions. After transport into the cells of the microorganism, ions are reduced using metabolic reactions mediated by enzymes such as nitrate reductase to forms MtNPs. The MtNPs accumulated in the periplasmic space can then be passed through the cell wall [ 28 , 29 ].

figure 1

Schematic representation of the mechanisms of extracellular and intracellular biosynthesis of MtNPs. Extracellular biosynthesis of MtNPs carried out by trapping metal ions on the cell wall and reducing them in the presence of secreted enzymes or metabolite. In the intracellular biosynthesis of MtNPs, after transfer of metal ions into cell cytoplasm, the metal ions are reduced as a result of metabolic reactions with enzymes, such as nitrate reductase

The extracellular biosynthesis of MtNPs is also a nitrate reductase-mediated synthesis in which the MtNPs are produced by reductase enzymes which are either located in the cell wall or secreted from the cell to the growth medium. In this process the nitrate reductase reduces metal ions to the metallic forms [ 27 , 29 ].

The presence of diverse components such as enzymes, proteins, and other biological molecules in microorganisms also play an important role in the process of reducing MtNPs [ 27 ]. Studies have shown that NADH-dependent enzymes are responsible for the MtNP synthesis. The reduction mechanisms seem to begin by transferring an electron from NADH by NADH-dependent reductases as the electron carrier [ 30 ]. In addition, proteins secreted by microorganisms can act primarily as a stabilizing agent and provides colloidal stability while preventing agglomeration of MtNPs [ 27 ].

For intracellular synthetic approaches microorganisms are cultured in a suitable growth medium with favorable pH and temperature conditions [ 23 ]. The biomass is harvested after an optimal incubation period and washed thoroughly with sterile water to minimize potentially undesirable effects of the culture medium. The resulting biomass is then incubated with metal salt solution. In addition to the use of whole microorganisms for intracellular synthesis of MtNPs an alternative is the use of cell-free (CF) approaches using either culture supernatant or cell-free extracts (CFE) [ 22 ]. In the CF approach using medium supernatant, after culturing the microorganisms in a liquid culture medium, the mixture containing the culture medium and biomass is centrifuged and the supernatant collected and incubated with an aqueous metal salt solution to synthesize the MtNPs. In this method, the compounds of the culture medium containing the appropriate enzymes and other essential secretory components produced by the microorganism are used to synthesize the MtNPs and also to act as reducing and capping agents. In approaches using cell-free extracts, the microorganisms are removed from the culture medium and resuspended in sterile distilled water for an approriate time. The resulting CFE is collected after centrifugation and is incubated with metal salt solutions, leading to the generation of MtNPs. In this approach the microorganisms and culture medium are removed through repeated washings, and only biomolecules released by cells due to autolysis or starvation conditions mediate synthesis of the MtNPs [ 19 , 22 , 25 , 31 ]. In all cell free processes a color change in the reaction mixture is frequently the first indication of nanoparticle synthesis with the color change being dependent on the precise nature of the MtNP being produced. For example, a change in color from pale yellow to dark purple indicates the formation of gold nanoparticles, a pale yellow to deep brown color change indicates the formation of silver nanoparticles and a yellow to yellowish-white color change indicates the formation of manganese and zinc nanoparticles [ 19 , 25 , 32 ].

Various physiological factors including microbial source, reaction temperature, pH, pressure, incubation time and metal salt concentration affect the synthesis of various MtNPs. Optimization of these physiological parameters is required for synthesis of nanoparticles with accurate size, morphology and chemical compositions [ 33 , 34 ]. After synthesis of MtNPs, purification before their use in any application is essential. Typically, repeated washing and high-speed centrifugation are performed to separate and enrich the produced MtNPs and to eliminate unreacted bioactive molecules [ 34 ]. In-cell synthesized nanoparticles require additional purification steps such as ultrasonication or reaction with appropriate detergents, which release the MtNPs after breakdown of the cell wall. These additional steps reduce the economic benefit of this approach [ 19 ].

Characterization of MtNPs synthesized from microorganisms is performed using various analytical techniques. UV–visible spectroscopy is generally used to confirm the synthesis and stability of MtNPs. Fourier-transform infrared (FTIR) spectroscopy is used to measure the properties of MtNPs such as chemical concentration, surface chemistry, surface functional groups and atomic arrangement [ 33 ] and transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) can be used to visualize the position, size and morphology of MtNPs [ 35 ]. X-ray powder diffraction (XRD) is used to determine the crystallographic structure [ 33 ]. The elemental composition of MtNPs is usually examine by energy dispersive x-ray spectroscopy (EDS) [ 36 ]. Dynamic light scattering (DLS) method is mainly used to evaluate the size as well as surface charge of MtNPs [ 33 ].

Application of green synthesized MtNPs in agriculture

Green-synthesized MtNPs have many potential applications in agriculture to increase the productivity of agricultural products. MtNPs are commonly used for generating products such as nanopesticides, nanofungicides, nanobiosensors and nanofertilizers. These nano-based products can help increase the quality and yield of agricultural products, reduce chemical pollution or even protect crops from environmental pressures [ 37 ].

The use of biosensors has revolutionized agricultural systems to increase the production of quality agricultural products due to their ability to quickly identify pathogens as well as their powerful monitoring and analytical capabilities [ 38 ]. Nanobiosensors are a modified version of a biosensor that can be described as an analytical unit by incorporating a biological sensitive element with a physicochemical transducer [ 39 ]. Nanobiosensors including enzymatic biosensors, genosensors, aptasensors, and immunosensors are made using a wide range of electrochemical, biological or physicochemical transducers. The use of these sensors has received much attention due to their fast, specific and selective performance in detection of toxins and plant pathogens [ 38 ]. Pesticides are used to protect plants from harmful agents such as plant pathogens and insects, to increase crop yield [ 40 ]. One of the most important challenges of using existing chemical pesticides is their negative effects on agricultural products in the food chain and ultimately on human health [ 37 ].

Nanopesticides represent an emerging nanobiotechnological development to encapsulate pesticides for controlled release and to improve the selectivity and stability of pesticides [ 37 , 41 ]. These nanopesticides can offer a wide range of benefits including increased efficiency, durability and reduced amount of active ingredient required in their formulation [ 42 , 43 ]. The nano-formulation of pesticides with MtNPs has shown a stronger effect against phytopathogens, insects and other pests that threaten crops. Fungi are the most common plant pathogens and cause more than 70% of major crop damage [ 40 , 44 ]. To control this damage common fungicides are currently used, the widespread use of which for long-term disease management leads to environmental pollution and dangerous effects on the ecosystem. The use of nanofungicides is an effective strategy against fungal pathogens. The use of MtNPs in the formulation of nanofungicides is the most common of their applications. These nanofungicides offer targeted delivery and greater bioavailability due to higher solubility and permeability, lower doses, lower dose-dependent toxicity, and controlled release [ 45 ].

Fertilizers are natural or synthetic substances that contain chemical elements necessary to improve plant growth and productivity and improve natural fertility by overcoming micronutrient deficiencies. The main problem of excessive and long-term use of chemical fertilizers in the agricultural sector is the reduction of soil fertility, which ultimately affects the production of agricultural products. Nanofertilizers are environmentally friendly fertilizers or smart fertilizers that deliver nutrients in small but effective amounts to plants. Nutrient uptake can be increased by encapsulating nanofertilizers, which ultimately reduces nutrient loss, promotes proper plant growth and improves crop quality [ 40 , 41 , 44 ]. Nano-formulations provide gradual and controlled release of nutrients to the target sites through direct internalization of products, which prevents nutrients from interacting with soil, water, air and microorganisms resulting in minimizing the risk of environmental degradation [ 43 ]. It has been frequently observed that the use of MtNP-based nanofertilizers has significant potential to increase crop productivity.

The application of synthesized green nanoparticle technology in the food or agricultural sector gives flexibility to conventional crop production systems, as it allows the controlled release of pesticides and fertilizers, as well as the targeted delivery of biological molecules. Interactions between MtNPs and plant responses are manifested by increase in breeding, and ultimately, it improves the quality and productivity of products [ 46 ]. In the following subsections, different species of microorganisms used for biosynthesis of MtNPs, and their perspective in agricultural applications are discussed.

Biosynthesis of MtNPs by probiotic bacteria and their application in agriculture

The use of probiotic microorganisms to produce MtNPs is an environmentally friendly as well as commercially attractive approach [ 47 ]. This is due to lower energy input, environmental sustainability, low costs, scalability and stability of MtNPs compared to the use of chemical synthesis methods. The non-pathogenicity of probiotics and their capacity to grow rapidly, regulating the expression of genes to produce various proteins and enzymes involved in the production of MtNPs is useful in many ways. Lactobacillus and Bifidobacterium are the most popular probiotics found in dairy products and natural flora in various parts of the body. These non-pathogenic gram-positive bacteria can be used in the production of a wide range of products [ 48 ]. The green synthesis of MtNPs, metal oxide nanoparticles (MONPs) and non-MtNPs by probiotics has been studied [ 49 ]. Probiotics exert their beneficial effects in a variety of ways, including direct effects on living cells and indirect effects on a wide range of metabolites. Probiotics have a negative electrokinetic potential that freely attracts cations, similar to other bacteria, which can be the starting point for the NP biosynthesis process [ 50 ].

The negative surface electrokinetic potential of Lactobacilli causes the rapid absorption of cations, which in turn plays a key role in the biosynthesis of MtNPs. Previous studies have reported biological adsorption and reduction of silver iodide by Lactobacillus sp. A09 [ 51 ] The tendency of lactobacilli to grow even in the presence of oxygen makes them metabolically highly viable. The bacterial redox potential decreases with the addition of reducing agents such as glucose. The oxidation–reduction potential represents the quantitative state of the degree of aerobiosis with the unit defined as rH2 (negative logarithm of the partial pressure of hydrogen gas). By adjusting the redox potential in the culture medium, the conditions can be changed in the desired direction. For example, suitable conditions can be created by lowering the rH2 for anaerobic conditions in the presence of oxygen, or by increasing the pH of the medium for creating aerobic conditions in an anaerobic environment. In this way, changing the different conditions of the culture medium plays an important role in the biosynthesis of MtNPs and/or MONPs. Various factors such as energy efficiency, glucose (which controls the value of rH2), ionic mean, pH, and total oxidation capacity (rH2) play an important role in the synthesis of NPs by Lactobacillus strains. Although Lactobacilli have a relatively weak metal detoxification system, a slightly acidic pH and a decrease in rH2 activates membrane-bound oxidoreductases and the metabolic pathway involved in MtONPs synthesis [ 52 ].

MtNPs such as silver, gold, cadmium, copper, zinc, iron and selenium have applications in agriculture such as plant growth stimulation, antimicrobial and antifungal effects, nanofertilizers, nanobiosensors, plant micronutrients and plant disease control [ 53 ]. Table 1 shows a collection of probiotic species used for the synthesis of different MtNPs and their potential application in agriculture. Silver NPs (AgNPs) are amongst the most studied in biological systems and their various inhibitory and antimicrobial effects have long been known [ 54 ]. Various probiotics including gram-positive bacteria such as lactic acid bacteria, bacillus , Staphylococcus , Brevibacterium and gram-negative sp. Including Pseudomonas and E . coli , used for AgNP production. Lactobacillus sp. have been studied significantly as potential systems for AgNP production and Sásková and colleagues have demonstrated high extracellular production of AgNPs from silver ions by Lactobacillus casei sp. [ 55 ]. Similarly AgNP synthesis by Lactobacillus acidophilus have been shown to provide capping and reducing activities [ 56 ]. Gold NPs (AuNPs) are widely used in agriculture as antifungal and antibacterial agents and as delivery vehicles of fertilizer and pesticide sensors. The use of probiotics in the synthesis of AgNPs and AuNPs also eliminates the use of toxic chemicals and solvents, thus following the principles of green chemistry [ 57 ]. Cadmium sulfide (CdS) NPs are used in a wide variety of approaches such as biological sensors that have applications in medicine as well as in agriculture [ 58 ]. CdSNPs for use as nanosensors can be synthesized by probiotic bacteria. Nanosensors are useful in pesticide residue detection and can also detect soil moisture and soil nutrient levels [ 58 , 59 ]. Copper is an essential micronutrient that is combined with many proteins and metalloenzymes and have a substantial role in plant metabolism and nutrition. CuNPs also have higher performance than bulk copper particles due to properties such as very small size and high surface-to-volume ratio compared to materials made from larger particles. The antifungal and antibacterial activity of CuNPs against gram-positive and gram-negative bacteria and pathogenic fungi has given them many applications in health and agriculture [ 60 ]. CuNPs have antifungal activity against plant pathogenic fungi such as Fusarium oxysporum , Fusarium culmorum , Fusarium graminearum and Phytophthora infestans [ 61 ]. They have also been reported to act as germinators and growth stimulants in some plants at concentrations below 100 ppm. So far, various chemical, physical and green synthesis methods have been used to synthesize CuNPs with different amounts, shapes and morphologies. Kouhkan et al. [ 62 ] reported that Lactobacillus casei is a promising source for the biosynthesis of CuNPs. Selenium is essential for the functions of most living organisms and is found in soil, water, seeds, livestock and food. Since SeNPs improve the plant’s ability to inhibit pathogens and activate antifungal properties, it is necessary to modify the Se content in plant nutrients by adding Se fertilizer to the soil and to balance Se in food [ 63 ]. Se-balanced food processing technology is a rapid process which helps to solve the Se imbalance issue in agriculture. Standardization of Se concentration in soil is very important and to achieve this pure Se compounds are used as fertilizer [ 64 ]. However, Se fertilizers remain in fertile topsoil during only one or few harvests and over a short period inorganic Se compounds are washed away by rain into the infertile horizons below the soil. Although the organic Se compounds are not actively leached, they are degraded quickly after applying. The advantage of SeNPs as nanofertilizers is that they do not leach slowly from the soil and do not dissolve in water or aqueous solutions [ 65 , 66 ]. Figure  2 shows the potential effect of MtNPs as nanofertilizers on plants. Several different methods for synthesizing SeNPs have been described including synthesis of SeNPs using various probiotics including Lactobacillus acidophilus , Lactobacillus casei and Bifidobacterium sp. The shape, size, and quality of NPs produced by these probiotics differ from those generated by other methods. SeNPs produced by probiotics have a homogeneous particle size distribution and regular spherical shape [ 65 , 67 , 68 ].

figure 2

Schematic representation of the entry of MtNPs into plants through soil and roots or through extra-soil parts of plants as nanofertilizers and their uptake, translocation and potential effects on plants

Biosynthesis of MtNPs by non-probiotics bacteria and their application in agriculture

Due to the growing need to develop new environmentally friendly technologies, the synthesis of MtNPs has received much attention as an advanced technology. Green synthesis of MtNPs by bacteria has become very important due to their relative ease of growth and lower production costs. Biosynthesis of AuNPs in three forms of spherical, triangular, and irregular (approximate size of 43.75 nm) has been reported using Deinococcus radiodurans [ 69 ]. In one study extracellular biosynthesis of AuNPs at room temperature using Escherichia coli K12 . Generated a product that could reduce the toxic substance 4-nitrophenol in the presence of NaBH 4 [ 70 ]. During the process of reducing 4-nitrophenol to 4-aminophenol, NaBH4 acts as a donor and prevents the formation of nitrophenolate (as a receptor). The rapid reduction of 4-nitrophenol to 4-aminophenol occurs when Ag/Au NPs are added to the reaction solution as a catalyst, which can be confirmed using the visible UV spectrum [ 71 ]. 4-Nitrophenol is a highly toxic organic compound and one of the most resistant contaminants in the effluents of various industries such as textile and dyeing. By spreading to the environment, this compound can contaminate soil and water leading to adverse effects on the central nervous system, liver and blood after ingestion of food grown in the contaminated areas. The development of a simple and effective method for the elimination or reduction of non-biodegradable bio pollutants into non-hazardous products is one of the serious challenges in environmental studies and agricultural systems. The product of chemical reduction of 4-nitrophenol is a useful and important compound called 4-aminophenol, which does not pose the risks of toxicity of 4-nitrophenol to the environment. The use of environmentally friendly green synthesis for produce nanoparticles as low-cost catalysts is a convenient method to chemically reduce toxic dyes such as 4-nitrophenol. MtNPs derive their catalytic capacity from their high surface-to-volume ratio. Due to their high adsorption level, MtNPs can provide conditions that increase the adsorption of the reactants on their surface and thus increase the reaction rate and reduce the activation energy level [ 72 ]. An Acinetobacter sp. species was able to synthesize AuNPs at 37 °C, pH 7, when treated with tetra-chloroauric acid (HAuCl 4 ). These AuNPs were monodisperse or spherical and had antioxidant activity [ 73 ]. In a study of the biosynthesis of AuNPs using Acinetobacter sp. SW30 addition of HAuCl 4 resulted in the biosynthesis of 10 to 20 nm polyhedral AuNPs. As the pH was increased to 9 and the temperature increased to 50 °C, more AuNPs were released into the solution [ 74 ]. Acinetobacter sp. SW30 has also been used at 30 °C and pH 7 to produce AuNPs with a monodisperse spherical shape and size of approximately 19 nm [ 75 ]. Reports indicate that filamentous cyanobacteria can biosynthesize AuNPs structures in various shapes, such as cubic, spherical, and octagonal, from the complexes of Au + -S 2 O −2 3 and Au 3+ -NaCl [ 76 , 77 ]. A Cyanothece sp. was able to synthesis AuNPs in the size range of 80 to 129 nm [ 78 ]. The first step in the interaction of cyanobacterium with Au3 + aqueous Cl − is the deposition of NP sulfur Au + on the cell wall and in the next step octagonal platelets forms of Au3 + are formed in solutions close to cell surfaces [ 77 ]. Plectonema boryanum UTEX 485, in the presence of S 2 O 3, was able to biosynthesize cubic form (sizes ranged from 10 to 25 nm) AuNPs in membrane vesicles. These bacteria also precipitated AuNPs in the form of octahedral platelets when incubated with AuCl 4 − [ 76 ]. Electron transfer in the process of photosynthesis affects the biosynthesis of AuNPs in cyanobacterium cell wall. Cell membrane compositions in cyanobacteria can produce AuNPs by affecting the re-accumulation of gold in the cell wall. In general, at neutral pH, the biosynthesis of AuNPs takes place mostly in the periplasmic region of cyanobacteria. As the pH becomes more acidic, the more the synthesized AuNPs show different sizes and morphologies. Small AuNPs are deposited on bacterial cell walls at pH 2.0, while larger particles could be observed in the extracellular matrix. In general, changes in solution pH are a very influential factor in appearance and structure, as well as deposition location (extracellular or intracellular) of AuNPs [ 79 ]. Extracellular AgNP biosynthesis was demonstrated using Pseudomonas DC5 and Pseudomonas CA 417 [ 11 ]. In one study, the specificity of metal ion accumulation in the biosynthesis of AgNPs by Pseudomonas stutzeri AG259 was used to produce a range of shapes and sizes [ 80 ]. In one study, Acinetobacter sp. GWRFH45 biosynthesized AgNps [ 81 ]. Rapid biosynthesis of AgNps by Enterobacteriaceae has also been reported [ 82 ]. The reduction of Ag + ions in Staphylococcus aureus led to the biosynthesis of AgNPs [ 83 ]. The use of bacterial cell culture supernatant to generate AgNPs of various shapes and sizes has been reported in several other studies [ 84 ]. In general, in the AgNPs biosynthesis cycle, the presence of nitrate ions in the presence of NADPH-dependent nitrate reductase enzymes (for free electron transfer) reduces the bioavailability of silver ions and ultimately causes spherical biosynthesis of AgNPs [ 79 ]. Au–Ag bimetallic NPs produced by a Deinococcus radiodurans synthesis system with a size of 149.8 nm showed the ability to decompose toxic triphenylmethane dye malachite green (MG) and convert it to the less toxic substance dimethylamino (benzophenone) [ 85 ]. The rapid and easy biosynthesis of a silver-gold double NPs functionalized with extremophilic Deinococcus radiodurans proteins (Drp-Au-AgNPs) led to the development of an environmentally friendly method for reducing polyphenyl from wastewater [ 85 ]. The ability of functionalized Drp-Au–Ag bimetallic MtNPs to degrade and reduce malachite green is attributed to a redox reaction as well as the alkaline conditions that amplify the electrostatic force between the functionalized Drp-Au–Ag bimetallic MtNPs and the malachite green molecules. Malachite green is a group of polyphenolic chemical dyes that are widely used in fishponds to repel pests and insects. Malachite green effluents, if released into the environment, in addition to proven mutagenic and carcinogenic effects in humans, can cause permanent dangerous and toxic effects. Nevertheless, the low price of green malachite is still a tempting factor to use this compound, so it can be considered an environmental problem. Although physical and chemical methods are used to remove polyphenyl compounds, the ability of nanoparticles as potential catalysts to absorb and then degrade polyphenol dyes is an efficient and environmentally friendly method for remediation [ 86 ]. In fact, nanobioremediation, is a new and efficient approach to clean up and remove contaminants and toxic compounds from the environment.

Extracellular biosynthesis of CdSNPs has been reported using Klebsiella aerogenes . The MtNPs ranged in diameter from 20 to 100 nm and their formation was highly dependent on the composition of the culture medium [ 87 ]. With the photosynthetic bacterium Rhodopseudomonas palustris , the extracellular biosynthesis of CdSNPs of approximately about 8 nm in diameter was dependent on cell growth stage and utilized the cysteine desulfhydrase located in the cytoplasmic space to stabilize the CdSNPs [ 88 ]. The results of a study on an intracellular CdSNP biosynthesized by E . coli showed that changes in growth phases affect the rate of biosynthesis and the size of CdSNPs. The biosynthesis rate of CdSNPs with a diameter of 2 to 5 nm in the stationary phase of E . coli was about 20 times higher than found in the logarithmic phase [ 89 ]. Extracellular biosynthesis of spherical CuNPs of 5–50 nm in size by Streptomyces griseus and 3.6–59 nm in size in e ndophytic actinomycetes has been reported [ 90 ]. A new species of Desulfuromonas palmitatis SDBY1 converts polycarbonate organic compounds to oxidized form in the presence of F 3+ , because F 3+ can play the role of H 2 receptor and be reduced [ 91 ]. Iron-reducing bacteria need electron-donating compounds during extracellular deposition of magnetite [ 92 ]. Shewanella oneidensis was used for the biosynthesis of Fe 2+ and Fe 3+ as extracellular magnetite. FeCl 2 , along with other salts, was used to reduce Fe 2+ and Fe 3+ . The reduction of Fe 2+ and Fe 3+ seems to be facilitated by the transfer of salts by electron donation [ 93 ].

Although bacteria, viruses, and fungi are used to produce nanobiosensors with different MtNPs, nanoparticles produced of bacterial origin are mostly used as nanobiosensors in agricultural systems due to advantages such as production control, lower cost and high quality [ 94 ]. Bacterial NP-based biosensors, such as nanowires, nanoparticles and nanocapsule substrates are used specifically to diagnose plant diseases and are also used in cleaning strategies related to the accumulation of pesticides and insecticides in the food sector. Quantitative detection of insecticides containing dangerous and prohibited compounds such as organophosphorus, carbamate compounds is also done using biosensors [ 19 ]. In a study on a SeNP-based agricultural sensor to detect heavy metal toxicity, Stenotrophomonas acidaminiphila was used for SeNPs biosynthesis. This study presented a colorimetric method for the detection of heavy metals during bioremediation. In the absence of heavy metals, this process takes place naturally and the color changes to red, but in the presence of toxic heavy metals the process of selenium green synthesis to SeNPs is inhibited and the color changes. This synthesis is dependent on NADH reductase and increasing the concentration of toxic heavy metals causes a gradual decrease in enzyme activity and discoloration [ 95 ].

Several studies have examined the importance of using NPs as a diagnostic tool to identify a wide range of pathogenic bacteria in plants [ 96 ]. The application of nanoparticles in new technologies used in non-laboratory rapid screening methods for the detection of plant pathogens has a significant impact on the quality of agricultural products. In a study by Panferov et al. [ 97 ], an enhanced and rapid method based on lateral flow immunoassay (LFIA) was developed to detect low levels of potato leaf roll virus (PLRV) in contaminated fields. In this method, AuNPs were used as labels and silver ions were reduced at the AuNP surface to increase sensitivity [ 97 ]. In another report, infection of potato tubers with Ralstonia solanacearum was detected using an AuNP-based immunoassay. In this study, enhanced AuNP biosynthesized approach was used to increase sensitivity in lateral flow immunoassay (LFIA). The special feature of this method was a significant reduction in time to diagnose the cause of the infection [ 98 ]. In another study, the diagnosis of Phytophthora infestans , the causative agent of late blight in potatoes and tomatoes was performed using a combination of AuNPs-based lateral stream biosensor and asymmetric PCR to amplify the portion of the Ph . infestans genome. This showed that rapid detection of Phytophthora infestans in the early stages of infection can lead to appropriate management decisions to prevent the progression and spread of infection [ 99 ]. In another report, a rapid and inexpensive biosensing method was developed to identify the tomato yellow leaf ring virus genome using a AuNP-based probe and the local surface plasmon resonance (LSPR) method. Color changes were detected by UV–Vis spectroscopy, which indicates the presence of viral infection in the sample, eliminating the need for PCR and ELISA-dependent methods [ 100 ]. Although there are reports of successful use of MtNPs synthesized by non-biological methods in agricultural-related nanosensors, the importance of environmental protection has given priority to the development of methods for green MtNPs synthesis. The working principles of MtNP-based sensors for the detection of plant pathogens and toxins shown in Fig.  3 .

figure 3

Schematic representation of the main constituents and working principle of MtNP-based biosensors for detection of plant pathogens and toxins

Bacterial-synthesized NPs such as AgNPs have shown remarkable antibacterial effects and their application increases crop productivity, reduces waste generation, and saves energy and water when compared with common pesticides [ 37 ]. AgNPs are well-known antibacterial agents that can penetrate the bacterial cell wall and change the structure of the cell membrane by continuously releasing silver ions. Accumulation of AgNPs after anchoring to the cell surface can cause denaturation of the cell membrane. The binding of AgNPs to the cell wall increases the permeability of the cytoplasmic membrane and affects bacterial cell wall cross-linkage. With the entry of free silver ions into the cell, inactivation of respiratory enzymes occurs and the production of reactive oxygen species (ROS) increases, which causes damage to DNA and intracellular macromolecules and disrupts the cell membrane. AgNPs interrupts the electron transport chain and thus disrupts the production of adenosine triphosphate. In addition, the affinity of AgNPs to sulfur and phosphorus in the DNA structure causes serious damage to the DNA replication process, which in turn results in impaired cell reproduction. AgNPs directly disrupt protein production in the cytoplasm by denaturing ribosomes and also indirectly affect the natural structure of the proteins by increasing ROS levels, which together can lead to bacterial cell death. In general, many nanoparticles induce their antimicrobial effect by similar mechanisms [ 101 ]. However, despite the specific properties of each MtNP, most nanoparticles due to their general properties include antibacterial activity, disruption of the cytoplasmic membrane and cell wall, disruption of the energy transfer chain and electron transfer chain, toxic ROS production or DNA/protein oxidation, and Inhibition of enzymes makes their use in fungicides and pesticides important. For example, AuNPs in addition to accumulation at cell surface can exert its antimicrobial effect on the bacterial cell wall through electrostatic interactions [ 102 ]. The positive feature of using bio-pesticides is that they do not have the environmental disadvantages of using synthetic pesticides, but their effect on pests compared to the chemical pesticides is slow and limited [ 103 ]. Encapsulation of antimicrobial polypeptides may help to the endocytosis of these polypeptides surrounded by MtNPS. In addition to inducing cell death in pests such as insects, herbs and fungi MtNPs also can help in the controlled release of polypeptides into cells [ 104 ]. This has the added benefit of providing an important strategy in protecting the environment by reducing the dispersion of nanopesticides while encapsulation of medicinal plant repellents in MtNPs increases controlled release and reduces the level of toxicity of synthetic pesticides [ 105 ]. As a result of these features, nanobiopesticides can overcome the limitations of synthetic pesticides and biopesticides. With the use of nanoparticles, the active ingredients can be stabilized and made available through sustained-released giving effective and sustainable management for a long time without the hazards of using synthetic chemicals [ 106 ].

Several reports have evaluated the successful use of biological nanoparticles against pests. In one such study, spherical AuNPs and AgNPs biosynthesized from Haloferax volcanii were successfully used for antibacterial applications against two gram-negative bacteria [ 107 ]. Extracellular biosynthesis of AgNPs with high antimicrobial properties has also been reported using Sporosarcina koreensis DC4 [ 108 ]. The antifungal activity against Fusarium graminearum of an AgNPs biosynthesized by Endophytic bacteria has also been reported. In one study, biosynthesis of AgNPs was performed using Pseudomonas poae strain CO, in which the AgNPs with a diameter of approximately 20–50 nm showed antifungal activity [ 109 ]. Successful biosynthesis of AgNPs was reported in three strains of Endophytic Streptomyces spp. The biosynthesized NPs were spherical in shape, varying in size from at least 11 to a maximum of 63 nm, and acted against a wide range of single-celled fungi [ 110 ]. AgNPs (20 to 100 nm) biosynthesized using Pseudomonas rhodesiae culture medium supernatant showed strong antibacterial activity against Dickeya dadantii infection in sweet potato roots [ 111 ]. A haloalkaliphilic bacterium Streptomyces sp. was able to biosynthesize spherical AgNPs (diameter 16 nm) with high fungicidal properties against Fusarium verticillioides , one of the main causes of infection in cornfields by inhibiting ergosterol biosynthesis leading to inhibition of conidia germination and destruction of the F . verticillioides membrane [ 112 ].

CuNPs biosynthesized by an actinomycetes sp. isolated from Convolvulus arvensis also showed significant antifungal and antibacterial activity [ 113 ]. In one study, the effect of foliar application of different concentrations of CuNPs on the accumulation of bioactive compounds and antioxidant capacity in tomato fruits was estimated. CuNPs reduced the formation of ROS by increasing the activity of superoxide dismutase and catalase enzymes. In addition, the content of vitamin C, lycopene and phenol was increased in the presence of CuNPs. The results of this study also showed that CuNPs increased the strength of tomato fruits [ 114 ]. To investigate the effect of CuNPs biosynthesized by Streptomyces griseus on fungi that cause red root rot disease, experiments were performed on infected tea plantations. Comparison of tea plants treated with the chemical fungicide carbendazim, biosynthesized CuNPs or bulk copper showed that fungal resistance and leaf yield were higher in tea plants treated with biosynthesized CuNPs than in tea plants treated with carbendazim or bulk copper. Soil nutrients were also increased after the use of CuNPs. This study suggests that these CuNPs can be used as fungicides in the formulation of nanobiofertilizers [ 46 , 90 ].

Several studies have examined the effect of MtNP size on their toxicity. Although factors such as size, concentration and zeta potential of MtNPs show various effects on different plants, there is a significant relationship between the size of MtNPs and the degree of toxicity created for the plant with the larger MtNPs being less toxic to plants than smaller ones. In addition, studies have shown that the concentration of nanoparticles also has a significant effect on their toxicity, for example, a concentration of more than 0.2 mg/ml CuNPs impairs plant growth and physiology [ 40 ].

The various MtNPs synthesized by non-probiotic bacteria with their potential applications in agriculture are summarized in Table 2 .

Biosynthesis of MtNPs by Fungi and their application in agriculture

Nanotechnology touches many fields, including agriculture and plant disease management. In recent years, fungi have been added to the list of microorganisms used in the production of nanoparticles. Among the various microorganisms used to synthesize nanoparticles, fungi are effective candidates for making intracellular and extracellular MtNPs. Nanoparticles made using fungi have good dispersion and stability characteristics. The attractiveness of using fungi in the production of nanoparticles is due to the presence of significant amounts of specific enzymes in these microorganisms, ease of working with them in the laboratory, scalability and financially economic growth of fungi even on an industrial scale making myconanotechnology an environmentally friendly and cost-effective option [ 115 , 116 ]. Although there are several methods for synthesizing MtNPs from fungi, little is currently known about potential drawbacks and limitations. Filamentous fungi can produce a wide range of MtNPs such as gold, silver, iron oxide, and even bimetallic nanoparticles [ 117 , 118 ]. Research has shown that several different species of fungi can be used in the green synthesis MtNPs with the desired size, surface charge and morphology, and desirable properties including Pestalotiopsis sp., Phoma sp., Humicola sp., Fusarium oxysporum, Aspergillus niger, Trichoderma sp., Hormoconis resinae, Phaenerochaete chrysosporium and Penicillium . Using fungi as reducing and stabilizing agents for the biosynthesis of AgNPs has been considered due to their high efficiency, ease of operation and low residual toxicity. The mechanisms of synthesis are not yet fully understood, but synthesis can be optimized by adjusting parameters such as silver salt concentration, biomass, temperature, pH and fungal cultivation time. As with bacterial produced AgNPs, similar structures synthesized using fungi, with low toxicity and good biological compatibility, can control pathogens [ 40 , 119 ].

These findings set the stage for future research into the use of these MtNPs as antimicrobials agent in agriculture sector. Among the various types of MtNPs studied to date, AgNPs stand out due to their wide range of antimicrobial potential [ 120 , 121 , 122 ]. These MtNPs attach to the cell wall and membrane of the microorganisms and may also enter the cell. They damage cellular structures, induce the production of ROS, and alter signal transduction mechanisms [ 123 , 124 ]. The use of fungi for the synthesis of AgNPs involves culturing the fungus on agar and then transferring it to a liquid medium. The produced biomass is then transferred to water to release the compounds that act in the synthesis of MtNPs. After filtration, the biomass is discarded and silver nitrate is added to the filter [ 125 , 126 ]. One of the first reports of the synthesis of AuNPs by fungi was shown by Verticillium sp. [ 127 ], though other fungi including Penicillium sp. Hormoconis resinae, Candida albicans, Alternaria alternate , Paraconiothyrium variable, Aspergillus sp., Volvariella volvacea, Colletotrichum sp. and Trichothecium sp. have also been used successfully for AuNP production. The living and dead cells of Aspergillus oryzae also produce AuNPs in a process that is economically viable for use in the food industry [ 128 ]. The fungus Colletotrichum sp, which has a parasitic life and grows on geraniums, produces AuNPs with rod-like and prism-like morphology when exposed to chlorate ions [ 129 ]. In addition to MtNPs, the production of Au–Ag bimetallic alloys is possible using F . oxysporum . In a recent study, it was shown that to exposure of F . oxysporum can stimulate accumulation of metal ions by physicochemical and biological mechanisms such as extracellular binding by polymers and metabolites, binding to specific polypeptides, and metabolism-dependent accumulation [ 130 ]. Exposure of F . oxysporum biomass to co-molar solutions of HAuCL 4 and AgNO 3 has also been shown to produce highly stable Au–Ag alloy nanoparticles with different molar ratios and it has been shown that NADH factors play a very important role in determining the chemical composition of Au–Ag alloy nanoparticles [ 129 ]. In addition, exposure of F . oxysporum to aqueous solution of CdSO 4 causes extracellular production of CdSNPs. The particles produced by this method have a uniform dispersion and their dimensions are in the range of 5 to 20 nm [ 131 ]. Cadmium quantum dot nanoparticles are produced by using fungi such as Coriolus versicolor , Schizosaccharomyces pombe , Candida glabrat and F . oxysporum [ 115 ]. Other important applications of fungi include the production of zirconia nanoparticles with many applications. Reaction of the aqueous solution of k2ZrF6 with F . oxysporum , hydrolysis of zirconium hexafluoride anions occurs extracellularly and crystalline zirconia nanoparticles are produced at room temperature [ 132 ].

Myconanotechnology has established a new field of research in the production of antifungal nanoparticles. The antifungal properties of AgNPs against rose powdery mildew caused by S phaerotheca pannos var. rosae were have been demonstrated by spraying a large contaminated surface area with nanosilver solution. Two days later, more than 95% of the rose powder had been eliminated and no recurrence was observed for a week [ 133 ]. In a related study, AgNPs had a toxic effects on the pathogen Colletotrichum gloesporioides , which causes anthracnose in a several fruits showing significant growth retardation of the C . gloesporioides . As a result, AgNPs can be introduced as a fungicide for the management of plant diseases [ 134 ]. AgNPs were synthesized using Epicoccum nigrum and their antifungal activity was observed against pathogenic fungi such as Fusarium solani, Sporothrix schenckii, C . albicans, Cryptococcus neoformans, Aspergillus flavus and Aspergillus fumigatus and AgNPs were synthesized using Guignardia mangiferae were active against the phytopathogenic fungi including Rhizoctonia solani , Colletotrichum sp. and Curvularia lunata [ 135 ]. Antifungal effects of AgNPs synthesized by the plant pathogen Fusarium solani isolated from wheat showed activity against various other species of phytopathogenic fungi that cause diseases of wheat, barley and corn kernels [ 136 ]. MtNPs are active against a wide range of pests and their use in the formulation of pesticides is easily achieved [ 137 , 138 ]. Porous hollow silica nanoparticles (PHSN) have been shown to be effective for controlled release of water-soluble pesticides and in improving their transport to target locations [ 139 ]. AgNPs synthesized using Aspergillus versicolor have been shown to be effective against infection with Botrytis cinerea and Sclerotinia sclerotiorum in strawberry plants [ 140 ]. Figure  4 a shows the various MtNPs can act as either plant protectants against pests or as carriers of pesticides. Figure  4 b shows the general mechanism of action of MtNPs as nanofungicide.

figure 4

Application of MtNPs as nanopesticides: a MtNPs act as nanopesticides targeting a wide range of pests and phytopathogenic agents and as a carrier for pesticides to provide crop protection, b Mechanisms of action of MtNPs as nanofungicides. MtNPs act on the fungus cell wall, leading to membrane damage. Disruption of the membrane by MtNPs causes pore formation. After internalization, MtNPs target main cellular organs such as the nucleus, ribosomes and mitochondria, causing cell death

Nanoparticles produced by fungi have coatings that are obtained directly from the fungi and which make them more stable. Depending on the fungus used, the cap may have biological activity and a synergistic effect with the nanoparticle core. These attributes contribute to the efficacy of nanofertilizers in achieving slow secretion or secretion due to biological and physical activation. At the same time, nanofertilizers improve plant nutritional efficiency and prevent excessive toxicity of chemical fertilizers. Thus, it helps developing countries in particular in establishing sustainable agricultural programs [ 141 ].

However, while there are several strong advantages for using fungi for green synthesis of MtNPs, there are also drawbacks that need to be addressed. These include determining which fungus is best for producing nanoparticles with the desired properties, determining the appropriate parameters for growth, the need for sterile conditions as well as the time required for the fungus to grow, and completing its synthesis. There may also be problems with scale-up production, including the need to further investigate the mechanisms by which cap layers are formed and the molecules contained in them. While more research is needed, studies showed that using fungi for the green synthesis of MtNPs has the potential to address a wide range of possible applications especially for the control of pests [ 135 ]. A summary of some fungal sources for the production of MtNPs with specific characteristics and potential applications in agriculture is shown in Table 3 .

Biosynthesis of MtNPs by yeasts and their application in agriculture

Yeasts are the unicellular microorganisms that reproduce during an asymmetric cell division process called budding and can be categorized as Ascomycetes such as Saccharomyces and Candida or Basidiomycetes such as Filobasidiella and Rhodotorula [ 142 ]. In addition to traditionally use of yeasts for production of several fermented food such as alcoholic beverages and bakery products modern application of yeasts include the production of heterologous compounds, single cell protein (SCP) and their use in the biofuels industry [ 142 ]. Yeasts also play an important role in agriculture as biological control agents, biological treatments and as indicators of a quality environment [ 143 ]. They grow easily on low-cost media and can adapt to harsh environmental conditions such as a wide range of temperature and pH and high concentrated organic and inorganic pollutants. Yeasts have the inherent ability to absorb and accumulate large concentrations of toxic metal ions from the environment and can adapt themselves to this environmental stress using various detoxification mechanisms such as mobilization, immobilization or metals transformation. These bioremediation mechanism of yeasts can play key roles for the green synthesis of MtNPs [ 144 ]. The stress caused by the presence of metal ions leads to activate a metabolic cascade of chemical reactions for the synthesis of stress-relieving compounds such as phytochelatin synthase and glutathione that have redox and nucleophilic features. These compounds bind to metal ions such as cadmium, zinc, silver, selenium, gold, nickel, copper, etc. reduce them to the respective MtNPs. Additional mechanisms take in this process include the activity of membrane-bound oxidoreductases and quinones. Adsorption of metal ions leads to an increase in pH and subsequent activation of pH-sensitive oxidoreductases, which act as both reducing and stabilizing agents for MtNP synthesis. Depending on the yeast species type, the biosynthesis of MtNPs can either be intracellular or extracellular [ 145 ].

Many Yeast species such as Saccharomyces cerevisiae , Saccharomyces boulardii , Candida utilis NCIM 3469 , Candida lusitaniae , silver-tolerant yeast strain MKY3 and a marine yeast Yarrowia lipolytica strain have been used for the biosynthesis of AgNPs [ 25 , 44 ]. In a recent study Elahian et al. [ 146 ] utilized a genetically modified strain of Pichia pastoris for AgNP biosynthesis. The yeast Pichia jadinii (formerly Candida utilis ), isolated from a metal-rich dump, has been shown to produce AuNPs from the metal [ 147 ]. The green synthesis of AuNPs using the tropical yeast Yarrowia lipolytica is also described by Agnihotri et al. [ 148 ]. It has also been demonstrated that extremophilic yeasts, isolated from acid mine drainage, are able to produce AuNPs and AgNPs [ 147 ]. Biosynthesis of other MtNPs such as CuNPs and Palladium nanoparticles (PdNPs) using Saccharomyces cerevisiae have been also reported [ 149 ].

Fernandez et al. [ 150 ], demonstrated antifungal activity of AgNPs synthesized using two epiphytic yeasts, Cryptococcus laurentii and Rhodotorula glutinis isolated from apple peel and its potential application as an efficacious nanofungicide against phytopathogenic fungi that cause postharvest diseases in pome fruits has been reported. Because epiphytic yeasts, like C . laurentii and R . glutinis , are harmless and are regard as GRAS (Generally Recognized As Safe) microorganisms, MtNPs production using these two yeasts has significant advantages in the application of agroecosystems [ 151 ].

Biosynthesis of MtNPs by microalgea and their application in agriculture

Microalgae, single-celled prokaryotic or eukaryotic predominantly aquatic microorganisms that undertake photosynthesis form colonies without any cell differentiation and can grow in a variety of environments, such as freshwater, saline, and sea, where their growth is directly related to temperature, light intensity, and nutrient concentration [ 152 ]. Microalgae have been widely used in a variety of industrial, health and biotechnological applications thanks to a wide range of potential biological applications, such as pigment overexpression, biological treatment, biofuel production and toxicity studies [ 153 ]. These photosynthetic microorganisms are very sensitive to environmental changes and can detect traces of contaminants, so they can be used as biosensors to detect contaminants such as herbicides, heavy metals and volatile organic compounds in the range of 1–10 ppb. Depending on their biological constituents, microalgae react selectively with some contaminants, which can result in electrical, thermal or optical signals which can be identified, processed and analyzed by microprocessors [ 154 ]. Microalgae-based synthesis of the MtNPs, known as "phyco-nanotechnology", is an emerging field with a wide range of potential applications [ 155 ]. Many phototrophic microorganisms belong to the microalgae, and can be used to produce secondary metabolites and substances with unique properties including carotenoids, enzymes, fatty acids, polymers, peptides, antioxidants, toxins and sterols [ 156 ].

Several reports have shown that some microalgae not only be able to accumulate heavy metals intracellularly or extracellularly, but they also have the ability to synthesize MtNPs such as silver, gold, cadmium and platinum [ 157 ]. In addition to the low cost of nanoparticles biosynthesis using microalgae, synthesis can also be performed at low temperatures with higher energy efficiency, lower toxicity and lower risk to the environment [ 158 ].

The mechanism of biosynthesis of MtNPs by microalgae is not yet well understood. However, it is clear that nanoparticles can be synthesized by extracellular and intracellular mechanisms from algal biomass. In the case of extracellular production the bioreduction of a metal ion MtNPs takes place on the surface of the microalgae cell whereas in the intracellular mechanism the process of enzymatic reduction takes place inside the cell [ 159 ]. It has been reported that intracellular polyphosphates and extracellular polysaccharides as well as carboxyl groups on the cell surface absorb metal ions through electrostatic interaction and then metal particles enter the cell and are captured during the processes used to form MtNPs [ 160 ]. Extracellular pathway synthesis of MtNPs by microalgae is carried out with the aim of eliminating the effects of toxic metals using reductase enzymes and shuttle quinones and by secreting extracellular enzymes or by electrostatic interactions between metal ions and cell surface constituents [ 160 ]. The synthesis of MtNPs also occurs through the activity of intracellular terpenoids, carbonyl groups, phenolic, flavonoids, amines, amides, proteins, pigments, alkaloids as reducing agents. Many methods have been described for synthesizing MtNPs from saline solutions using microalgae to improve the size, shape of nanoparticles and higher quality [ 161 ]. These include the use of biological molecules extracted from lysed microalgae cells, the use of cell-free supernatant, or the biological synthesis of nanoparticles from living microalgae. Several microalgae species have been used for the biological synthesis of MtNPs using their extracted biomolecules [ 160 ]. To obtain AuNPs, the algal biomass is first lyophilized and then reverse-phase-high performance liquid chromatography (RP-HPLC) carried on to purify the gold-shaped protein (GSP) which is responsible for guiding the shape of the nanoparticles. This protein is then placed in aqueous HAuCl 4 solution for the synthesis of nanoparticles of different shapes. In the case of AgNPs low molecular weight proteins (PLW) and high molecular weight proteins (PHW) in algal biomass are responsible for reducing silver ions in their metallic type. Spirogyra insignis (Charophyta) fine powder is used for biosynthesis of both AgNPs and AuNPs [ 162 ]. AgNPs have also been synthesized using cell-free supernatants of cyanobacterium and chlorophyta cell lysates [ 160 ].

One of the problems of using microalgae in biosynthesis of MtNPs in bioreactors on an industrial scale is their precipitation in the culture medium. However, immobilization of microalgae in organic matrices (polyvinyl alcohol, polysulfone) and polymers matrices (alginate, carcinogen, chitosan and silica gel) is one of the solutions to this problem and recycling of microalgae [ 163 ]. Once stabilized in organic matrices, microalgae retain their ability to synthesize nanoparticles after which they are released into a matrix in a complex culture medium. Biosynthesis of AgNPs from different microalgae species such as chlorophyta, haptophyta and ocrofita has also been reported by different groups [ 164 , 165 ]. A summary of reports of the biosynthesis of MtNPs by microalgae is presented in Table 4 .

The synthesis of AgNPs by microalgae has great potential due to the high growth of algal microbiomes during the biosynthetic process and also the increase in the surface area of silver in the nanometer range [ 166 ]. AgNPs synthesized by microalgae may exhibit their antibacterial effect by altering the permeability of cell membranes and airways [ 167 ]. Antifungal activity of AgNPs by inhibiting the growth of fungal hyphae have been reported [ 168 ]. However, nanoparticles biosynthesized by microalgae show a greater inhibitory effect [ 169 ]. El-Moslamy et al. [ 170 ] showed the effective role of AgNPs synthesized by Chlorella vulgaris in controlling plant diseases with strong antifungal activity against Alternaria alternata , the causative agent of leaf spot disease and plant rot. AgNPs produced by the microalga Chlorococcum humicola with the help of microalgal biomass activity against Candida albicans, Aspergillus niger and Aspergillus flavus showed significant growth inhibition against C . albicans . Biomass containing Chlorella sp. and Haematococcus Candida albicanspluvialis inhibited the growth of Penicillium expansum , the main cause of loss of quality and quantity of fruit after harvest [ 152 , 171 ].

Challenges and future direction of using MtNPs in agriculture

Green synthesis of MtNPs using microorganisms is a promising and environmentally friendly approach for agricultural applications such as nanofertilizers, nanopesticides and nanobiosensors. Given their potential widespread use in the future it is likely that large volumes of MtNPs produced by different methods will enter ecosystems [ 172 ]. Despite the favorable physical and chemical properties of MtNPs, the complexity of soil-crop ecosystems means that the environmental behaviors of these nanoparticles are not yet fully predictable after use, and this remains an important challenge [ 173 ]. Therefore, before fully utilizing their potential, it is necessary to evaluate the effects and interaction with living systems. At this stage, screening of nanomaterials is essential to assess their potential toxicity and to understand their mechanisms of action to prevent their adverse effects in the future [ 174 ].

The nanoscale dimensions of MtNPs, which determines many of their beneficial properties, can potentially also increase their potential adverse effects [ 172 ]. The toxicity of MtNPs is influenced by various factors such as solubility and their binding specificity to biological sites [ 175 ]. Several studies have shown the unpleasant aspect of long-term exposure to some MtNPs such as AuNPs and AgNPs. In a study by Vecchio et al. [ 174 ] the in vivo toxicity of AuNPs in Drosophila melanogaster was evaluated. Due to the mutations that can be passed on to offspring, significant phenotypic changes were observed in later generations of Drosophila after treatment with AuNPs, indicating the potential severity of AuNP toxicity. These findings provide important evidence of the adverse effects of AuNPs on the growth and development of organisms. These studies also demonstrate the need for reliable evaluation of the toxicological properties of nanomaterials and the need for significant efforts by the nanoscience community to produce biocompatible nanomaterials without any adverse effects on human health and the environment [ 174 ].

AgNPs are primarily produced for antiseptic applications and have potential antimicrobial activity against many pathogenic microorganisms. However, together with this favorable feature, AgNPs also show impermissible toxic effects on human health and ecosystems. Ecologists have warned that if these nano-antimicrobials are released into the environment, their spread could have serious negative consequences for other microorganisms in natural ecosystems. There is ample evidence that AgNPs are not only toxic to bacteria, but also to the cells of other organisms such as brain cells, liver cells, and stem cells, which can lead to severe damage [ 175 ]. MtNPs cause toxicity through important cellular processes such as increased levels of ROS, decreased intracellular glutathione levels, and decreased mitochondrial membrane potential. AgNPs can adversely affect on cells and embryos of freshwater fish. In one study, the toxic effects of AgNPs on adult Japanese rice fish ( Medaka, Oryzias latipes ) were evaluated by exposure to these nanoparticles. The results showed a decrease in the activity of lactate dehydrogenase and antioxidant enzymes in the liver, glutathione depletion and lipid peroxidation in the liver and gills, with varying degrees of histological lesions in the tissues [ 176 ].

Several studies have shown that MtNPs can also have an adverse effects on key major elements (plant, soil and water) in agroecosystems [ 25 ]. Generally MtNPs can enter the agricultural ecosystem through both direct and indirect routes [ 173 ]. MtNPs used for agricultural applications can enter soil, climate, and atmosphere through washing, rainfall, airflow, and trophic transfer. Various studies have shown that these MtNPs may be absorbed by microorganisms in the soil, sediments and plant roots. These MtNPs are then transferred from the roots to other parts of the plants where they can accumulate [ 25 ]. Accumulation as a key behavior of MtNPs can significantly affect their fate and toxicity in the agricultural system [ 173 ]. Standardization of MtNPs use is therefore required for their safe and sustainable use in agriculture [ 25 ]. Biogenic MtNPs can be potentially toxic directly to plants, to plant-related beneficial microbes and eventually to human. Therefore, when using MtNPs directly in crops special attention must be paid to the interaction between nanoparticles and the treated plants [ 25 , 172 , 177 ]. The interaction between MtNPs and plants leads to numerous physiological, morphological and genotoxic changes that must be fully understood to ensure effective application of nanotechnology in agriculture. The effects of MtNPs on plants vary according to the growth stage of the plant, the time of exposure to nanoparticles, the adsorption method as well as the different physical and chemical properties of the plants themselves [ 178 ]. However, some MtNPs have a positive effect on the plant system and can improve seed germination and stimulate growth parameters, though these effects can differ between different plants [ 178 ]. Several studies have also reported significant phytotoxicity of a group of MtNPs such as AgNPs, AuNPs, and CuONPs to certain plant species by inhibiting germination and root growth [ 173 , 179 , 180 ]. Different MtNPs have been assessed for plant toxicity based on their uptake, deposition and accumulation in plant cells or organs [ 25 ]. The results showed that the uptake and deposition of MtNPs depended on various factors including MtNP characteristics such as size, composition, surface characteristics, dose, delivery methods and plant species. The results also showed that bioaccumulation may affect plant physiology and plant growth [ 25 , 181 ]. Deposition of MtNPs in the edible part of plants can cause a risk to human and animal health [ 173 , 182 , 183 ].

At the cellular level, MtNPs can enters to various organelles and interfere with the mitochondrial and chloroplast electron transport chains. In these cases they can activate metabolic pathways related to oxidative stress, which is associated with increased concentrations of reactive oxygen species and leads to cytotoxicity and genotoxic effects such as membrane damage, chlorophyll degradation, vacuole shrinkage, DNA damage and chromosomal aberrations [ 182 , 184 ]. Excessive exposure of MtNPs to crop plants such as tomatoes, wheat, onions, etc. may cause oxidative bursts by interference with the electron transfer chain and can disrupt the ROS detoxifying, resulting in genotoxic implication. As a result, the production of secondary metabolites and phytohormones are affected and plant growth retardation occurs [ 25 ]. The phytotoxity and side effects of MtNPs that have been reported so far in crops include disturbances in water transfer, decreased photosynthetic rate, decreased growth hormone production, metabolic disorders, increased oxidative stress, chromosomal abnormalities, decreased growth, transcriptional changes in several genes and hypersensitivity to natural toxins such as arsenic [ 172 , 185 ]. MtNPs can also affect beneficial plant-associated microbes in the surrounding soil when used to control phytopathogens. Microbes are associated epiphytically and endophytically with plants in the rhizosphere and soils near the plant root and may significantly promote plant growth through nitrogen fixation and phosphate solubilization [ 25 , 186 ]. MtNPs used for plants crops and soil may have toxic effects on these beneficial microbes in the same way that they have on plant pathogens. These effects on the soil microbial community can be evaluated by measuring respiration and enzymatic activities in the soil [ 25 ]. For example, AgNPs have been shown to have potential antibacterial activity against soil microbial growth at levels below the concentrations of other heavy metals. Studies have also shown that AgNPs have toxic effects on beneficial microbial communities, including nitrogen-fixing bacteria, ammonifying bacteria and chemolithotrophic bacteria. These bacteria are able to form symbiotic relationship with leguminous plants and in addition to fixing nitrogen, affect plant yield and growth by secreting substances [ 175 ].

One of the main sources of indirect input of MtNPs, particularly AgNPs, is through discharge into wastewater which then leads to accumulation of these molecules into sewage sludge [ 173 ]. The main concern is the land application of this sewage sludge for agricultural or remediation purposes since the soil may receive a large source of silver contamination which can then affect plants and crops. Exposure of soil to MtNPs may lead to changes in microbial biomass, which in turn can affect plant growth and have physiological, biochemical, and molecular effects on them [ 172 , 187 ]. With this risk of increased concentrations of potentially damaging materials, sustainable use of green synthesized NPs in agriculture will require further work to identify and address these issues. The development of less phytotoxic MtNPs must be examined in future studies and the effects of different MtNPs on plant growth at working concentrations must be determined coupled with clarification of the different effects of MtNPs application on plants and soil microbiota. Further research is also needed on the removal and clearance of MtNPs from agricultural soils and sewage sludge linked with experimental studies to understand the long-term effects of MtNPs on ecosystems and plant physiology.


Green synthesis technology offers a potentially easy, efficient, clean, non-toxic and environmentally friendly method for the synthesis of MtNPs and has received much attention in recent years due to its economic prospects. A variety of microorganisms and plant extracts can be used for the efficient biosynthesis of MtNPs. While the synthesis of MtNPs using plants extracts is easier than that of microorganisms, the use of microorganisms to produce MtNPs is more cost-effective. Changing attitude of the international community towards sustainable development, improving environmental conditions and minimizing harmful man-made waste, provides a promising future for green synthesis of MtNPs and their application in various technologies, including agriculture.

Nanotechnology is an effective tool for improving the agricultural industry. The implementation of nanotechnology in modern agriculture, helps to boost the global economy. Given the various challenges posed by population growth and global climate change, the use of MtNPs in agriculture significantly helps to overcome the damage caused by excessive use of pesticides and chemical fertilizers for increasing crop production. More appropriate use of pesticides and fertilizers enclosed in various nanoformulations provides better application and controlled release and prevents environmental pollution. There are numerous studies on the successful use of various MtNPs in agriculture sector as nanobiosensors, nanopesticides and nanofertilizers. However, there is still not much knowledge about the adsorption capacity, permissible limit and environmental toxicity of these MtNPs.

Regardless of their origin as products with a specific purpose for agriculture as or the possibility of introducing them into the environment through the mismanagement of wastes containing MtNPs, it is necessary to carefully evaluate the toxicological effects of the MtNPs on the ecosystem. Therefore, in-depth studies are needed to investigate and determine their long-term effects, and if proven safe, they can be valuable as alternatives to conventional products used in agriculture. Nanotechnology is considered as one of the main components of sustainable agricultural development, but the promise of significant use of nanotechnology can only be achieved if ecotoxicity of these nanomaterials are fully assessed and properly managed.

Availability of data and materials

Not applicable.


  • Metal nanoparticles

Gold nanoparticles

Silver nanoparticle

Metal oxide nanoparticles

Cell-free extract

Fourier transform infrared

Transmission electron microscopy

Scanning electron microscopy

Atomic force microscopy

X-ray diffraction

Energy-dispersive spectroscopy

Dynamic light scattering

Cadmium NPs

Selenium NPs

Malachite green

Cadmium sulfide NPs

Lateral flow immunoassay

Potato leaf roll virus

Local surface plasmon resonance

Copper oxide NPs

Hollow silica nanoparticles

Single cell protein

Palladium nanoparticles

Generally recognized as safe

Reverse-phase-high performance liquid chromatography

Gold-shaped protein

Reactive oxygen species

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This research was supported by the National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.

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Howra Bahrulolum and Saghi Nooraei contributed equally to this work

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Department of Industrial Environmental and Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O.BOX: 14155-6343, 1497716316, Tehran, Iran

Howra Bahrulolum, Saghi Nooraei, Nahid Javanshir, Vasighe Sadat Mirbagheri & Gholamreza Ahmadian

Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

Hossein Tarrahimofrad

Faculty of Fisheries and Environment Science, Gorgan University of Agriculture Science and Natural Resources, Gorgan, Iran

Vasighe Sadat Mirbagheri

School of Life Sciences, Gibbet Hill Campus, University of Warwick, Coventry, UK

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The nanotechnology and biomedical sciences opens the possibility for a wide variety of biological research topics and medical uses at the molecular and cellular level. The biosynthesis of nanoparticles has been proposed as a cost-effective and environmentally friendly alternative to chemical and physical methods. Plant-mediated synthesis of nanoparticles is a green chemistry approach that connects nanotechnology with plants. Novel methods of ideally synthesizing NPs are thus thought that are formed at ambient temperatures, neutral pH, low costs and environmentally friendly fashion. Keeping these goals in view nanomaterials have been synthesized using various routes. Among the biological alternatives, plants and plant extracts seem to be the best option. Plants are nature’s “chemical factories”. They are cost efficient and require low maintenance. The advantages and disadvantages of nanotechnology can be easily enumerated. This study attempts to review the diversity of the field, starting with the history of nanotechnology, the properties of the nanoparticle, various strategies of synthesis, the many advantages and disadvantages of different methods and its application.

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Green Synthesis of Zinc Oxide Nanoparticles Using Plant Extracts and Their Antimicrobial Activity

  • Published: 06 June 2024

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green synthesis method of nanoparticles

  • D. C. Bouttier-Figueroa 1 ,
  • M. Cortez-Valadez 2 ,
  • M. Flores-Acosta 3 &
  • R. E. Robles-Zepeda 1  

The synthesis of zinc oxide nanoparticles (ZnO NPs) through the use of plant extracts is a remarkably simple, cost-effective, efficient, and environmentally friendly approach. In recent years, there has been a surge in the exploration of eco-friendly methods for synthesizing ZnO NPs, with researchers addressing the potential of extracts derived from various plant components, including leaves, stems, roots, and fruits. This comprehensive review aims to encapsulate and delve into the extensive research surrounding the green synthesis of ZnO NPs, emphasizing their diverse antimicrobial applications while encompassing the latest advancements documented in the literature. Furthermore, this review meticulously examines the sizes and morphological characteristics of the synthesized nanoparticles, offering valuable insights into their structural properties. Finally, a thorough exploration of the potential interaction mechanisms between ZnO NPs and bacterial cell walls was conducted, elucidating how such interactions may induce cell death and highlighting the consequential antimicrobial activity exhibited by these nanoparticles.

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D. C. Bouttier-Figueroa acknowledges the postdoctoral position at the Universidad de Sonora.

D. C. Bouttier-Figueroa acknowledges the grant from CONAHCYT.

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Extraction and Identification of the Essential Oil of Russian knapweed Compounds and their Valorization in Green Synthesis of Iron Oxide Nanoparticles During a Surfactant-free Nano-emulsions System

  • Azizi, Amir
  • Nazari, Mahboobeh
  • Roozbahani, Pouria Alaei

This study subjected the essential oil extracted from the aerial parts of the Russian knapweed plant, sourced from natural habitats in Markazi Province, Iran, to rigorous analysis. The Clevenger method facilitated the extraction, and standard laboratory techniques, including gas chromatography-mass spectrometry (GC-MS), were employed for quantitative and qualitative chemical compound identification. The reducing and stabilizing capabilities of the extracted essential oil were evaluated for the first time during a surfactant-free nano-emulsion system in the synthesis of iron oxide nanoparticles as a green and environmentally-friendly approach. The characteristics of the synthesized nanoparticles were comprehensively explored using conventional methods such as XRD, SEM, DLS, and VSM. The efficiency of the essential oil extraction was estimated to be 0.11%, and the gas chromatograph identified twenty-one chemical compounds, constituting 67.38% of the total essential oil. The major constituents included 1,8-cineole (17.18%), camphor (16.32%), beta-caryophyllene (14.14%), caryophyllene oxide (10.99%), and alpha-pinene (8.75%). The study chose 4:1 ratio of the organic phase (essential oil) to the aqueous phase with the smallest emulsion droplet size for synthesizing the iron oxide nanoparticles. The X-ray diffraction (XRD) results indicated successful formation of the mixture of iron oxides (Fe 3 O 4 and Fe 2 O 3 ) through the prepared nano-emulsion reactor. SEM, DLS, and VSM analyses conducted at 200 °C for 2 h revealed the key characteristics of the prepared nanoparticles, including an average particle size of 12.3 nm, a surface charge of + 19.26 mV, and a magnetic property of 30 emu/g, respectively. These findings underscore the potential of the essential oil of Russian knapweed as a green and environmentally-friendly agent in the synthesis of iron oxide nanoparticles.

  • Essential Oil;
  • Russian knapweed;
  • Green Synthesis;
  • Nano-emulsions;


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