Palladium, Pd, is a chemical element with atomic number 46. It is a silvery-white lustrous rare metal that was discovered in 1803 by the English chemist William Hyde Wollaston. Pd was named after the asteroid Pallas that had previously been discovered, in 1802. Pd, together with platinum (Pt), rhodium (Rh), ruthenium (Ru), iridium (Ir), and osmium (Os) form a group of metals that are referred to as Platinum Group Metals (PGMs). These elements have similar chemical properties overall, but Pd is the least dense and has the lowest melting temperature amongst them.

Pd is mostly extracted from ore deposits in a few countries. In 2020, the overall mine production of Pd reached 210,000 kg; major producers are illustrated in Figure 1A (Geological Survey, 2020). Recycling Pd forms an additional source of supply. In 2020, 96,000 kg of Pd was recycled, mainly from automotive catalytic converters, jewelry, and electronic scrap (Key trends in the Palladium, 2022). The production of Pd is limited and cannot keep up with the increased use. Consequently, the price for Pd has dramatically increased over the last years (Figure 1B) (Geological Survey, 2020; Palladium Prices, 2022).

The largest application of Pd today is in automobile catalytic converts. Nobel metals are used in catalytic converters to reduce carbon monoxide, hydrocarbons, and nitrous oxide in automotive emissions by converting them to carbon dioxide and nitrogen. 90% of these pollutants are eliminated from emissions with the use of autocatalysts (Zereini and Alt, 2006). In 1993, the catalytic converter industry began producing Pd-based catalytic converts as an alternative to the traditional Pt-Rh catalyst used. Formerly, Pd had not been considered for use in automobile catalysts by the industry for two reasons: it is not as stable as Pt, and it is more prone to contamination by impurities contained in automobile emissions. However, due to technical improvements and economic influences, Pd has been increasingly used instead of Pt (Zereini and Alt, 2006).

In 2020, 302 tons of Pd were industrially consumed, and 82% of this consumption was in the automobile industry, followed by 7% in electronics, 5% in chemical catalysis, 3% in dental alloys, 2% in jewelry, and 1% in other industries (summarized in Figure 2A) (Palladium consumption worldwide in 2020, 2022). The current Pd price remains above acceptable levels for several large-scale commercial applications; Pd-based nanomaterials, and especially NPs, thus have the potential to provide more efficient and cost-effective solutions to meet the requirements of present and evolving applications (Chen and Ostrom, 2015).

Pd NPs and Pd-based alloy clusters have been extensively investigated in the last decades. Pd NPs form a versatile catalyst that has a wide range of applications in organic reactions such as in hydrogenation (Favier et al., 2019), carbon–carbon bond forming [Heck reaction (Heck and Nolley, 1972) and Suzuki coupling (Miyaura et al., 1979)], as well as in petroleum cracking (Cagran and Pottlacher, 2006). In fact, the Nobel Prize in Chemistry in 2010 was awarded to Richard F. Heck, Ei-ichi Negishi, and Akira Suzuki for the discovery of Pd-catalyzed cross-coupling reactions in organic synthesis (The Nobel Prize in Chemistry, 2010). In addition to the catalysis of organic reactions, applications of Pd NPs include fuel cells (Antolini, 2009) and hydrogen sensing and storage (Adams and Chen, 2011). Potential electronics applications also include sensors (Silva et al., 2011). Moreover, several applications in biomedical therapies including photothermal, antibacterial, and anticancer therapies have been developed or discussed (Phan et al., 2020). The applications of Pd NPs are summarized in Figure 2B and are described in more detail in Section 5. In this review, we describe and compare the existing physical, chemical, and biological methods for Pd NP production.

Physical vapor deposition involves different techniques that provide the conversion of the material from a condensed phase (solid or liquid) to a vapor phase and then back to a condensed phase (all carried out in vacuum) (Faraji et al., 2018; Prabakaran and Rajan, 2021). In thermal evaporation deposition technique, the material to be deposited is heated to evaporation temperature using resistive heating (the production of heat by passing an electric current through a conductor). The evaporated particles travel directly to the deposition target (substrate) where they condense and form a thin film of NPs (Rajput, 2015) (Figure 3A). Another technique is based on arc deposition, in which the surface of a cathode is struck with a high-current, low-voltage arc that vaporizes the material of the cathode producing vapor that travels to the substrate where it condenses and forms NPs (Rajput, 2015) (Figure 3B). In physical vapor deposition techniques, the particle size and film thickness can be controlled by varying the deposition parameters (Mehl et al., 2015).

Sputtering is a physical process in which atoms or molecules are ejected out of a material to a substrate by the bombardment of high-energy particles. Before the 1970s, this method used high electric fields (also known as diode sputtering). However, an enhanced variant of the method emerged later in which an additional strong magnetic field is used (known as magnetron sputtering) (Dinca and Suchea, 2019). In this process, a high electric field is applied in a vacuum chamber containing an inert gas such as argon, where the cathode is attached to the metal source and the anode is attached to the substrate. Due to the collisions of electrons (released from the cathode) with the available gas molecule, positively charged (ionized) argon atoms are formed. Once formed, they are strongly attracted to the cathode resulting in high-energy collisions with the metal leading to the ejection of one or more atoms of the material. The ejected metal atoms are accelerated toward the anode bombarding its surface. As a result, small particles of that metal are formed on the substrate. The magnetic field used in this method helps in confining and trapping the electrons released from the cathode around the source (Pal et al., 2001; Musil et al., 2006) (Figure 3C). In this method, Ar pressure influences the particle size in an inverse manner, i.e., the particle size of Pd decreases with increasing Ar pressure (Slavcheva et al., 2014). The effect of using a mix of inert gases (Ar and He) on the particle size was studied by Alexeeva et al., 2015 (Alexeeva and Fateev, 2016). The researchers found that Pd particle size decreases with increasing helium flow rate due to the more frequent occurrence of three-body collisions. In addition, the He stream is extracted out of the vacuum chamber more effectively than for Ar, and nucleation time decreases in this case.

This technique is based on the irradiation of a material with a laser beam resulting in the removal of small particles from that material. The laser flux, laser wavelength, and the duration of irradiation are some of the parameters that are used to precisely control the size of released particles of the material (Chichkov et al., 1996). This technique has different variants based on the atmosphere in the chamber. A common variant of this method, known as pulsed laser ablation in liquid, is based on the immersion of the source material in a solvent. The atomized material released from the target interacts with the solvent and nucleates to form NPs (Cristoforetti et al., 2011) (Figure 3D).

The effect of different salts on the size of Pd NPs produced by pulsed laser ablation in liquid was evaluated by Marzun et al. (2015). The ablation of Pd in deionized water results in bimodal size distribution where large NPs lead to colloidal instability. It was found that the use of salts with low ionic strength (0.005 mM phosphate, 0.01 mM carbonate, 0.03 hydroxide) significantly inhibits the formation of large NPs (>30 nm). Moreover, Mendivil et al. (2015) studied the effect of variation of ablation fluence on the size of Pd NPs formed in different liquids. It was found that in distilled water the average size of NPs increased with the decrease in ablation fluence, while the opposite effect was found in sodium dodecyl sulfate (SDS) solutions.

In this method, the metal cations are reduced and deposited on the electrode of an electrolytic cell by a direct electric current. The electrolytic cell is filled with a salt solution of the metal to be deposited, in which the positive metal cations are attracted to the cathode of the cell and reduced on its surface to zerovalent metal by gaining the required electrons (Diculescu et al., 2007; Shendage et al., 2013). In some cases, the opposite reaction may occur on the surface of the anode (oxidation of the metal to form cations), thus, the anode should be made of a material that is resistant to electrochemical oxidation, such as carbon or lead (Dufour, 2006). Figure 4A graphically illustrates this method.

For the synthesis of size-controlled Pd NPs using this method, a chemical stabilizing agent should be added into the solution. The stabilizing agents, such as poly (N-vinylpyrrolidone) (PVP), promote the formation process of Pd NPs, inhibit the electrodeposition process of Pd onto the cathode, and stabilize the formed NPs (Pan et al., 2008). The size of the NPs can by controlled by adjusting the applied current and the concentration of the stabilizing agent. For instance, for the synthesis of Pd NPs using tetrachloropalladate as the precursor, the concentration of PVP was found to be inversely proportional with the mean diameter of the NPs formed. Changing the concentration of PVP from 15 to 30 g dm⁻³ resulted in lowering the mean diameter of the NPs formed from 22.2 to 10.1 nm. For controlling the size of NPs by adjusting the applied current, the researchers found that the higher the current density, the higher the overpotential, and thus the smaller the size of the NPs formed (Pan et al., 2008).

This process is based on the application of powerful ultrasound frequencies (20 kHz–10 MHz) on a solution containing the ions of the metal in question in order to generate NPs (Gedanken, 2004). In this range of frequency, the ultrasonic waves have wavelengths ranging from 10 cm to 100 μm, much larger than the molecular size. Consequently, the effects on the chemistry of molecules do not arise from the direct interaction between sound waves and chemical species, but rather from a physical phenomenon called acoustic cavitation, which is defined as the formation, growth, and the implosive collapse of bubbles (Xu et al., 2013). When sound waves propagate through a liquid, alternating expansive and compressive waves produce dynamic tensile stress changing the density of the liquid, which creates bubbles from pre-existing impurities. At a critical size, these bubbles collapse adiabatically, producing the characteristic extreme and transient conditions of sonochemistry: temperatures above 5000 K and pressures exceeding 1000 bar (Xu et al., 2013). Under these extreme conditions in so called hot spots, high-energy chemical reactions occur such as the dissociation of chemical bonds. At the same time, nonvolatile precursors may undergo chemical reactions with radicals or other high-energy products of sonolysis inside the collapsing bubbles, providing electrons for the reduction of metal cations. When these atoms are injected into the liquid phase, they nucleate and form NPs (Xu et al., 2013). In some cases, chemical reducing and stabilizing agents such as ethylene glycol and poly (vinylpyrrolidone), respectively, are added to the liquid phase in order to control the size and the shape of Pd NPs produced (Nemamcha et al., 2006). Figure 4B graphically illustrates this method.

Similar to electrochemical deposition, a chemical stabilizing agent should be added into the solution in order to synthesize size-controlled Pd NPs sonochemically. The size and shape of the NPs formed can be controlled by varying the concentration of the stabilizing agent, the current density, the ultrasonic intensity, and the interval between two continuous ultrasonic pulses (Qiu et al., 2003). Sonochemical preparation method is also used to synthesize substrate-supported Pd NPs, such as alumina-supported Pd NPs. For that, an alcohol additive is typically added to the solution in order to accelerate the reduction of Pd(II) ions. The size of supported-NPs can be controlled by varying the alcohol additive, the concentration of alcohol additive, and the concentration of the support particles (Okitsu et al., 1999; Okitsu et al., 2000).

This method is based on the saturation of a supercritical fluid with a solid substrate, and the depressurization of the solution through a nozzle into a low-pressure chamber. This process results in the nucleation of the material of the substrate, generating NPs that are collected from the gaseous stream (Byrappa et al., 2008). There are different variants for this method, however, rapid expansion of supercritical solution (RESS) is the common variant used for Pd NPs synthesis, in which supercritical carbon dioxide is usually used as the supercritical fluid (Kameo et al., 2003). Figure 4C graphically illustrates this method.

Supercritical fluid nucleation is used to synthesize substrate-supported and functionalized Pd NPs (Cansell and Aymonier, 2009; Tang and Shim, 2015). For the synthesis of substrate-supported Pd NPs, the substate is mixed together with Pd precursor in a pressure reactor. The size of the NPs can be easily controlled by adjusting the concentration of the Pd precursor, for instance, adjusting the concentration of palladium (II) hexafluoroacetylacetonate in the process of synthesizing RGO (reduced graphene oxide)-supported Pd NPs (Tang and Shim, 2015). In addition, supercritical fluid nucleation has been used to synthesize Pd NPs functionalized with fluorinated and non-fluorinated alkyl moieties, Pd NPs capped with thiols, and Pd-impregnated Nafion membranes (Jiang et al., 2005; Moisan et al., 2008; Cansell and Aymonier, 2009). The synthesis of functionalized NPs using this approach is comparatively simple, as it limits the number of steps in the preparation of functional NPs. The size of the NPs formed can also be controlled by varying the solvent, and the operating parameters such as the temperature (Cansell and Aymonier, 2009).

This technique is based on the conversion of a colloidal solution (sol) into a gel composed of discrete metal NPs. This method involves five different stages: hydrolysis, condensation, ageing, drying, and calcination. Firstly, the NP precursor (a metal alkoxide) is dissolved in water or organic solvents to produce a homogenous solution. Then, an acid or base is added to the solution to achieve the hydrolysis/alcoholysis of the precursor, forming a colloidal solution. In a next step, the solvent is removed to achieve the condensation of adjacent molecules through metal linkages. Condensation occurs via two processes; the formation of a (-OH-) bridge between two metal centers (called olation), or the formation of (-O-) bridge between two metal centers (called oxolation). Both types of condensation increase the viscosity of the solution forming a porous structure called gel. Next, an aging step is done where the condensation of the solution continues slowly, decreasing the porosity and increasing the distance between colloidal particles. Then, the gel is thermally dried to evaporate the remaining solvent. Lastly, the dried gel is calcinated and grinded to form NP powders (Kumari et al., 2019). Figure 4D graphically illustrates this method.

The sol-gel method is used to synthesize Pd composites such as Pd@SiO₂ core-shell NPs (Hu et al., 2013; Li et al., 2014). The nanosized Pd cores are formed by reducing tetrachloropalladate with sodium borohydride reducing agent and tetradecyl trimethyl ammonium bromide (TTAB) as a stabilizing agent, then the silica shells were formed by using tetraethyl orthosilicate (TEOS). The size of NPs and the thickness of the shells can be controlled by varying the concentration of the chemicals, the molar ratios, and the pH value of the solutions (Hu et al., 2013).

Biogenic synthesis of NPs can be achieved through plant-mediated, bacteria-mediated, algae-mediated, or fungi-mediated processes. For the synthesis of Pd NPs, a Pd precursor (Pd salt solution) is mixed with either a plant extract or with bacterial/algal/fungal biomass, usually at room temperature. The synthesis process immediately starts and frequently lasts no more than 2 hours, which is indicated by the color of the reaction medium (the color typically changes to dark brown or black) (Fahmy et al., 2020).

The exact mechanism of the biogenic synthesis of Pd NPs is not fully understood and requires more research, however, three major steps have been suggested. Firstly, Pd²⁺ ions need to be reduced to zerovalent Pd by acquiring electrons from a donor biomolecule or metabolite. Then, the reduced ions nucleate and start to grow and agglomerate together forming thermodynamically stable Pd NPs. Lastly, the synthesized NPs are capped by a variety of functional groups such as aldehydes, ketones, alcohols, amines, or carboxylic acids, which help in the stabilization of the NPs. The synthesized NPs can be of different sizes or form different shapes depending on the reaction conditions (Qazi et al., 2016; Vishnukumar et al., 2017; Fahmy et al., 2020; Manjare et al., 2021). Figure 5 graphically illustrates the three major steps of the biogenic synthesis of Pd NPs.

Plants possess natural mechanisms for the detoxification of metal ions in the soil. These processes can be exploited for the purification of ground water and for coping with soil salinity (Manjare et al., 2021). The term phytoremediation has been coined for plant-based approaches for the extraction, removal, or for lowering the availability of soil pollutants (Yan et al., 2020). Phytoremediation includes several mechanisms depending on the plant in question and on the pollutant, such as phytoaccumulation, phytofiltration, phytostabilization, and phytodegradation (Ali et al., 2013). These mechanisms are the basis for the development of plant-mediated synthesis methods of NPs.

Plant-mediated synthesis of NPs is based on the bioactive compounds in plant extracts, which include polyphenols, alkaloids, terpenoids, flavonoids. These compounds are believed to be responsible for the reduction of metal ions into metal NPs (Vishnukumar et al., 2017). Extracts of various plant parts, e.g., leaves, seeds, fruits, and roots, have been successfully employed to biologically synthesize Pd NPs with different sizes and shapes (Table 1). The size and morphology of plant-mediated Pd NPs are affected by Pd precursor concentration, the concentration of the plant extract, and the duration, temperature, and pH of the reaction (Qazi et al., 2016; Vishnukumar et al., 2017; Fahmy et al., 2020; Manjare et al., 2021).

Examples: leaf extract of Anacardium occidentale was used for the synthesis of 2.5–4.5 nm spherical Pd NPs. Fourier-transform infrared spectroscopy (FTIR) analysis showed the involvement of soluble polyols in the reduction of Pd ions, and of negatively charged carboxylate ions together with protein molecules in the stabilization of Pd NPs (Sheny et al., 2012). Several other plant extracts were also successfully used to produce small spherical Pd NPs with a narrow size distribution. A fruit extract of Gardenia jasminoides Ellis yielded 3–5 nm spherical Pd NPs. In this case, FTIR analysis showed that the antioxidants present in the crude water extract of the fruit (such as geniposide, chlorogenic acid, crocins and crocetin), were involved in the production of Pd NPs, and that the properties of the Pd NPs are temperature-dependent. At different temperatures ranging from 40 to 90°C, the synthesis reaction produced Pd NPs where the size, shape, and dispersity change systematically (Jia et al., 2009). Other examples include the tuber extract of Gloriosa superba (Rokade et al., 2018), the leaf extract of Parthenium hysterophorus (Ahsan et al., 2020), and the seed extract of Piper nigrum (Kandathil et al., 2018) that also produced small spherical NPs with narrow size distributions between 5 and 8 nm, 2 and 6 nm, and 2 and 7 nm, respectively.

Other plant extracts can yield larger spherical Pd NPs or NPs populations with wide size distributions. The Pd NPs produced by the fruit peel extract of Annona squamosa L. (Roopan et al., 2012) and rind extract of Citrullus lanatus (Lakshmipathy et al., 2015) were 80 nm and 96 nm, respectively. For Annona squamosa L., the exact reducing molecule was not identified, however, aldehyde and hydroxyl functional groups were found to be involved as capping agent for the synthesized NPs. For Citrullus lanatus, the reduction and capping of Pd NPs was dependent on polyhydroxylated molecules. Regarding the large size distribution, leaf extracts of Pelargonium graveolens (Li et al., 2017) and Prunus × yedoensis (Manikandan et al., 2016) yielded 50–140 nm NPs and 50–150 nm NPs, respectively. The reason why some plant extracts produce Pd NPs with a narrow size distribution and others with a broad size distribution is unknown. However, we speculate that the large size distribution is due to the presence of several biomolecules that could act as reducing and capping agents in parallel, each resulting in a differently sized Pd NPs.

The shape of plant-mediated NPs is not limited to spherical particles. Several plant extracts were found to synthesize Pd NPs with distinct shapes. The bark extract of Bauhinia variegata produced 1–9 nm cylindrical Pd NPs (Vaghela et al., 2018), the leaf extract of Lithodora hispidula (Sm.) Griseb produced 22 nm rod-shaped Pd NPs (Turunc et al., 2017), the leaf extract of Rubus glaucus Benth. produced 55–60 nm decahedral Pd NPs (Kumar et al., 2015), and the fruit extract of Terminalia chebula produced 80–100 nm cubic Pd NPs (Kumar et al., 2013). The shape of the biogenically synthesized Pd NPs in these examples was attributed to the biomolecules acting as reducing and capping agents, or to the specific reaction conditions.

Only in some cases, the specific reducing agent/s responsible for reducing Pd²⁺ to Pd⁰ in the extract solutions were identified or at least investigated in some detail. For the leaf extract of Sapium sebiferum, the phenolic compound 6-O-galloyl-D-glucose was suggested as the reducing agent of Pd²⁺. This compound donates one electron per molecule from one of its hydroxyl groups (Figure 6A). As a result, the leaf extract of this plant produces 5 nm spherical Pd NPs (Tahir et al., 2016a). Another phenolic compound was found in the leaf extract of Phoenix dactylifera; catechin also donates one electron per molecule from one of it hydroxyl groups (Figure 6B), producing 2–25 nm spherical Pd NPs (Tahir et al., 2016b). For the leaf extract of Glycine max, the amino acid tyrosine was suggested to be the main reducing agent of Pd²⁺ (Figure 6C). The leaf extract of this plant produces 15 nm spherical Pd NPs (Kumar Petla et al., 2012). For the leaf extract of Delonix regia, different phenolic compounds, namely gallic acid, protocatechuic acid, 3-hydroxybenzoic acid, and catechin, were suggested as electron donors for the reduction of Pd²⁺ ions. Unlike the aforementioned compounds, gallic acid donates two electrons per molecule from two of its hydroxyl groups, thus, only one molecule is needed for the complete reduction of one Pd²⁺ ion (Figure 6D). The leaf extract of Delonix regia produces 2–4 nm Pd NPs (Dauthal and Mukhopadhyay, 2013).

In addition to monometallic Pd NPs, bimetallic, trimetallic, and substrate-supported Pd NPs can also be made using plant extracts (Table 2). For bimetallic Pd NPs, 5–40 nm spherical AuPd NPs were produced by Myrica gale tannin in a one-step reduction reaction. The synthesized NPs had an Au core and Pd shell structure. Interestingly, the researchers found that changing the molar ratio of Au³⁺/Pd²⁺ and the concentration of Myrica gale tannin results in changes on the morphology of the Pd shell from spherical to cubic. Adding Au³⁺ and Pd²⁺ in a 1:1 M ratio with 0.4 g/L Myrica gale tannin resulted in the synthesis of AuPd NPs with a cubic Pd shell, in which some of these bimetallic NPs had multiple Au cores per Pd shell. By decreasing the concentration of Myrica gale tannin to 0.1 g/L, spherical Pd shells were generated. Modifying the molar ratio of Au³⁺/Pd²⁺ from 1:1 to 2:1 also resulted in the synthesis of spherical Pd shells (Huang et al., 2011). Many other examples for the production of bimetallic Pd NPs using various plant extracts exist. The seed extract of Peganum harmala L. produced 24–44 nm spherical PtPd bimetallic NPs (Fahmy et al., 2021). Camellia sinensis and Vitis vinifera leaf extracts produced 20–30 nm and 2–20 nm FePd bimetallic NPs, respectively (Smuleac et al., 2011; Luo et al., 2016). The leaf extract of Cacumen platyclade produced 7–13 nm AgPd bimetallic NPs (Lu et al., 2014).

Plant extracts can also be used to produce monometallic or bimetallic Pd NPs supported on different oxide substrates. The root extract of Euphorbia condylocarpa M. Bieb. (Nasrollahzadeh et al., 2015a). and Euphorbia stracheyi Boiss. (Nasrollahzadeh and Sajadi, 2016a). and the flower extract of Fritillaria imperialis (Veisi et al., 2021) were used to produce monometallic Pd NPs supported on Fe₃O₄. The parameters of the biosynthesized NPs were different in each case, being 39 nm with irregular shapes, 10 nm spherical, and 20–30 nm quasi-spherical, respectively. The fruit extract of Berberis vulgaris produced 18 nm spherical Pd NPs supported on RGO (reduced graphene oxide) (Nasrollahzadeh et al., 2016), while the fruit extract of Piper longum produced 6–26 nm spherical Pd NPs supported on natrolite zeolite (Hatamifard et al., 2015). The seed extract of Theobroma cacao L. was used to produce 40 nm Pd NPs supported on CuO (Nasrollahzadeh et al., 2015b). The leaf extract of Myrtus communis L. yielded 17–25 nm spherical Pd NPs supported on TiO₂ (Nasrollahzadeh and Sajadi, 2016b). Other plant extracts were successfully used to synthesize bimetallic Pd NPs supported on different oxide substrates. For instance, the leaf extract of Cacumen platycladi was used to produce 20–30 nm spherical AuPd NPs supported on TiO₂ (Hong et al., 2014), and 9.7 nm spherical AuPd NPs supported on MgO (Zhan et al., 2012). Even trimetallic Pd NPs can be synthesized using plant extracts. For this, the extract of two plants was used to provide the reducing and capping agents required for the biosynthesis of trimetallic-alloy NPs. The bud extract of Syzygium aromaticum together with the leaf extract of Aegle marmelos was used to produce 8–11 nm spherical AgAuPd NPs (Rao and Paria, 2015).

Unlike in the case of plant extracts, bacteria-mediated synthesis of Pd NPs requires live bacteria for the reduction and formation of Pd NPs. The first report of bacteria able to produce Pd NPs was on the anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans (Lloyd et al., 1998). The bacterial biomass was mixed with a Pd precursor and with external, bioavailable electron donors (sodium pyruvate, formate, or H₂ gas) simultaneously. The color of the reaction medium changed to black within 10 min and the color intensified within up to 24 h. The controls (heat-killed bacterial biomass and live biomass lacking the electron donors) did not undergo any color change. The examination of bacteria under Transmission electron microscopy (TEM) revealed the deposition of 50 nm Pd NPs in the periplasmic space, whereas the controls did not show any NPs. This strongly suggested that the reduction of Pd²⁺ ions to elemental Pd is based on the action of enzymes. The researchers suspected the involvement of hydrogenase enzymes in this process. Therefore, they inhibited those enzymes by adding 0.5 mM Cu²⁺. They found that Cu²⁺-treated bacterial solution did not change in color and did not show any Pd NPs in TEM. This supported the direct involvement of hydrogenase enzymes in the reduction of Pd²⁺ ions (Lloyd et al., 1998).

A second bacterium able to synthesize Pd NPs is the dissimilatory metal-reducing species Shewanella oneidensis MR-1 (De Windt et al., 2005). In that study, three main variables were tested: different electron donors, alternative electron acceptors, and the atmospheric conditions (aerobic or anaerobic). It was found that all tested electron donors resulted in the bioreduction of Pd²⁺ and formation of Pd NPs. However, formate and H₂ resulted in larger amounts of NPs than pyruvate, lactate, and ethanol. The addition of alternative electron acceptors such as nitrate and the exposure to oxygen did not affect the formation of Pd NPs. The synthesized Pd NPs were deposited on the outer membrane and in the periplasmic space of the bacteria. Pd NPs synthesized in the presence of H₂ as an electron donor were smaller than those formed in the presence of formate. In this paper, the researchers also suggested the involvement of hydrogenase enzymes in the process of Pd NPs synthesis.

Another sulfate-reducing bacterium, Desulfovibrio fructosivorans, was reported for its ability for synthesize Pd NPs (Mikheenko et al., 2008). In that study, the deposition of Pd NPs was tested for the wildtype and for mutants lacking the periplasmic NiFe hydrogenase gene, the periplasmic Fe hydrogenase gene, or both. It was found that the wildtype strain deposited small Pd NPs on both the cytoplasmic and outer membranes, while the mutant lacking both hydrogenase gene deposited large Pd NPs only on the cytoplasmic membrane. The researchers suggested that the role of periplasmic hydrogenases is to serve as a nucleation site assisting Pd NPs growth, where their gene knock-out experiments result in the absence of Pd NPs in the outer membrane.

Bacteria-mediated Pd NPs synthesis is not limited to sulfate- and metal-reducing bacteria. Two strains of cyanobacteria (Calothrix pulvinate and Anabaena flos-aquae) (Brayner et al., 2007), Clostridium pasteurianum (Chidambaram et al., 2010), and different gram-negative alpha-, beta- and Gamma-proteobacteria bacteria, including Paracoccus denitrificans, Cupriavidus necator H16, and Pseudomonas putida (Bunge et al., 2010), were reported to have the ability to synthesize Pd NPs. For the latter three species, 3–30 nm Pd NPs were synthesized in the periplasmic space when using formate as electron donor. In these cases, Pd NPs synthesis also proceeds in autoclaved cells where all proteins are inactivated, thus ruling out the direct involvement of hydrogenases in the synthesis of Pd NPs. For cyanobacteria, the nitrogenase enzyme complex was suggested to be responsible for the reduction of Pd²⁺ ions to metallic Pd.

Also the model bacterium E. coli can synthesize Pd NPs (Deplanche et al., 2010). In general, the biogenic synthesis process of Pd NPs is divided into two separate steps: the bacterial biosorption or uptake of Pd²⁺ ions from the reactions medium, and the reduction of Pd²⁺ ions to elemental Pd inside the bacteria (in the periplasm and/or the cytoplasm). In this study, the ability of E. coli wildtype and mutants lacking the genes encoding hydrogenase enzymes were assessed for their ability to take up and reduce Pd²⁺ ions. None of the mutants lost its ability to take up Pd²⁺ ions from the medium, suggesting that the three hydrogenases present in E. coli (Hyd-1, Hyd-2, and Hyd-3) are not involved in this step. For the reduction step, the mutants having only Hyd-1 or Hyd-2 did not show any significant difference compared to the wildtype. The mutant having only Hyd-3 was significantly slower in the reduction of Pd²⁺ ions. Hyd-1 and Hyd-2 hydrogenases are periplasmic hydrogenases (the catalytic subunit is exposed to the periplasmic side of the membrane), and apparently the presence of one of these hydrogenases is enough to reduce Pd²⁺ in the periplasm (where Pd²⁺ ions are expected to be). The catalytic subunit of Hyd-3 is facing the cytoplasm, and thus cannot fully reduce the Pd ions in the periplasm efficiently. The mutant lacking all the three hydrogenases was found to lose the ability to reduce Pd²⁺ into Pd NPs completely. Therefore, the hydrogenases are key enzymes for the reduction of Pd²⁺ ions in E. coli. Under TEM, the mutants having only Hyd-1, Hyd-2, or Hyd-3 hydrogenases showed somewhat different patterns of Pd NP deposition. For the mutant lacking these three hydrogenases, all the periplasmic and cytoplasmic small Pd NPs were not present. Instead, big clumps of Pd were deposited on the surface of the bacteria. These big clumps of Pd were suggested to be formed chemically rather than biologically at a slower rate compared to the biogenic process (Deplanche et al., 2010). After reports that Pd NPs can be synthesized under fermentative conditions in Clostridium pasteurianum (Chidambaram et al., 2010) and E. coli (Deplanche et al., 2010), Hennebel et al. (2011) tested five additional bacterial species (Bacteroides vulgatus, Citrobacter braakii, Clostridium butyricum, Enterococcus faecium, and Klebsiella pneumoniae) under fermentative conditions for their ability to produce biogenic Pd NPs. All species were able to reduce Pd²⁺ and to form crystalline Pd⁰ NPs under fermentative conditions. The researchers suspected that the H₂ produced during the fermentation process was the reducing agent of Pd²⁺ ions. However, in a control experiment where the cells were centrifuged and resuspended in fresh medium to remove H₂ before the addition of Pd precursor, Pd NPs were also observed at the cell surfaces. Therefore, it was concluded that Pd²⁺ ions are reduced enzymatically, by H₂, or by a combination of the two processes. The selected bacterial species have different metabolic pathways and produce different amounts of H₂ gas per 1 molecule of glucose. However, no correlation between the reduction of Pd²⁺ and the amount of H₂ produced was found. The difference in Pd NP deposition patterns among the species was attributed to the differences in cell structure and composition, enzyme production, Pd toxicity resistance, and fermentation product type and its distribution.

New reports of bacterial strains and species able to synthesize Pd NPs (Table 3), along with methods to optimize the reaction conditions and control the yield or the properties of Pd NPs are continuously published. Great efforts have been made in order to understand the influence of different abiotic factors on the biogenic synthesis of Pd NPs, and to characterize Pd NPs produced by different microorganisms (Omajali et al., 2015; Chen et al., 2018; Chen et al., 2019; Bachar et al., 2020; Tan et al., 2020). Other studies demonstrate the successful production of bimetallic Pd NPs such as Pd/Au, Pd/Ag, Pd/Pt, and Pd/Ru NPs using various bacterial strains (Murray et al., 2018; Omajali et al., 2019; Kimber et al., 2021). But despite these efforts, we are not much closer to understanding the underlying biological mechanisms for the formation of Pd NPs. No other enzymes or proteins except for the hydrogenases have been reported to be involved until recently.

In 2020, Matsumoto et al. (2020) performed a high-throughput experiment for the identification of genes responsible for the reduction of Pd ions in E. coli. The researchers screened the Keio collection, a single-gene knockout library of E. coli BW25113 (Baba et al., 2006). The difference in Pd-ion reduction ability for approximately 4000 mutants was measured through the change in the optical density (OD₆₀₀) between the mutants and the wildtype (relative reduction rate). The results showed that seven knock-out strains had a relative reduction rate increased more than 1.5-fold, suggesting that the genes missing in those knock-outs were repressing Pd reduction. At the same time, 261 knock-outs had a relative reduction rate decreased more than 0.5-fold (73 of which had a relative reduction rate lower than 0.3-fold), suggesting that the genes missing in those knock-outs are participating in Pd ion reduction. No single-gene knock-out had a relative reduction rate of zero, suggesting that the reduction process of Pd ions is complicated and is not carried out by only a single enzyme or protein.

The genes repressing Pd reduction were related to membrane transport, transcriptional regulation, and metabolism. The researchers believe that the deletion of transcriptional regulators has broad effects on metabolism and physiology, while the deletion of membrane transport or metabolic enzyme genes can directly affect Pd ion reduction. Therefore, these genes can be candidate genes for repressing Pd reduction. The list of the genes promoting Pd reduction included 3 genes from formate metabolism. The authors highlight that formate metabolism genes could be directly involved in Pd ion reduction since formate was used as an external electron donor for the synthesis of Pd NPs. Therefore, their knock-out directly affected the reduction of Pd ions (Matsumoto et al., 2020). However, these results provide just a list of candidate genes that might be involved in Pd reduction, therefore, more work is needed to find out the role of each of these genes.

In addition to plants and bacteria, other organisms such as algae, yeast, and fungi are also able to biologically synthesize Pd NPs with different sizes and shapes (Table 4). Several algal species have been successfully used to produce biogenic Pd NPs: Botryococcus braunii produces 2–7 nm Pd NPs (Anju et al., 2020), Chlorella vulgaris produces 5–20 nm spherical Pd NPs (Arsiya et al., 2017), Dictyota indica produces 19 nm spherical Pd NPs (Yazdani et al., 2018), Padina boryana produces 5–20 spherical Pd NPs (Sonbol et al., 2021), and Sargassum bovinum produces 5–10 nm octahedral Pd NPs (Momeni and Nabipour, 2015). Yeast and fungi can also biosynthesize Pd NPs. Dry granules of Saccharomyces cerevisiae yeast produced 32 nm hexagonal Pd NPs (Sriramulu and Sumathi, 2018), while the fruit extract of Agaricus bisporus mushroom produced 13–18 nm triangular Pd NPs (Mohana and Sumathi, 2020).

As an alternative to using the extract of various plant parts or the biomass of different microorganisms, specific bio-derived materials or substances for the synthesis of Pd NPs can be employed (see bottom of Table 4). For instance, a wide range of sugars and polysaccharides has been intensively utilized for this purpose. Using the products generated from acidic/alkaline treatment of glucose (Monopoli et al., 2010) and sucrose (Amornkitbamrung et al., 2014) as reducing agents for Pd²⁺ produced 15 nm and 4.3 nm spherical Pd NPs, respectively, while the use of a mix of glucose and starch produced 1–20 nm quasi-spherical and octahedral Pd NPs (Gioria et al., 2020). The acidic/alkaline treatment of glucose and sucrose generates chemical intermediates with aldehyde functionality, which act as reducing species for Pd ions. To produce Pd NPs with narrow size distribution and to prevent them from aggregation, a stabilizing agent should be used. For the production of Pd NPs using glucose, a chemical surfactant (tetrabutylammonium hydroxide) was used to stabilize the NPs (Monopoli et al., 2010). For the production of Pd NPs using sucrose, no additional stabilizing agent was needed as sucrose itself or its degradation products in the reaction act as a stabilizing agent preventing the NPs from aggregation (Amornkitbamrung et al., 2014). In a comparison of glucose and starch, glucose acted as a reducing agent while starch acts as a stabilizing agent (Gioria et al., 2020). The polysaccharides of different plant and bacterial gums have also been used for the biosynthesis of Pd NPs. For instance, guar gum produced 70–80 nm spherical Pd NPs (Anjum et al., 2020), gum acacia produced 9 nm spherical Pd NPs (Devi et al., 2011), and xanthan gum produced 10 nm spherical Pd NPs (Venkatesham et al., 2015). In addition, other sugars such as glucosamine (Ullah et al., 2018) and glucomannan (Chen et al., 2021) resulted in the synthesis of 3–9 nm and 6 nm spherical Pd NPs, respectively. Interestingly, gums, glucosamine, and glucomannan acted as both reducing and stabilization agents for the production of Pd NPs. These carbohydrate polymers act natural templates for the nucleation and growth of Pd NPs, acting as both reducing and stabilizing agents due to their functional and structural properties (Chen et al., 2021).

In addition to polysaccharides, other biological compounds such as vitamins and hormones were successfully used for the synthesis of Pd NPs previously. Ascorbic acid is a well-known reducing agent, and the use of ascorbic acid as a reducing agent together with sodium alginate as a stabilizing agent produced 13–33 nm spherical Pd NPs (Ameri et al., 2020). However, when chitosan was used as a stabilizing agent together with ascorbic acid, 30 nm flower-shaped Pd NPs were formed (Phan et al., 2019). The use of oxytocin hormone produced 15–20 nm polygonal Pd NPs; in this case, the amino acids present in oxytocin acted as both reducing and stabilizing agents (Bendre et al., 2020).

Many other bio-derived materials can act as both reducing and stabilizing agents and have been successfully used for the biosynthesis of Pd NPs, including compounds such as hyaluronic acid (Yin et al., 2021), lentinan (Han et al., 2019), lignin (Coccia et al., 2012), sodium alginate (Xiong et al., 2020), and tannic acid (Kumari et al., 2013). The synthesized Pd NPs were 1.5–5 nm, 2–5 nm, 16–20 nm, 2 nm, and 11.3 nm spherical Pd NPs, respectively.

Biogenic NPs, due to their unique physicochemical properties combined with properties of capping materials, display interesting catalytic reactivity, selectivity, and product yield in numerous organic reactions (Mughal et al., 2021; Palem et al., 2022). There are also extensive studies confirming their environmental advantage over physiochemically produced NPs (Palem et al., 2022). Specifically, Pd is a key catalyst in organic cross-coupling reactions such as Heck and Suzuki coupling reactions (Cui et al., 2017; Miller et al., 2017; Biffis et al., 2018). The unique electronic ground-state structure of Pd (4d¹⁰5s⁰) and the square-planar geometry of Pd(II) complexes with additional coordination sites at the axial positions, which can accommodate various ligands, give Pd unique properties in C-C bond formation and C-O bond cleavage (Jbara et al., 2017). Therefore, a wide range of biogenically produced Pd NPs is being tested for their catalytic performance.

Examples: Pd NPs produced by Camellia sinensis leaf extract showed potent catalytic activity for the synthesis of biaryls via Suzuki cross-coupling reaction and for the reduction of 4-nitrophenol. At the same time, the NPs displayed high reusability where the catalytic activity was not affected after the reuse of catalyst for nine cycles (Lebaschi et al., 2017). Pd NPs produced by Citrullus lanatus rind extract also showed potent catalytic activity for Suzuki cross-coupling reaction, in which the catalyst displayed high product yield for 12 different coupling reactions (Lakshmipathy et al., 2015). The biogenic Pd NPs produced by Hibiscus sabdariffa L. were tested for 23 different Suzuki cross-coupling reactions. The reaction time and yield were high except for three reactions. The reusability of the catalyst was also high, the yield was not significantly affected after 6 catalytic cycles (Hekmati et al., 2017). Interestingly, Pd NPs produced by Colocasia esculenta leaf extract exhibited superior catalytic activity for Suzuki cross-coupling reactions over conventional catalysts. Here, the reaction with the biogenic catalyst required a shorter time and less catalyst. However, the reusability of the biogenic catalyst was not high, the yield reduced 20% after five catalytic cycles (KumaráBorah et al., 2015).

For Heck cross-coupling reactions, the biogenic Pd NPs produced by Rosmarinus officinalis leaf extract were tested for 22 reactions between different combinations of aryl halides and olefins. The product yield was high for all aryl halides expect for aryl chlorides. However, in all Heck olefination reactions, the selectivity for the trans-product was 100%. At the same time, the researchers optimized the conditions for Heck cross-coupling reaction between iodobenzene and methyl acrylate using the biogenic catalyst and achieved >99% product yield. This yield was higher than the yield of four conventional catalysts used for comparison in the same study (Rabiee et al., 2020). Bacterial Pd NPs also exhibit potent catalytic performance in Heck cross-coupling reactions. The biogenic Pd NPs produced by Desulfovibrio desulfuricans were tested for Heck cross-coupling reaction between iodobenzene and ethyl acrylate in different reaction conditions. The researchers found that the reaction rate can reach values up to 95% with the biogenic catalyst compared to only 82% with the conventional one (Bennett et al., 2013). Aside from cross-coupling reactions, biogenic Pd NPs exhibited also potent catalytic performance in hydrogenation reactions (Jia et al., 2009; Dauthal and Mukhopadhyay, 2013), reduction reactions (Veisi et al., 2016a; Sharmila et al., 2017a; Gopalakrishnan et al., 2017; Veisi et al., 2021), and photocatalysis (Tahir et al., 2016a; Turunc et al., 2017).

Metal NPs are widely known for their antibacterial activity. Several mechanisms of action for this activity were proposed, including the production of reactive oxygen species, DNA damage, cellular transport disruption, cellular membrane disruption, ATP depletion, and protein denaturation through the interaction with thiol groups (Rawashdeh and Haik, 2009; Rana and Kalaichelvan, 2011; Tahir et al., 2016a; Slavin et al., 2017), and probably a combination of these mechanisms makes the bacteria fail to establish a defense mechanism for adaptation and survival. Therefore, metal NPs are considered a promising agent for treating multi-drug resistant bacteria (Slavin et al., 2017). Due to the similarity of biomolecules between human and bacterial cells, metal NPs would be similarly cytotoxic to human cells. Interestingly, in a meta-analysis and statistical study of 71 published papers discussing the cytotoxicity of biogenic metal NPs on normal versus cancerous cells, it was found that biogenic metal NPs are 9 times more cytotoxic to human cancer cells than to normal cells (Barabadi et al., 2018). This huge difference was suggested to be a result of the high metabolic activity of cancer cells, which leads to the uptake of higher number of metal NPs compared to normal cells (Rai et al., 2016; Barabadi et al., 2018).

Examples: Biogenic Pd NPs display antimicrobial and anticancer activities. Some have a narrow range of activity, such as in the cases of Pd NPs produced by Solanum nigrum leaf extract (Vijilvani et al., 2020) and by Cissus quadrangularis stem extract (Anjana et al., 2019) exhibit antibacterial activity against Escherichia coli. Other biogenic Pd NPs were tested against a wider range of bacteria, and it was found that Pd NPs produced by Filicium decipiens leaf extract displayed antibacterial activity against both gram-positive (Bacillus subtilis and Staphylococcus aureus) and gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The inhibition zone of gram-negative bacteria was larger than of gram-positive bacteria (twofold), which may suggest that Pd NPs can penetrate the thin outer membranes of gram-negative bacteria more easily than the thicker cell walls of gram-positive bacteria (Sharmila et al., 2017b). However, Pd NPs produced by Sapium sebiferum leaf extract demonstrated higher antibacterial activity against gram-positive than gram-negative bacteria (Tahir et al., 2016a). This suggests that different biogenic Pd NPs have different antibacterial mechanisms of action attributed to their different sizes, shapes, and their capping compounds.

The antimicrobial activities of Pd NPs are not limited to bacteria. For example, Pd NPs produced by Piper betle leaf extract exhibited antifungal activity against Aspergillus niger (Mallikarjuna et al., 2013), while Pd NPs produced by Melia azedarach leaf extract showed larvicidal activity against Aedes aegypti (Bhakyaraj et al., 2017). Moreover, Pd NPs produced by Cocos nucifera coir extract displayed anti-feedent and ovicidal activity against Callasobruchus maculates agricultural pest (Elango et al., 2017). At the same time, other biogenic Pd NPs exhibited anticancer activity, as those produced by Evolvulus alsinoides leaf extract, which were efficient against A2780 ovarian cancer cells (Gurunathan et al., 2015), and the NPs produced by Urtica, which were efficient against MDA-MB-231 human breast cancer cells, HT-29 human colon cancer cells, and MIA PaCa-2 human pancreatic cancer cells (Gulbagca et al., 2021).

Some biogenic Pd NPs displayed a wide range of activities. For instance, Pd NPs produced by Bauhinia variegate bark extract exhibited antibacterial activity against Bacillus subtilis and Stayphylococcus aureus, antifungal activity against Candida albicans, and anticancer activity against MCF-7 breast cancer cells (Vaghela et al., 2018). Pd NPs produced by Moringa oleifer flower extract demonstrated antibacterial activity against Enterococcus faecalis (and not against Bacillus cereus, Staphylococcus aureus, or E. coli) and anticancer activity against A549 lung cancer cells (Anand et al., 2016). Interestingly, Pd NPs produced by Rosmarinus officinalis leaf extract exhibited a wider range of activities. They displayed antibacterial and antifungal activities against all tested species. Pd NPs produced by Camellia sinensis leaf extract exhibited antioxidant activity, antibacterial activity against Escherichia coli and Staphylococcus epidermidis, and anticancer activity against MOLT-4 T-lymphoblastic leukemia cells (Azizi et al., 2017). Additionally to that, Pd NPs produced by Dioscorea bulbifera tuber extract showed antioxidant activity and anticancer activity against HeLa cervical cancer cells (Ghosh et al., 2015). In the same study, the researchers produced bimetallic PtPd NPs using the same plant extract. Remarkably, the bimetallic NPs exhibited higher antioxidant and anticancer activities compared to the monometallic NPs (Ghosh et al., 2015). In the majority of these studies, a general mechanism of action was suggested for the biogenic Pd NPs. However, these studies lack a detailed evaluation of the role of the surface molecules capping the biogenic NPs on both the cellular uptake of these NPs and on their mechanism of promoting toxicity.

In addition to having a wide range of antimicrobial and anticancer properties, Pd NPs have also shown promising results in several biomedical therapy approaches. Due to the diversity in their size and shape, photothermal conversion efficiency, and photostability, Pd NPs are emerging as potentially efficient photothermal therapy agents (Phan et al., 2020). NP-mediated photothermal therapy (cancer cell death by heat generated in tumor tissue) has shown great potential for the treatment of cancer. However, the development of small-size biocompatible photothermal therapy agents with high photothermal conversion efficiency is a big challenge (Xiao et al., 2014). Porous 20 nm Pd NPs have displayed photothermal conversion efficiency of 93.4% when dispersed in water and illuminated with an 808 nm laser. Using these NPs for the in vitro photothermal heating of HeLa cells led to 100% cell death when using 808 nm laser irradiation and 70% cell death when using 730 nm laser after 4 min of irradiation (Xiao et al., 2014). Theoretically, other nanomaterials exhibit higher photothermal conversion efficiencies, such as Au rods, which exhibit a 98.6% photothermal conversion efficiency. However, the large size of Au rods is a huge disadvantage and results in lowering the theoretical 98.6% photothermal conversion efficiency to 85.6% when irradiated with a 730 nm laser. Despite having a higher theoretical photothermal conversion efficiency, the large size of Au rods (60.8 nm on average) compared to 20 nm porous Pd NPs resulted in showing lower experimental photothermal conversion efficiency due to the higher fraction of scattering in the extinction (Xiao et al., 2014).

In another study, porous 22 nm Pd NPs were coated with chitosan oligosaccharide to improve their biocompatibility and further functionalized with RGD peptide (Arginylglycylaspartic acid) to increase their accumulation in MDA-MB-231 breast cancer cells. In an animal model, they have shown very promising results in the in vivo photothermal therapy of breast cancer. The researchers injected these Pd NPs intravenously in BALB/c nude mice bearing MDA-MB-231 xenograft tumors, irradiated the tumor region with 808 nm laser for 5 min, and measured the tumour progression rate for 20 days. Impressively, on the 20th day, the thermally destructed tumor was reduced and healed completely (Bharathiraja et al., 2018). Interestingly, besides their photothermal conversion efficiency, Pd NPs also produce large signals in photoacoustic imaging, which is an emerging new non-invasive biomedical technique that combines ultrasound-based signals with optical contrast to image treated cancer tissues. In (Basavegowda et al., 2016), the researchers injected the coated and functionalized Pd NPs intravenously in the animal model and diagnosed the tumor region using photoacoustic imaging. After accumulating in the tumor region, the injected NPs acted as an excellent contrast agent and generated a clear image of the tumor tissue.

Pd NPs could also play an important role in improving current cancer treatment technologies such as chemo- and radiotherapy. Song et al. (2018) have demonstrated that porous hollow Pd NPs can deliver ¹³¹I (a radioisotope that is commonly used in radiotherapy) and DOX (a chemotherapy drug that is commonly used in chemotherapy) in vivo in mice injected with MCF-7 breast cancer cells. Compared to other mesoporous nanocarriers, these NPs exhibited high drug loading and controllable drug release promoted by laser irradiation and acidic pH in tumor microenvironments. Moreover, Pd NPs have also shown promising results in other biomedical areas such as gene therapy (Kang et al., 2018), drug delivery (Gil et al., 2018), prodrug activation (Weiss et al., 2014), and biosensors applications (Yi et al., 2017).