Enhanced photocatalytic degradation and antimicrobial activities of biogenic Co₃O₄ nanoparticles mediated by fenugreek: sustainable strategies

Abstract

The present study introduces Trigonella foenum-graecum (TFG, fenugreek)-mediated Co₃O₄ nanoparticles (NPs) as an innovative solution for eliminating industrial azo dyes from contaminated water. The novelty lies in their rapid, cost-effective synthesis and excellent photocatalytic and antimicrobial performance, which mark a significant advancement in environmental remediation. The NPs are synthesized using a co-precipitation method and characterized through advanced techniques. UV-visible absorption spectroscopy revealed two prominent direct bandgap transitions, surpassing previous reports and enhancing light absorption for efficient photocatalysis. FTIR analysis confirmed the successful incorporation of TFG phytochemicals, while XRD and SAED patterns indicated high crystallinity, a small crystallite size (1.6 nm), and ultrafine average particle size (5.5 nm) as observed by HRTEM. XPS analysis validated the synthesis with controlled oxidation states and defect sites featuring Co²⁺ and Co³⁺ ions. The optimized synthesis process led to outstanding photocatalytic performance, achieving 100% degradation of Congo red dye in just 60 minutes at a concentration of 120 mg L⁻¹. This efficiency underscores their capability to treat CR-contaminated water under specific conditions. The synergy between TFG phytochemicals and Co₃O₄ NPs demonstrates significant potential for water pollution remediation. Additionally, these NPs exhibit strong antimicrobial activity against Gram-negative and Gram-positive bacteria, highlighting their broader environmental significance and potential applications in various ecological fields.

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1. Introduction

Green synthesis of nanoparticles, an advanced process with applications spanning energy, cosmetics, and more, offers distinct advantages over traditional methods mainly concerned with environmental sustainability and human health.^1–4 The traditional methods, including hydrothermal, sonochemical, sol–gel, and other techniques, involve the use of hazardous chemicals, high energy-inputs, and complex techniques that can have detrimental environmental impacts. These synthesis methods frequently employ toxic reagents and solvents resulting in the release of hazardous waste, more like the use of hydrofluoric acid, reducing agents such as hydrazine or sodium borohydride, and capping/stabilizer agents like polyvinyl alcohol, which can lead to the formation of by-products that are difficult to manage and dispose of.⁵ Furthermore, techniques like the hydrothermal method, Chemical vapor deposition technique, Physical vapor deposition, etc. often require high temperatures and pressures, which increase energy consumption.^5,6 In contrast, the green synthesis of nanoparticles offers a more environment-friendly alternative by utilizing natural resources and minimizing the use of toxic substances.⁷ For green synthesis, plants that are rich in biomolecules such as flavonoids, terpenoids, and alkaloids, serve as valuable resources.^8–10 These biomolecules effectively combine with metal ions to facilitate nanoparticle production, with flavonoids playing a crucial role in ion reduction.¹¹ Derived from plants, these biomolecules are vital for capping nanomaterials, providing stability and biocompatibility, and participating in bio-reduction processes.^12,13 Thus, this method aligns with the principle of green chemistry, which emphasizes the reduction of hazardous (into eco-friendly products) and capping agents, energy efficiency, and the use of renewable sources, thereby controlling the environmental impact associated with the traditional synthesis methods.⁷ Amid global concerns about antibiotic resistance, driven by overuse and environmental factors, there is an urgent need for novel approaches.^14–16 Nanoparticles, with their potential to enhance antibiotic effectiveness and mitigate resistance, offer promising solutions, especially in medical devices and implants.¹⁷

Various metals, including zinc (Zn),¹⁵ copper (Cu),¹⁶ silver (Ag),¹⁷ iron (Fe),¹⁸ titanium (Ti),¹⁹ and gold (Au)²⁰ have been widely explored for plant-mediated NP synthesis from different plant species for photocatalytic degradation. However, their higher toxicity limits their use in medical applications.²¹ Cobalt oxide nanoparticles (Co₃O₄ NPs), with spinel structure, antiferromagnetic properties, and p-type semiconductor characteristics, are gaining attention.²² Co₃O₄ NPs have diverse applications in electrochemical devices, solar cells, energy storage, gas sensors, catalysis, and biomedicine. Also, Co₃O₄ NPs exhibit antibacterial, anticancer, antioxidant, antifungal, and enzyme-inhibitory properties, making them ideal for biomedical applications.²³ In addition, Co₃O₄ NPs serve as effective photocatalysts for dye degradation and environmental remediation, offering cost-effective alternatives to noble metals.²⁴ The green synthesis of Co₃O₄ NPs using plant extracts is considered a safe, inexpensive, and environmentally friendly method, resulting in stable materials. Various plants, such as Calotropis procera, Sageretia thea, Moringa oleifera, etc. have been explored for the green synthesis of Co₃O₄ NPs.^25–29 For instance, Diallo et al.,³⁰ utilized Aspalathus linearis leaf extract, while Dubey et al.,²⁶ employed Calotropis procera latex for cobalt oxide NP production. Bibi et al.³¹ synthesized cobalt oxide NPs using Punica granatum peel extract.

Even with these advances, the biosynthesis of Co₃O₄ NPs utilizing Trigonella foenum-graecum (TFG, fenugreek) seed extract as a reducing and stabilizing agent remains unexplored. This study introduces a straightforward method for the green synthesis of Co₃O₄ NPs using TFG seed extract. Trigonella foenum-graecum, commonly known as methi or fenugreek, belongs to the Fabaceae family and holds significant value in traditional medicine.³² It is widely cultivated in countries such as India, Egypt, France, Iran, Nepal, etc.^33,34 TFG seeds, known for treating various conditions, contain saponins with digestive enzyme inhibition properties and exhibit pharmacological qualities such as antilipidemic, anthelmintic, anti-inflammatory, and neuroprotective effects.^35,36 TFG seed extract, rich in triterpenoids, flavonoids, steroids, cardenolides, alkaloids, and other compounds, serves as a dual-functional agent, acting as both a reducing and capping agent during nanoparticles synthesis. This helps regulate NP shape, minimize aggregation, and stabilize the NPs.³⁷ Therefore, TFG-leaf extract has been utilized in the synthesis of various metal and metal oxide NPs (such as Ag, ZnO, and TiO₂) for applications in antimicrobial activity and dye degradation.^38–42 Specifically, for metal NPs, studies by Rizwana et al., and Moond et al., have demonstrated that silver (Ag) nanoparticles synthesized using TFG leaf extract were effective in inhibiting the growth of both Gram-positive and Gram-negative bacteria and in degrading different dyes, including Methylene Blue, Methyl Orange, and Rhodamine B.^39,40 Regarding metal oxides, Kermani et al., evaluated the dye degradation capabilities of ZnO nanoparticles synthesized using TFG leaf extract, demonstrating significant degradation of Methylene Blue (MB) and Eriochrome Black T (EBT) under UV light.⁴¹ Subhapriya et al., synthesized spherical TiO₂ nanoparticles (20–90 nm) using TFG leaf extract, which exhibited excellent antimicrobial properties.⁴² These above-mentioned findings highlight the potential of TFG extract (leaf extracts rather than seeds) mediated nanoparticles for photocatalytic degradation and antimicrobial applications. Thus, the present study aims to fill this gap by exploring the potential of TFG-seed extract as a novel, sustainable resource that can serve as an effective reducing and capping agent for the green synthesis of Co₃O₄.

To the best of our knowledge, this study is the first to report the use of TFG-seed extract for the synthesis of Co₃O₄ NPs for photocatalytic degradation and antimicrobial activities. In this study, these hypotheses are tested through a comprehensive analysis of the structural, morphological, and surface properties of the synthesized Co₃O₄ nanoparticles, demonstrating their exceptional photocatalytic degradation of Congo Red dye at two different catalyst doses and antimicrobial effectiveness against both Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. The successful synthesis of nanoparticles and findings reveals a sustainable strategy for nanoparticle production, demonstrating a commonly available and economically viable plant resource. This technique not only reduces reliance on toxic chemicals but also addresses pressing challenges such as water pollution and antibiotic resistance. Therefore, the present work underscores the novelty and potential for utilizing plant-based resources (using TFG seed extract) in developing advanced materials (Co₃O₄ NPs), promoting sustainable practices.

2. Experimental details

The details of chemicals/materials used in the whole experiment, including the procedure of synthesis of the nanoparticles, and photocatalysis and antimicrobial activity are given in the supplementary file. A systematic synthesis process of Trigonella foenum-graecum-mediated Co₃O₄ nanoparticles is depicted in Fig. 1.

Fig. 1 Synthesis of Trigonella foenum-graecum-mediated Co₃O₄ nanoparticles.

2.1. Formation mechanism of Co₃O₄ using TGF seed extract

The formation mechanism of Co₃O₄ nanoparticles using TFG seed extract begins with the preparation of an aqueous cobalt nitrate (Co(NO₃)₂) solution, where 0.1 M Co(NO₃)₂·6H₂O is dissolved in distilled water and stirred for 30 minutes. This ensures the complete dissolution of cobalt ions, providing a sufficient concentration of precursor ions for nanoparticle synthesis. Then, the seed extract comprised of phytochemicals, is added to the cobalt solution and heated (60–70 °C). The phytochemicals in the seed extract, such as alcohols, carboxylic acids, and ethers (FTIR), act as both reducing and capping agents.^1,43 Thus, the phytochemicals acting as reducing agents, start the reduction of Co²⁺ ions to Co⁰, initiating the nucleation of Co₃O₄ nanoparticles, and temperature accelerates the reaction. Then pink to dark brown color change of the mixture indicates the successful reduction of cobalt ions and the formation of nanoparticles. After that, the interaction between cobalt particles and the phytochemicals takes place and it has a key role in influencing the shape of formed particles.^7,43 As the reaction proceeds, the phytochemicals acting as capping or stabilizing agents, adsorb onto the Co₃O₄ nanoparticle surface and influence the growth and stabilization of the particles. Due to uniform growth rates in all directions, facilitated by evenly distributed capping agents on the Co₃O₄ nanoparticle surface, the spherical shape is formed (FESEM). In contrast, non-spherical shapes emerge when the capping agents bind selectively to certain crystal facets, leading to anisotropic growth. As the Co₃O₄ nanoparticles continue to grow, their aggregation is minimized due to the presence of capping agents that prevent excessive particle–particle interactions. So, depending upon the excess or less availability of phytochemicals acting as stabilizing agents, an aggregation phenomenon occurs. This controlled aggregation leads to the development of distinct shapes both spherical and non-spherical due to the selective adsorption of different phytochemicals on various facets of the Co₃O₄ nanoparticles. These shapes are confirmed by FESEM and TEM analysis, which shows a mixture of semi-spherical and irregularly shaped particles. At last, the resulting Co₃O₄ nanoparticles were washed with distilled water and ethanol to remove any residual plant extract and impurities, ensuring their purity. And, the drying process at 100 °C for 4 hours eliminates moisture, and subsequent calcination at 180 °C for 2 hours enhances the crystallinity and stability of the Co₃O₄ nanoparticles.⁴³ The whole probable formation mechanism is as follows:

Co(NO₃)₂·6H₂O → Co²⁺ + 2NO³⁻ + 6H₂O (1)

Co²⁺ + TFG-Phytochemicals (reduced form) → Co⁰/TFG + TFG-Phytochemicals (oxidized form) (2)

Co/TFG + H₂O → Co(OH)₂ + 2H-TFG compound (3)

6Co(OH)₂ + 2H-TFG compound + Heat → 2Co₃O₄ + 6H₂O + 2H-TFG compound (4)

Co₃O₄ + TFG-Phytochemicals → TFG-Capped Co₃O₄ nanoparticles → Crushing → washing → calcination (180 °C) → Crystalline-TFG-Capped Co₃O₄ nanoparticle (5)

3. Results and discussion

3.1. X-ray diffraction analysis

The crystallinity of nanoparticles was examined through X-ray diffraction (XRD) spectroscopy. Fig. 2 depicts the XRD spectra of Co₃O₄ NPs that were made from an ethanolic extract of TFG seeds. In Fig. 2a, the (220), (311), (222), (400), (422), (511), (440), and (531) planes of the Co₃O₄ NPs should be represented by distinct peaks at 2θ values of 31.61°, 37.21°, 39.23°, 45.28°, 56.11°, 59.73°, 65.61°, and 68.42° respectively. The results from the literature (JCPDS card No. 01-074-1657 (Fig. 2b)) are in good agreement with all the reflections in Fig. 2a and can be easily indexed to a face-centered phase of Co₃O₄. The absence of distinctive peaks of impurity phases like CoO and CoOOH shows the purity of the final products. The synthesized Co₃O₄ crystallites were confirmed to be pristine and devoid of any crystallographic anomalies by X-ray patterns of the particles, which showed sharp as well as narrow width peaks and agreed with previous reports in the literature.^44–46

Fig. 2 (a) XRD pattern of TFG mediated Co₃O₄ NPs and (b) standard JCPDS of Co₃O₄.

The average crystalline size of the cobalt oxide nanoparticles was calculated from the width of the XRD peaks, using the Debye–Scherrer method (eqn (6)).⁴⁷

D = kλ/β cos θ (6)

where, D = the size of the particle, k = Scherrer's constant, β = full width at half maxima of peaks in XRD, θ = corresponding angle for peaks, and λ = the X-ray wavelength. Table 1 shows the crystallographic parameters (i.e., FWHM, d-spacing, and crystallite size of the peaks), and Bragg's law (eqn (7)) was used to find the inter-planner spacing between the atoms for every diffraction peak.⁴⁸

2d sin θ = nλ (7)

where n = integer, θ = Bragg's angle, d represents the inter-planar spacing and λ is the X-ray wavelength. FWHM was determined for each observed peak utilizing a Lorentz best-fit curve (R² > 0.99).

Table 1 Crystallographic parameters of TFG-mediated Co₃O₄ NPs

S. no.	2 Theta (degree)	Theta (radians)	FWHM β (degree)	FWHM β (radians)	d-Spacing	Crystallite size (D) (nm)
Average crystallite size = 1.6.
1	31.6194	0.275931319	8.20829	0.14326	2.82738	1.05
2	37.2124	0.324739451	5.11700	0.08931	2.41427	1.71
3	39.2354	0.342393457	3.41700	0.05964	2.29432	2.58
4	45.2874	0.39520712	11.88300	0.20740	2.00078	0.76
5	56.1164	0.489707972	4.72600	0.08248	1.63765	1.99
6	59.7374	0.521307158	4.62400	0.08070	1.54674	2.07
7	65.6194	0.572637292	4.19900	0.07329	1.42161	2.35
8	68.4244	0.597115534	12.71600	0.22194	1.37001	0.79

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From the most intense peak (311), the crystallite size was calculated to be 1.71 nm, which was closely related to the average crystallite size (1.6 nm). In a previous report, a diameter (d) value of 26 nm was documented for Co₃O₄ NPs synthesized using Curcuma longa plant extract after drying at 100 °C for 60 min.⁴⁹ Another investigation reported a d value of 29.10 nm for Co₃O₄ NPs synthesized using Piper nigrum leaves extract with heating at 60 °C for 30 minutes and calcined at 600 °C and also reported a d value of 42.05 nm after calcined at 900 °C.⁵⁰ For low crystallinity, Co₃O₄ NPs made by aqueous leaf extract of Mollugo oppositifolia L. and calcined at 500 °C for 3 h, the crystallite size was reported 22.70 nm.⁵¹ Consequently, it was inferred that the biomolecules present in the TFG seed extract function both as reducing and stabilizing agents, contributing to the synthesis of Co₃O₄ NPs with smaller crystallite sizes. This, in turn, will enhance the activation of the Co₃O₄ surface, promoting improved dye adsorption and photocatalytic activity.

Considering the above, the X-ray diffraction (XRD) investigation of the TFG-mediated Co₃O₄ NPs revealed detailed insights into their structural properties. As a result, the XRD analysis not only verified the effective synthesis of Co₃O₄ NPs utilizing TFG-mediated techniques, but it also revealed critical information regarding their crystalline nature, purity, and size.

3.2. FTIR spectroscopic analysis

FTIR spectroscopy is often used to assess the structure and purity of metal or metal oxide nanoparticles (NPs). Each peak in the FTIR spectrum represents a particular vibrational mode of a chemical bond inside a molecule. These frequencies have been used to find functional groups or types of chemical bonds present in a sample. FTIR spectra of cobalt nitrate hexahydrate (Co(NO₃)₂·6H₂O) salt solutions were measured between 500 and 4000 cm⁻¹ (Fig. 3a). The peaks in the range of 3200–3600 cm⁻¹ were corresponding to the O–H stretching vibration, which suggests the presence of water molecules in the hexahydrate. The carbonyl group (C=O), which is found in aldehydes, ketones, and carboxylic acids, was associated with the band at 2366 cm⁻¹. Furthermore, C=O ester, C=O carbonyl group, C=C bond in an aromatic ring, C–H bending vibrations in alkanes, and C–H bending vibrations in aromatic compounds were assigned to the bands at 1692, 1640, 1529, 1358, and 1013 cm⁻¹, respectively.^49,52

Fig. 3 FTIR analysis of (a) aqueous solution of the cobalt salt, (b) ethanol TFG seeds extract, and (c) TFG/Co₃O₄ NPs.

Fig. 3b represents the FTIR spectrum of an ethanolic extract of TFG seeds in the 500–4000 cm⁻¹ range. The symmetric and asymmetric C–H stretching vibrations in alkanes correlated with the peaks at 2972 and 2875 cm⁻¹, respectively. Where the peaks at 1056 and 987 cm⁻¹ were directed to the stretching vibrations of C–O (alcohol, anhydrides, carboxylic acid, and ether) and C–H bending vibrations in aromatic compounds. The peak at 883 cm⁻¹ corresponded with the C–H bending vibration of alkenes.⁵³ Bands can be noticed in an FTIR spectrum of TFG seed extract mediated-Co₃O₄ NPs with a slight decrease or increase in peak intensity (Fig. 3c), indicating the interaction between TFG seed extract and cobalt oxide NPs. In the FTIR spectra, a prominent absorption peak occurred at 660 cm⁻¹ which is equivalent to the metal–oxygen stretching vibration, which forms the spinal structure of Co₃O₄ NPs.^54,55 The peak at 1698 cm⁻¹ was the standard peak of aromatic rings as well as the in-plane bending of C=O.^45,56 These peaks revealed the production of Co₃O₄ NPs. Peaks in the 3400–3900 cm⁻¹ range were attributed to the intermolecular bond of O–H stretching.^57,58 The presence of hydroxyl groups on the particle surface offers enhanced photocatalytic activity. The peak at 1698 cm⁻¹ was the characteristic peak of aromatic rings and the in-plane bending of C=O.^45,56 These peaks indicated the formation of the Co₃O₄ NPs.

The peaks in the range of 3400–3900 cm⁻¹ are attributed to the O–H stretching of intermolecular bonds. This O–H group is likely associated with chemically adsorbed water on the surface of Co₃O₄. Given the physical and chemical fractions of water adsorbed on Co₃O₄, the physical fraction can be disregarded because the sample was calcined at 200 °C. At this temperature, only the chemically adsorbed fraction remains. The low transmittance value suggests the presence of a significant number of OH groups on the Co₃O₄ NPs surface. A high number of hydroxyl groups on NP surfaces is known to enhance photocatalytic activity.⁵² For comparison, a broad peak at approximately 3225 cm⁻¹, indicative of hydroxyl groups from polyphenolic compounds, was previously reported for Co₃O₄ NPs synthesized using P. granatum peel extract.³¹

3.3. UV-visible Analysis of TFG-Mediated Co₃O₄ NPs

By observing the UV-visible spectrum of biosynthesized Co₃O₄ NPs, the optical absorption characteristics were elucidated. Fig. 4 displays the UV-visible spectrum and Tauc plot of Co₃O₄ NPs produced by ethanolic extract of TFG seed. According to the UV-vis spectra of the sample (Fig. 4a), there is a particular absorption at two separate wavelengths in the 200–650 nm range, focused at 267 nm and 524 nm, respectively, revealing the phenolic chemicals present that are in charge of the green production of Co₃O₄ NPs. The existence of the first band close to the UV dimension, which is due to the O²⁻ → Co²⁺ charge transfer process, and the second absorption band close to the green region, which is assigned to the O²⁻ → Co³⁺ charge transfer, indicates that Co₃O₄ NPs have formed. They observed two distinct absorption peaks at different frequencies as a result of the ligand-to-metal charge transfer (LMCT) method, such as the O²⁻ → Co²⁺ charge transfer transition for the first absorption band at 267 nm and the O²⁻ → Co³⁺ charge transfer transition for the second absorption band at 524 nm, respectively.^59,60

Fig. 4 UV-vis. analysis of (a) TFG-mediated Co₃O₄ NPs (b) Tauc plot of Co₃O₄ NPs.

The UV-visible spectra of M. charantia leaf extract exhibit two absorption bands at 270 nm and 316 nm, as previously described. In contrast, the results of the current study are not consistent with the reported absorption bands by David et al.⁶¹ when it comes to the activity of phenolic compounds that are responsible for the formation of Co₃O₄ NPs. This could be attributable to the impact of bioactive compounds found in the various plant materials employed in various research. The optical band gap of the Co₃O₄ NPs was estimated by the Tauc relation given below:⁶²

αhγ = A(hγ − E_g)ⁿ (8)

where A is constant, E_g is the energy band gap, and n depends on whether the transitions are direct or indirect. For the permitted direct band gap (n = 2), Tauc plot of (αhγ)²vs. (hγ) is drawn in Fig. 4b, and the energy band gap of the constructed sample was determined to be 1.89 eV and 3.2 eV, respectively, the values are considerably higher than the one that is reported in the literature for bulk Co₃O₄ and Co₃O₄ nanomaterials.^45,63–65

In a recent investigation, values of bandgap of 3.35 eV and 3.18 eV have been reported using Phytolacca dodecandra leaf extract and chemically synthesized Co₃O₄ NPs, respectively.⁶⁶ A previous study reported an E_g value of 3.4 eV for Co₃O₄ NPs synthesized through the auto-combustion method with fresh lemon juice with heat treatment at 350 °C.⁶⁷ Similarly, an E_g value of 3.4 eV was reported for Co₃O₄ NPs synthesized using Curcuma longa leaf extract.⁴⁹ In this context, the organic components present in TFG seed extract are likely to exert superior control over the optical properties, resulting in a smaller E_g value. As a result, the synthesized Co₃O₄ NPs are expected to exhibit enhanced photocatalytic performance in the UV-visible range.

Thus, the UV-visible analysis of TFG-mediated Co₃O₄ NPs revealed unique absorption peaks, with a prominent peak observed in the visible region. The electronic transitions within the Co₃O₄ NPs are responsible for the position and intensity of the absorption peaks. The observed absorption behavior underscores the distinctive optical characteristics conferred by the TFG-mediated synthesis method. The existence of a prominent absorption band in the visible area suggests that the nanoparticles have the potential to be used in visible-light-driven catalysis or photocatalysis. The absorbance characteristics of the TFG-mediated Co₃O₄ NPs are consistent with their crystalline structure and purity, as validated by supplementary tests such as X-ray diffraction. Furthermore, the UV-visible analysis provides critical information about the bandgap of the synthesized Co₃O₄ NPs, revealing their electronic structure. This knowledge is critical for comprehending the reactivity of nanoparticles and their prospective uses in photovoltaics, sensors, and other optoelectronic devices. In conclusion, UV-visible analysis has been useful in characterizing the optical characteristics of TFG-mediated Co₃O₄ nanoparticles, revealing information on their prospective uses in a variety of domains, including photocatalysis and optoelectronics.

3.4. Morphology and elemental analysis

Scanning electron microscopy was used to analyze the morphology of the produced nanoparticles. Fig. 5a and b exhibit the surface morphology of TFG-mediated Co₃O₄ NPs at various magnifications. Individual Co₃O₄ NP agglomerations can be observed in the SEM images. A deeper examination of these aggregated pictures reveals that some of the particles are semi-spherical (Fig. 5a), while a few are monoclinic non-spherical (Fig. 5b). The formation of the sphere-like shape of Co₃O₄ NPs can be shown in Fig. 5c.

Fig. 5 FESEM images of green synthesized Co₃O₄ NPs at different magnifications: (a) at 30 000 (b) at 50 000, (c) at 100 000 magnifications, and (d) EDX spectrum.

The elemental composition of the synthesized tricobalt tetra-oxide nanoparticles was determined using an energy-dispersive X-ray diffractive (EDX) analysis. The energy dispersive spectra (EDS) of the prepared sample showed that the sample generated by the above method contains pure Co₃O₄ phases. As shown in Fig. 5d, EDX validates the presence of cobalt and oxygen signals in cobalt oxide nanoparticles, and this analysis revealed the peaks that matched the optical absorption of the generated nanoparticle. The optical absorption peaks of Co₃O₄ NPs are seen in the EDS analysis, and these absorption peaks are caused by the surface plasmon resonance of cobalt oxide nanoparticles. These elements are derived from biological components, namely alga and Co₃O₄ NPs. The elemental analysis of the nanoparticle revealed 73.23% cobalt and 26.77% oxygen, indicating that the created nanoparticle is in its purest form. The EDX experiments yielded equivalent findings to previous work, with elemental analysis of the nanoparticle providing 59.27% cobalt and 40.73% oxygen reported by Nagal et al. in 2023, whilst Marimuthu et al. reported weight of 67.56% cobalt and 32.44% oxygen in 2020.^68,69

The FESEM analysis revealed that TFG functional groups appear to create a distinct coating around the Co₃O₄ NPs. This suggests that TFG might be more effective than other green extracts in minimizing NP aggregation and achieving better size control. Further investigation is needed to fully understand the mechanisms by which TFG interacts with the NPs and influences their morphology.

3.5. Transmission electron microscopy

Transmission electron microscopy (TEM) serves as an indispensable tool, providing profound insights into the dimensions, configuration, and crystalline composition of nanomaterials. In the context of this research, we employed both high-resolution TEM (HRTEM) and selected area electron diffraction (SAED) techniques to precisely characterize the morphological attributes and crystallinity of Co₃O₄ NPs synthesized through a TFG-mediated approach. Fig. 6 provides a comprehensive analysis of TFG-mediated Co₃O₄ NPs, utilizing high-resolution transmission electron microscopy (HRTEM), a size distribution histogram, fringe patterns, and selected area electron diffraction (SAED) patterns. The aggregates of TFG-mediated Co₃O₄ NPs depicted in Fig. 6(a) find support in the literature, where similar aggregation phenomena have been observed in the bio-mediated synthesis of metal oxide nanoparticles.^70,71 The size distribution histogram (Fig. 6b) and the average particle size of 5.5 nm are indicative of controlled synthesis, a phenomenon attributed to the influence of phytochemicals in TFG. This is consistent with the findings of Zuhrotun et al. (2023), who reported that the phytochemicals present in plant extracts act as effective capping agents, regulating the size and morphology of nanoparticles during biosynthesis.⁷²

Fig. 6 Morphological exploration of TFG-mediated Co₃O₄ NPs: (a) TEM image, (b) particle size distribution, (c) d-spacing HRTEM, and (d) SAED pattern.

Phytochemicals within TFG, including compounds such as saponins and polyphenols, emerge as proficient capping agents in the Co₃O₄ NPs synthesis.⁷³ These molecules form a protective sheath around the nascent nanoparticles, acting as molecular guards that thwart unbridled growth and aggregation. This capping prowess is fundamental in conferring a distinct and uniform size distribution to the nanoparticles, as discerned from the accurately illustrated histogram. Flavonoids and alkaloids found in TFG's chemical repertoire serve as stalwart stabilizing agents.⁷⁴ Their presence is pivotal in curbing the tendency of NPs to coalesce or agglomerate, ensuring the stability of the nanoparticle dispersion. This stability, governed by the chemical balance struck by these phytochemicals, is paramount for maintaining the individual identity of nanoparticles and guarding against undesirable alterations in size or shape over temporal scales.

Phytochemicals, notably terpenoids, and tannins, actively engage in the reduction and nucleation phases of nanoparticle synthesis. Their intervention in the reduction kinetics and nucleation processes orchestrates a delicate control mechanism, influencing the initiation and subsequent growth of Co₃O₄ NPs. The discernible outcome is the meticulous formation of NPs within a specific size range, a manifestation exemplified by the average particle size of 5.5 nm. The dynamic interplay between TFG phytochemicals, such as coumarins and sterols, and the nanoparticle surface plays a pivotal role in shaping crystal growth and morphology. These surface interactions serve as molecular orchestrators, guiding the arrangement of atoms during the crystal growth phase. The result is prominently displayed in the HRTEM micrograph, where well-defined lattice fringes bear witness to the highly crystalline nature of the nanoparticles. TFG's phytochemical composition, including compounds like alkaloids and saponins, assumes the role of biological templates in the formation of Co₃O₄ NPs. The inherent chemical blueprint of these compounds provides a template for the nucleation and subsequent growth of nanoparticles, imparting a specific morphology. This templating effect is notably evident in the observed spherical shape of the nanoparticles, a testament to the guiding influence of TFG's chemical constituents in the nanomaterial synthesis process. The well-resolved lattice fringes observed in the HRTEM micrograph (Fig. 6c) align with the literature on crystallinity analysis. The study by Hirpara et al. (2022) and Mourdikoudis et al. (2018)^75,76 demonstrated that lattice fringes in HRTEM images are crucial for determining the crystalline nature of nanoparticles and extracting information about interplanar spacings. The distinctive d-spacing of 0.28 nm, indicative of atomic planes in Co₃O₄, is consistent with known values reported in the literature.^57,77

At 500 KX magnification, the HRTEM provides an intricate view into the crystallinity of TFG-mediated Co₃O₄ NPs, unveiling a captivating narrative supported by empirical evidence. The captured image vividly presents well-defined lattice fringes, serving as a visual testament to the exceptional crystalline nature of the synthesized material. This observation aligns with established literature emphasizing HRTEM's capability to reveal nanoscale features with remarkable precision.⁷⁸ The discernible lattice fringes in the HRTEM micrograph disclose a characteristic d-spacing of 0.28 nm, a key feature indicative of atomic planes in Co₃O₄. This finding is consistent with known values reported in the literature, as demonstrated by the study conducted by Al Jahdaly et al. (2022)⁵⁷ highlighting the significance of lattice fringes in HRTEM images for determining the crystalline nature of nanoparticles and extracting information about interplanar spacings. Of particular significance is the presence of singular, isolated crystallites within the TFG-mediated Co₃O₄ NPs, serving as a compelling confirmation of their monocrystalline nature. This observation not only reinforces the precision of the synthesis process but also contributes to the broader understanding of the material's structural intricacies. Each crystallite emerges as a miniature masterpiece, characterized by an ordered arrangement of atoms, as corroborated by existing research on monocrystalline nanoparticles.⁷⁹ In essence, the detailed examination at 500 KX magnification through HRTEM not only unravels the crystalline nature of TFG-mediated Co₃O₄ NPs but also provides valuable insights that contribute to the scientific understanding of their structural intricacies, reinforcing the significance of employing advanced microscopy techniques in nanomaterial characterization.

3.6. X-ray photoelectron spectroscopy (XPS)

XPS is a powerful analytical technique that plays a pivotal role in unravelling the chemical composition and oxidation states of elements residing on the surface of materials. In the context of this investigation, XPS is employed as a key tool to illuminate the intricate surface chemistry of Co₃O₄ NPs synthesized through a TFG-mediated approach. The detailed findings are depicted in Fig. 7, providing a comprehensive view of the elemental and chemical landscape of these nanoparticles. The survey spectrum (Fig. 7a) in XPS reveals crucial information about the elemental constituents of TFG-mediated Co₃O₄ NPs. The distinct peaks in the spectrum unequivocally showcase the presence of cobalt (Co), carbon (C), and oxygen (O). Specific binding energies are employed to precisely pinpoint the location of various Co species, including Co 2s at 926.90 eV, Co 2p_1/2 at 837.1 eV, and Co 2p_3/2 at 775.66 eV. Additional peaks corresponding to O 1s (530.24 eV) and C 1s (284.60 eV) further consolidate the elemental composition of the Co₃O₄ NPs.

Fig. 7 XPS analysis of Co₃O₄ NPs: (a) Holistic elemental survey (b) cobalt chemical environment – Co 2p spectra, and (c) oxygen's role in Co₃O₄ Structure – O 1s Spectra.

Fig. 7b provides a closer examination through the high-resolution Co 2p spectrum, offering deeper insights into the chemical environment of cobalt within the nanoparticles. The characteristic doublet structure of Co 2p_1/2 and Co 2p_3/2 reveals distinct peaks. The Co 2p_3/2 peak at 779.43 eV signifies the presence of Co³⁺ in Co₃O₄, while the Co 2p_1/2 peak at 794.24 eV corroborates the formation of CoO NPs. The observation of intense satellite peaks further suggests the coexistence of Co ions in the +2 and +3 oxidation states. Importantly, the absence of a peak at 777.10 eV confirms the absence of metallic cobalt impurities, aligning with complementary analyses such as XRD and SAED. Some studies using hydrothermal or solvothermal methods report the presence of mixed oxidation states (+2 and +3) alongside metallic cobalt impurities.⁸⁰ In contrast, our XPS results for TFG-mediated Co₃O₄ NPs showcase a well-defined coexistence of Co³⁺ and Co²⁺ without any detectable metallic cobalt peaks. This suggests a superior control over the oxidation state and purity achieved through the TFG-mediated synthesis, potentially leading to enhanced properties for specific applications. In Fig. 7c, the high-resolution O 1s spectrum is presented, deconvoluted into three peaks to provide a nuanced understanding of oxygen's role in the Co₃O₄ structure. The dominant O₁ peak at 529.34 eV signifies oxygen integrated into the Co₃O₄ structure through metal-oxygen bonds. The broader O₂ peak at 530.82 eV indicates defect sites with lower oxygen coordination, as reported by Zhang et al.⁸¹ These peaks collectively confirm the successful formation of Co₃O₄ NPs, with oxygen atoms bonded to both cobalt and other oxygen atoms.

In essence, XPS serves as a valuable tool in uncovering the surface composition and oxidation states of TFG-mediated Co₃O₄ NPs, providing essential insights that contribute to a deeper understanding of their properties and potential applications in various fields, including catalysis, energy storage, and biomedical applications. Further research avenues may involve theoretical calculations to validate XPS findings and explore the influence of TFG phytochemicals on the surface characteristics of these nanoparticles.

3.7. Photocatalytic analysis

Photocatalysis, the sun-driven engine for unlocking efficient and sustainable chemical reactions, offers immense potential for environmental remediation.⁸² This work delves into this exciting field, exploring the fascinating capabilities of newly synthesized TFG-mediated Co₃O₄ NPs in promoting the breakdown of the notoriously persistent Congo Red (CR) dye. Congo Red's alluring appeal lies in its vibrant red color, a signature characteristic of azo dyes. This captivating hue originates from the robust –N=N– chromophore group, a molecular bridge connecting two aromatic rings (Christie, 2003; Zollinger, 2003). Beyond its aesthetic charm, this intricate structure imbues CR with remarkable stability, making it highly resistant to degradation. These unique properties elevate CR to a formidable benchmark for evaluating the efficacy of photocatalysts. To comprehensively assess the performance of our Co₃O₄ NPs, we establish a crucial baseline by examining the behavior of CR under UV light in the absence of any catalyst (Fig. 8a). This initial exploration provides a null reference point, allowing us to precisely quantify the enhanced degradation facilitated by our TFG-mediated Co₃O₄ NPs when introduced to the system.

Fig. 8 Photodegradation of CR dye using Co₃O₄ NPs.

Interestingly, under UV light exposure alone for 80 minutes, CR dye displayed remarkable stability, with its concentration and vivid color remaining virtually unchanged. This resilience underscored the limitations of UV light alone in effectively degrading persistent dyes. However, the introduction of our synthesized TFG-mediated Co₃O₄ NPs as photocatalysts marked a crucial turning point in the experiment (Fig. 8b). In stark contrast to the UV-only scenario, CR encountered a formidable adversary. Its vibrant hues swiftly succumbed to degradation, and its concentration significantly plummeted within the same 60-minute timeframe. This notable decline serves as compelling evidence for the exceptional efficacy of TFG-mediated Co₃O₄ NPs in cleaving the intricate aromatic rings constituting CR's robust backbone.

The outstanding performance of our TFG-mediated Co₃O₄ NPs can be ascribed not only to their inherent photocatalytic properties but also to the pivotal role played by TFG phytochemicals employed in their synthesis. These bioactive molecules contribute to the enhanced performance in several key ways: (a) morphology guidance: TFG phytochemicals act as surfactants, influencing the growth and shape of the Co₃O₄ NPs during synthesis.⁸³ This influence can lead to the formation of specific crystal facets with higher photocatalytic activity due to their increased surface area and exposure to active sites. (b) Nanoparticle Stabilization: TFG molecules coat the surface of Co₃O₄ NPs, preventing aggregation and agglomeration. This coating improves their dispersion in the reaction medium and increases their availability for interacting with dye molecules, thereby boosting the overall reaction rate.⁸⁴ (c) Enhanced charge separation: TFG molecules can modify the electronic structure of Co₃O₄ NPs, facilitating the separation of photogenerated electrons and holes.⁸⁵ This separation minimizes recombination losses, allowing the charges to participate in subsequent redox reactions with the dye molecules and improving the efficiency of the photodegradation process and (d) photoinduced electron transfer: the positive charge of the TFG head group attracts negatively charged dye molecules to the Co₃O₄ surface, promoting close contact, and enhancing the transfer of photoexcited electrons from the dye to the Co₃O₄ conduction band. This initiation of the degradation process of the dye molecule.⁸⁶ Therefore, TFG phytochemicals play a multifaceted role in enhancing the photocatalytic performance of Co₃O₄ NPs by influencing their morphology, stabilizing their dispersion, improving charge separation, and facilitating electron transfer. This synergistic effect translates to the remarkable breakdown of CR dye observed in our study.

Fig. 8(b)–(d) research the photocatalytic prowess of our TFG-mediated Co₃O₄ NPs against CR under various conditions. Encompassing both darkness and UV light exposure at different NP dosages (100 and 200 mg L⁻¹), these analyses illuminate the dynamic dance between catalyst concentration and degradation efficiency. A consistent decline in CR concentration over time is evident across all scenarios, manifested by the diminishing peak at 497 nm in the UV-Vis spectra. However, the tempo quickens under UV irradiation, particularly at the higher NP dosage. With 100 mg L⁻¹ of NPs, a remarkable 79% drop in the peak is observed after just 60 minutes, signifying extensive dye degradation (Fig. 9(b)). This accomplishment is further echoed by the mesmerizing transformation of the solution, its vibrant red hue yielding to pristine transparency – a testament to the thorough dismantling of the CR molecule.

Fig. 9 Proposed photocatalytic degradation mechanism of Co₃O₄ NPs.

Fig. 8(c) sheds light on the pivotal role played by Co₃O₄ NP concentration in this intricate symphony. At higher dosages (200 mg L⁻¹), a greater density of active sites graces the NP surface, effectively acting as magnet-like attractants for CR molecules. Conversely, at lower concentrations, the scarcity of active sites can bottleneck the process, imposing mass transfer limitations that impede dye molecules’ access.⁸⁷ Boosting the NP concentration overcomes these limitations by narrowing the average distance between dye molecules and active sites, resulting in a smoother, more efficient transfer of reactants.⁸⁸ This optimization transcends mere mass transfer, igniting a synergistic interplay that propels the degradation rate to dizzying heights. Increased adsorption elevates the probability of a CR molecule encountering an active site, while the abundance of available sites ensures a readily accessible pool of charge carriers for immediate degradation reactions.⁸⁹ This dynamic cooperation culminates in a significantly accelerated degradation rate, culminating in complete dye removal at the 200 mg L⁻¹ dosage. This potent combination becomes the key conductor in the orchestra of breakdown at higher NP concentrations.

These profound insights illuminate the delicate interplay between NP concentration and degradation efficiency. Optimizing the dosage plays a critical role in achieving peak performance, ensuring the ideal balance between the dance of nanoparticles and CR molecules. By understanding this delicate orchestration, we unlock the full potential of Co₃O₄ NPs as potent photocatalysts for environmental remediation.

3.7.1. Mechanisms of TFG-mediated Co₃O₄ NPs in degradation of CR dye. The remarkable ability of TFG-mediated Co₃O₄ nanoparticles to efficiently catalyze the degradation of CR dye is attributed to a complex interaction of processes resulting from the synergistic effect between the nanoparticles and the integrated phytochemicals (Fig. 9). When exposed to UV radiation, the Co₃O₄ nanoparticles capture light, causing the electrons to move from the valence band to the conduction band and creating pairs of electrons and holes.⁹⁰ TFG phytochemicals significantly influence these photocatalytic processes via many crucial mechanisms: (a) improved charge separation: the electrically charged parts of TFG molecules work as very effective electron acceptors, efficiently trapping photoexcited electrons from the Co₃O₄ conduction band, surpassing the effectiveness of conventional surfactants such as CTAB.⁹¹

The fast transport of electrons reduces the occurrence of electron–hole recombination, hence extending the lifetime and enhancing the presence of very reactive charge carriers for later degradation events.⁹² (b) Production of reactive oxygen species (ROS): the TFG molecules grab electrons which then interact with dissolved oxygen, triggering the creation of powerful ROS such as superoxide and hydroxyl radicals.⁹⁰ These extremely reactive species have a crucial function in chemically attacking and breaking down the organic dye molecules, specifically targeting the azo linkages and aromatic rings included in CR's structure.⁹³ (c) Direct hole oxidation: the residual vacancies in the Co₃O₄ valence band engage in direct oxidation reactions with CR molecules that have been adsorbed onto the surface of the catalyst. The holes in this frontal assault efficiently disrupt the azo link and aromatic rings present in the dye molecule, therefore commencing its disintegration into smaller organic pieces. (d) Enhanced adsorption and electron transfer: the polar functional groups and aromatic rings present in TFG phytochemicals engage in hydrophobic and electrostatic interactions with dye molecules, hence facilitating their effective adsorption onto the surface of Co₃O₄. The close closeness between dye molecules and the Co₃O₄ conduction band improves the efficiency of electron transport, hence speeding up the degradation process.⁹⁴ (e) Enhanced stability and dispersion: unlike most artificial surfactants, TFG molecules often display amphiphilic characteristics, acting as stabilizers for Co₃O₄ NPs in the reaction media. This hinders the accumulation and clustering of particles, guaranteeing enhanced scattering and availability of reactive sites for interacting with dye molecules, consequently enhancing the overall efficiency of photocatalysis.⁹⁵ (f) Precise selectivity of azo bonds: specific functional groups in TFG phytochemicals exhibit preferential interaction with the azo bond found in CR molecules. The precise targeting in question improves the efficiency of degradation by directly triggering the breaking of chemical bonds and impeding the reformation of polymers throughout the process.⁹⁶ At last, molecules of CR dye were degraded to H₂O, CO₂, and other harmless molecules.⁹⁰

Essentially, TFG-mediated Co₃O₄ NPs act as a precisely controlled photocatalytic system, where phytochemicals coordinate important processes to achieve effective degradation. These biomolecules interact together to increase the breakdown of the persistent dye by enhancing charge separation, generating reactive oxygen species (ROS), facilitating adsorption, increasing stability, and specifically targeting the azo bond of the dye. The complex interaction shown here emphasizes the possibility of using natural extracts like TFG as sustainable and efficient substitutes for manufactured surfactants in photocatalytic applications aimed at treating organic pollutants like CR.

3.7.2. Investigation of CR degradation kinetics. The photocatalytic degradation of CR dye has been systematically studied at a fixed concentration of 120 mg L⁻¹, employing varying doses of the photocatalyst (TFG-mediated Co₃O₄) at 100 and 200 mg L⁻¹ (Fig. 10). To elucidate the reaction kinetics, pseudo-zero-order, pseudo-first-order, and pseudo-second-order kinetic plots are made using the provided equations, and pertinent parameters are extracted.⁹⁷

Fig. 10 Kinetics of CR dye: (a) Pseudo-zero-order, (b) Pseudo-first-order, and (c) Pseudo-second-order, (d) reusability of TFG-Co₃O₄, and (e) stability of TFG-Co₃O₄.

Zero-order kinetics. In zero-order kinetics, the reactant concentration diminishes linearly with time, irrespective of its initial concentration. The equation is represented as:

C_t = C₀ − k₀ × t (9)

First-order kinetics. In first-order kinetics, the reaction rate is directly proportional to the concentration of the reactant, expressed as:

ln(C_t) = ln(C₀) − k₁ × t (10)

Second-order kinetics. Second-order kinetics states that the reaction rate is proportional to the square of the reactant concentration. The equation is written as:

1/C_t = 1/C₀ + k₂ × t (11)

In these equations, k₀, k₁, and k₂ denote the rate constants for zero-order, first-order, and second-order kinetics, respectively. The rate constant values specific to CR degradation are provided in Table 2. Evaluation of the regression coefficients (R²) indicated that the pseudo-first-order kinetic model (ln(C₀/C_t) vs. irradiation time) best fits the CR degradation pattern (Fig. 10(b)). This suggests a direct proportionality between the degradation rate and the remaining CR concentration. Observing the kinetic results, the reaction-rate constant substantially increases from 0.02698 min⁻¹ to 0.05049 min⁻¹ as the Co₃O₄ dose escalates from 10 mg to 20 mg. This underscores the enhanced performance of the 20 mg photocatalyst. Even after five cycles of reusability, the photocatalyst retains its effectiveness, removing more than 90% and 85% of CR dye at concentrations of 200 mg L⁻¹ and 100 mg L⁻¹, respectively (Fig. 10(d)). Notably, these results surpass reported photocatalytic outcomes in the literature, emphasizing the superior efficiency of the synthesized Co₃O₄ (Table 2). The stability of the synthesized TFG-Co₃O₄ nanoparticles was confirmed by XRD analysis conducted after the photocatalytic degradation of CR dye, which showed no significant change in the crystalline structure, indicating that the nanoparticles maintain their structural integrity during and after the catalytic process (Fig. 10(e)).

Table 2 Reaction rates and regression coefficients for degradation of CR dye

S. no.	Catalyst dose (mg L^−1)	Pseudo-zero-order		Pseudo-first-order		Pseudo-second-order
		k 0 (mg L^−1 min^−1)	R ^2	k 1 (min^−1)	R ^2	k 2 (L mg^−1 min^−1)	R ^2
1	100	0.04683	0.89	0.02698	0.97	0.03112	0.63
2	200	0.06816	0.95	0.05049	0.98	0.07152	0.57

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3.7.3. Comparison of photocatalytic results with literature. The present study demonstrates a significantly higher photocatalytic removal efficiency compared to previous studies, as summarized in Table 3. The present photocatalyst, Co₃O₄, synthesized using a green method, achieved a 98% removal efficiency of CR dye in just 60 minutes under UV light. This performance is notably superior to other reported studies utilizing similar conditions and materials. CoO synthesized via a green method removed 98% of Acid Blue 74, but it required 150 minutes under UV light.⁹⁸ Additionally, Co₃O₄ synthesized through the green method showed a 91% removal of Malachite Green under simulated sunlight, but it took 100 minutes.⁹⁹ Other studies with Co₃O₄ also reported lower removal efficiency and longer durations, such as the 96% removal of CR in 240 minutes using visible light⁶⁵ and a 78% removal of Remazol Brilliant Orange in 50 minutes under solar light.³¹ Moreover, ZnO-based photocatalysts exhibited varied efficiencies depending on dye and conditions; for example, ZnO achieved a 99% removal of Methyl Red in 180 minutes under solar light,¹⁰⁰ but this took significantly longer than our study. TiO₂ photocatalysts also showed high efficiencies, such as 97% removal of CR under UV light in 150 minutes,¹⁰¹ but still required more time compared to our findings.

Table 3 Comparison of present photocatalytic results with previous reports

S. no.	PC	Synthesis method	Dye	PC dose	Light source	RE (%)	Time (minutes)	Ref.
Note: PC: Photocatalyst, AB: Acid blue, RBO: remazol brilliant orange, McG: malachite green, DB: direct blue, MR: methyl red, MO: methyl orange, IC: indigo carmine, BG: brilliant green, CBB: coomassie brilliant blue, RG: reactive green, MB: methylene blue, MG: methyl green, RE: removal efficiency.
1	CoO	Green	AB74	20 mg	UV	98	150	98
2	Co3O4	Green	RBO	500 mg	Solar	78	50	31
3	Co3O4	Green	McG	50 mg	Simulated sunlight	91	100	99
4	Co3O4	Green	CR	30 mg	Visible	96	240	65
5	Co3O4	Green	DB71	20 mg	UV	79	80	102
6	Co3O4	Green	MR	125 μg mL^−1	Sunlight	53	360	103
			MO			56	360
7	Co3O4	Green	CR	20 mg	UV	98	60	Present study
8	Fe3O4	Green	IC	5 mg	Visible	77.9	180	104
			BG			80.4
			MR			73.9
9	ZnO-60	Green	CBB	—	UV	80	60	105
	ZnO-RT
10	ZnO	Green	MO	75 mg L^−1	UV	87	270	106
11	ZnO	Green	MR	50 mg	Solar	99	180	100
12	TiO2	Green	CR	20 mg	UV	97	150	101
13	TiO2	Green	RG	20 mg	UV	96	180	107
14	CuO	Green	CR	50 mg	Sunlight	90	60	108
15	CuO	Green	MB	100 mg	Sunlight	71	200	109
16	CuO	Electro-chemical	CR	10 mg	Sunlight	85	120	110
			MB			93
17	CuO	Mechano-chemical	MG	10 mg	Sunlight	65	60	111
			MO			65

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Thus, the present study stands out due to its remarkable photocatalytic activity, achieving near-complete dye degradation in a shorter time frame than most of the previously reported photocatalysts, underlining its potential for efficient environmental remediation applications. While the study demonstrates the efficacy of TFG-mediated Co₃O₄ nanoparticles in degrading Congo Red dye, the photocatalytic performance was tested only under specific conditions (UV irradiation, concentration of 120 mg L⁻¹). Future studies could explore the efficiency of these nanoparticles across a broader range of environmental conditions, including varying pH levels, different light sources (e.g., visible light), and other dye pollutants to validate the generalizability of the results.

3.7.4. Investigation of the degraded fragmentation products of CR dye via GC-MS. The metabolites released during the breakdown of CR dye by TFG-Co₃O₄ NPs under UV irradiation were identified using the GC-MS analysis (Fig. 11). There were multiple peaks in the GC of the degraded dye metabolites, but only the distinctive peaks at m/z 418, 245, 207, and 137 have been associated with the degradation products A–D. Fig. 11 suggests the structure of the detected compounds based on the fragmentation pattern as well as the entire fragmentation pattern. The GC-MS results are consistent with the formation of salicylic acid and benzidine as the breakdown metabolites with the lowest molecular weights, which further degrade into harmless compounds.

Fig. 11 The GC-MS predicted fragmentation pattern of CR dye photodegradation in the presence of bio-active CP-Co₃O₄ NPs.

3.8. Antibacterial efficacy of TFG-mediated Co₃O₄ NPs against P. aeruginosa and S. aureus

The antibacterial efficacy of TFG-mediated Co₃O₄ NPs against both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus was systematically investigated using the agar well diffusion technique (Fig. 12).

Fig. 12 Antibacterial activity of TFG-mediated Co₃O₄ NPs.

In this experimental setup, Nutrient Broth (NB) medium obtained from HiMedia Laboratories was chosen as the growth medium for the bacterial cultures and the agar plates. Rigorous sterilization of both the media and plates was achieved through autoclaving at 121 °C for 15 minutes. Post-sterilization, 20 mL of autoclaved NB medium was carefully dispensed into sterilized Petri dishes and allowed to solidify, providing a stable substrate for bacterial growth. Cultures of P. aeruginosa and S. aureus were meticulously prepared in NB broth. Subsequently, aliquots from each culture were separately inoculated onto NB agar plates, followed by a 24-hour incubation period at 37 °C with continuous shaking at 80 RPM. After incubation, bacterial cultures were diluted to a standardized cell density of 1 : 100 in fresh NB medium. Sterilized well borers were then employed to create wells in the solidified NB agar plates already seeded with bacteria. Distinct volumes (e.g., 10, 25, 40 μg mL⁻¹) of the TFG-mediated Co₃O₄ NPs solution were accurately pipetted into the respective wells. To serve as controls, solutions such as sterile water or TFG suspension without NPs may have been included for comparative analysis. The plates underwent a subsequent incubation period of 24 hours at 37 °C. Finally, the diameters of the clear zones surrounding the wells, indicative of inhibition zones, were accurately measured and recorded in Table 4. This systematic assessment provides crucial insights into the antibacterial potential of TFG-mediated Co₃O₄ NPs at varying concentrations against the tested bacterial strains, contributing to the understanding of their practical applications in microbial control and biomedical fields.

Table 4 Zone of inhibition (ZOI) of TFG-mediated Co₃O₄ NPs against P. aeruginosa and S. aureus

S. no.	Bacteria	ZOI (mm) at different concentrations
		10 μg mL^−1	25 μg mL^−1	40 μg mL^−1
1	S. aureus	11.8	23.2	26.6
2	P. aeruginosa	8.0	13.4	19.6

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3.8.1. Antibacterial activity against S. aureus and P. aeruginosa. The antimicrobial efficacy of TFG-mediated Co₃O₄ NPs against both Gram-positive S. aureus and Gram-negative P. aeruginosa is illustrated in Fig. 13, presenting a multifaceted attack mechanism. The unique interaction is initiated by the negatively charged bacterial cell walls, drawing the NPs towards them. TFG-mediated Co₃O₄ NPs, a vital component in this process, are postulated to disrupt the bacterial membrane structure, impairing permeability, interfering with crucial transport processes, and compromising the bacterial defense mechanisms. It has been reported that the antibacterial effectiveness of nanomaterials is closely affected by their physicochemical characteristics, such as their ability to generate reactive oxygen species (ROS), as well as their surface area, particle size, solubility, and surface charge.¹¹² For example, Co3O4 nanoparticles can accumulate in the outer membrane/cytoplasm. This accumulation may lead to the release of metal (cobalt) ions, which bind to biomolecules in the bacterial cell membrane through electrostatic interactions, causing depolarization and leading to cellular leakage.¹¹³ For. Additionally, the excess production of ROS generated by these metal (cobalt) ions can penetrate the bacterial cell membrane and are capable of inducing substantial damage to critical cellular components such as DNA and proteins, ultimately resulting in bacterial cell death. In some instances, the NPs may breach the bacterial defenses, directly engaging with internal structures like mitochondria, leading to additional damage and cellular leakage. This intricate and multi-faceted assault is reflected in the substantial antibacterial activity observed against both S. aureus and P. aeruginosa. However, the efficacy of TFG-mediated Co₃O₄ NPs is not solely reliant on their intrinsic properties; rather, it is intricately linked to factors such as size, dosage, surface properties, TFG content, and treatment duration. Optimizing these parameters presents exciting prospects for further enhancing their antibacterial performance.

Fig. 13 Antimicrobial action mechanism of TFG-mediated Co₃O₄ NPs.

Beyond their inherent potency, the versatility of these NPs positions them as promising candidates for various applications. Their broad-spectrum effectiveness against both Gram-positive and Gram-negative bacteria makes them attractive for use in disinfectants and antimicrobial coatings, thereby safeguarding surfaces and materials from harmful microbial contamination. Moreover, in the context of escalating antibiotic resistance, TFG-mediated Co₃O₄ NPs emerge as a potential eco-friendly and effective alternative for bacterial control across diverse sectors, heralding a new era of antibacterial solutions with far-reaching implications. This innovative approach holds promise for addressing contemporary challenges in microbial control, opening avenues for sustainable and impactful antibacterial interventions.

The antimicrobial activity was assessed against two bacterial strains (Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa). Although these are significant pathogens, the spectrum of antimicrobial activity should be further tested against a wider array of microbial species, including fungi and resistant bacterial strains, to fully understand the potential applications of these nanoparticles in diverse environments.

3.8.2. Comparison of antibacterial results with literature reports. The present study reveals a significantly enhanced antibacterial activity of Co₃O₄ synthesized using TFG compared to previously reported catalysts, as shown in Table 5. In this study, the catalyst exhibited an impressive ZOI against S. aureus (ZOI: 11.8 mm, 23.2 mm, and 26.6 mm at concentrations of 10 μg mL⁻¹, 25 μg mL⁻¹, and 40 μg mL⁻¹, respectively). This represents a substantial increase in antibacterial efficacy compared to other Co₃O₄ catalysts synthesized using different methods. For example, Co₃O₄ prepared with Curcuma longa achieved a maximum ZOI of 14 mm at 100 μg mL⁻¹,⁴⁹ while the same catalyst synthesized with Psidium guajava showed a ZOI of 18 mm at 200 μg mL⁻¹.¹⁰² In comparison to other antibacterial agents, such as Ag NPs synthesized using Solanum melongena, which achieved a ZOI of 15 mm against S. aureus at a concentration of 20 μg mL⁻¹,¹¹⁴ the present study demonstrates a comparable or superior antibacterial effect at a lower concentration of 25 μg mL⁻¹. Additionally, for P. aeruginosa, the present Co₃O₄ catalyst also showed enhanced activity with ZOI measurements of 8.0 mm, 13.4 mm, and 19.6 mm at concentrations of 10 μg mL⁻¹, 25 μg mL⁻¹, and 40 μg mL⁻¹, respectively. This is considerably higher than the ZOI achieved by ZnO synthesized using Passiflora caerulea, which recorded a ZOI of only 10 mm at 50 μg mL⁻¹.¹¹⁵ Overall, the antibacterial performance of the present Co₃O₄ catalyst against both S. aureus and P. aeruginosa surpasses those of previously reported catalysts at comparable or even lower concentrations, underscoring its potential as a highly effective antimicrobial agent.

Table 5 Comparison of present antibacterial results with previous reports

S. no.	Catalyst	Method/Plant	Bacteria	Concentration (μg mL^−1)	Approx. ZOI (mm)	Ref.
1	Co3O4	Curcuma longa	S. aureus	10	3	49
				20	5
				50	8
				100	14
2	Co3O4	Psidium guajava	S. aureus	50	9	102
				100	12
				150	16
				200	18
3	Co3O4	Chemical	S. aureus	500	21.17	116
4	ZnO	Aspergillus niger	S. aureus	—	10	117
5	Ag NPs	Solanum melongena	S. aureus	20	15	114
6	Ag	Actinobacteria	S. aureus	100	8	118
			P. aeruginosa	100	100
7	ZnO	Passiflora caerulea	P. aeruginosa	50	10	115
8	ZnO	Trigonella foenum-graecum	S. aureus	50 × 10^3	10.3	113
				150 × 10^3	11.7
			P. aeruginosa	50 × 10^3	14.3
				150 × 10^3	17.0
9	Co3O4	Trigonella foenum-graecum	S. aureus	10	11.8	Present work
				25	23.2
				40	26.6
			P. aeruginosa	10	8.0
				25	13.4
				40	19.6

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4. Conclusion

This study presents a novel and efficient method for synthesizing Co₃O₄ nanoparticles using TFG seed extract as a dual-functional agent. The TFG phytochemicals play a crucial role in stabilizing and directing the nanoparticle synthesis, resulting in well-defined morphologies and enhanced photocatalytic and antimicrobial properties. The synthesized Co₃O₄ NPs demonstrated exceptional photocatalytic efficiency, achieving 100% degradation of Congo Red dye within 60 minutes under UV irradiation, and showed strong antibacterial activity against both Gram-positive and Gram-negative bacteria. These findings highlight the potential of TFG-mediated Co₃O₄ nanoparticles in environmental remediation, particularly for wastewater treatment and combating antibiotic-resistant bacteria. Future research should focus on expanding the scope of photocatalytic testing under varying environmental conditions, exploring the antimicrobial activity against a broader range of microbial species, and assessing the scalability of the synthesis method for industrial applications.

Data availability

The data is available upon request.

Conflicts of interest

The authors asserted that they have no known financial or interpersonal conflicts that might have influenced the research provided in this paper.

Acknowledgements

The authors, Dr Sanjeev Kumar, Dr Supreet, and Dr Gautam Singh, would like to thank supporting Project No. (SUR/2020/004020), Science & Engineering Research Board (SERB), Govt. of India, for the financial support.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ma00795f

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