1 Introduction

Due to the damage of fossil fuels to environmental ecosystems and the rapid depletion of fossil fuel resources, research in the field of sustainable and renewable energy generation technology has accelerated [1]. Biofuels, which emerged as alternative energy sources, not only reduce harmful emissions, but also provide sustainable waste management within the scope of circular bio-economy. Today, biofuels such as bioethanol, biodiesel, biogas, biomethane and biohydrogen are generated in large-scale facilities and are used in many fields. Among these, hydrogen draws attention as the most environmentally friendly fuel, thanks to its zero carbon emission [2]. It is a clean energy source that does not cause extra pollution during hydrogen production and during its use as fuel and can be produced easily. There are many different hydrogen production techniques and one of the most sustainable methods is bio-hydrogen production by biological means [3,4,5]. However, the efficiency of biological hydrogen production is generally lower compared to other methods [6]. Nanoparticles possess an extensive reactive surface area due to their fine particle size and exhibit altered electrical properties [7, 8]. Numerous studies on bio-energy production have explored the advantageous characteristics of metallic nanoparticles [9]. Specifically, during dark fermentation, metallic nanoparticles play a pivotal role in expediting e- transfer by permeating cell walls of microorganisms and targeting hydrogenase enzymes [10, 11]. Acting as catalysts, metallic nanoparticles enhance enzyme activity, thereby directly augmenting hydrogen production [12]. Notably, iron oxide nanoparticles distinguish themselves among other metal nanoparticles for their ability to enhance hydrogen production by oxidizing reduced ferredoxin [13, 14]. In nanoparticle synthesis, several types of feedstock and classical methods such as sol-gel and precipitation are generally used [15]. Despite various methods for nanoparticle production, recent years have witnessed the emergence of plant-mediated sustainable nanoparticle production methods, which offer a more accessible, cost-effective, and environmentally friendly alternative compared to traditional approaches [16,17,18].

Green synthesis involves utilizing potentially pathogenic bacteria, fungi, various plants, and microalgae as biosynthesis agents. Microalgae, being non-pathogenic and harnessing sunlight for energy, carbon dioxide for carbon, and ammonium salts for nitrogen, find extensive application in biotechnological processes like nanoparticle production [19]. The foremost advantage of employing plant extracts and microalgae for nanoparticle biosynthesis lies in the ready accessibility of these plant resources. Their abundant presence in nature makes this approach cost-effective and ideal for large-scale production [20]. A pivotal factor in the production of nanoparticles by green pathway is the antioxidant capacity of the chosen plant extract, which serves as a reducing agent. Some researches proved that plants with high antioxidant capacity possess strong reducing properties. Hence, the level of antioxidants directly correlates with the reducing potential [21].

In general, continuous efforts are being made in the scientific world to strengthen technological infrastructures in the processes in the field of bioenergy production, to discover new production methods or to increase the efficiency of classical bioenergy production methods. Developments in the field of nanotechnology, a wide variety of nanomaterials produced by different methods have begun to be used to contribute to developments in all areas of industry. There are many nanomaterial-based studies in the literature in the last 10 years. Studies using nanomaterials are not only in the field of increasing bioenergy production efficiency, but also include areas such as nanomaterial-based enzyme production, treatment of lignocellulosic wastes, and providing antibacterial properties to materials [22, 23]. However, there are limited studies published in the literature, especially in the field of NP production with the green synthesis method. Determining the optimum chemical and reducing agent ratio required for NP synthesis from different plant-derived materials will contribute to the use of similar materials in future studies. It is not possible to synthesize NP from every plant-based material, but as studies on this subject become widespread, the limits of the green synthesis method will be determined.Many plant extracts can be used in nanoparticle synthesis as well as olive leaves could be used in nanomaterials production systems because of having high anti-oxidant content [24,25,26]. In previous studies, our working group also synthesized different nanoparticles using olive leaf extract (OLE) for reduction reaction [27]. In this study, nickel ferrite and cobalt ferrite nanoparticles production by green route and their effects on dark fermentation yield was investigated. To the best of authors knowledge there is no study on the potential of nickel ferrite and cobalt ferrite nanoparticle produced from OLE with green synthesized method on bio-hydrogen fermentation in the literature. In this study, hydrogen yield increasing effect was observed in dark fermentation studies to control the functionality of the produced nanoparticles following the use of olive leaf in nanoparticle production. This study holds significant implications for advancing eco-friendly and efficient bioenergy generation methods.

2 Materials and methods

2.1 Nanoparticle synthesis

OLE was used as a reducing agent for NPs synthesis similar to our previous study [27]. Olive leaves samples underwent a cleansing process with DW and kept at 40oC for 24 h. For reducing agent preparation 10g of leaves were putted into100 ml DW for 20 minutes at 100oC. The boiled leaves solution filtered and the liquid phase was used for NPs production. For the nickel ferrite nanoparticles production Fe (NO3)3·9H2O and NiCl2·6H2O with 2:1 (M) was dissolved in deionized water (25 mL) and mixing at 80 °C for metal solution preparation [28]. 25 ml of OLE was mixed with the prepared metal solution. Then the mixed solution autoclaved for 2 h at 200°C for the reaction. After the reaction solid phase was washed with DW and dried at 40oC for 24 h. For the cobalt ferrite nanoparticles same procedure was used except metal solution. The metal solution was prepared using Fe(NO3)2•9H2O and Co(NO3)2·6H2O with M ratio of 2:1 [29]. Finally, produced NPs were stored at room temperature until further usage.

2.2 Characterization of nanoparticles

The dimensions and shape of the nanoparticles were assessed through the application of scanning electron microscopy (SEM). Fourier Transform Infrared spectroscopy (FTIR), performed using equipment from Perkin Elmer Inc. in Wellesley, MA, was utilized to analyze the chemical composition of both the nanoparticles and the reducing agent. The EDX spectrum was generated through the application of energy-dispersive X-ray spectroscopy. The SEM and EDX analyses were conducted in Yildiz Technical University Central Laboratory.

2.3 Inoculum and experimental set-up

The experimental sets for fermentation were performed in serum bottles with a working volume of 50 mL. The preparation of fermentation media and Clostridium sp. was enriched in accordance with the methods detailed in our prior research [30]. Bottles were inoculated with 10% (v/v) inoculum. The medium was subjected to a 5-minute infusion of nitrogen gas that was free from oxygen. Nanoparticle concentrations in the range of 50 to 500 mg/L were applied for NiFe2O4 NPs containing reactors (N1, N2, N3, N4, N5, and N6) and CoFe2O4 containing reactors (C1, C2, C3, C4, C5, and C6). Reactors were stirred at 130 rpm at mesophilic conditions. The generated biogas was measured by syringe. All experiments were performed repetitively.

2.4 Analytical procedures and kinetic analysis

During experiments the produced Volatile fatty acids (VFAs) were analyzed in Gas Chromatography (GC) (Shimadzu GC2010, Tokyo, Japan). Hydrogen generation analysis was conducted using also GC (Shimadzu GC2014, Tokyo, Japan). The detailed procedure for the analyses has been given in previous studies. [30]

The hydrogen production data was simulated by the Modified Gompertz model (Eq. (1)).

$$P(t)={P}_m\times \mathit{\exp}\left[-\mathit{\exp}\right[\frac{R_m\times e}{P_m}\ \left(\lambda -t\right)+1\left]\ \right]$$
(1)

where P is the cumulative bio-hydrogen production (mL), Pm is the maximum bio-hydrogen production potential (mL), Rm is the maximum HPR (mL h-1), λ is the lag phase time(h), t is the incubation time (h).

3 Results and discussion

3.1 Examination of nanoparticles synthesized via green methods

The SEM technique was employed to investigate the morphology and structural characteristics of the NiFe2O4 nanoparticles that were synthesized. Figure 3a shows the formation of irregular cubic shape and presence of some large particles. The SEM results of synthesized CoFe2O4 NPs at are presented in Fig. 1b and the particles have uniform structure. Their average particle sizes were 385 and 292 nm for NiFe2O4 and CoFe2O4 nanoparticles, respectively. According to the EDX spectrum the the NiFe2O4 NPs sample includes nickel, iron and oxygen elements and iron, oxygen, and cobalt elements for CoFe2O4 nanoparticles. The EDX spectrum of samples indicating the purity of synthesized nanoparticles (Fig. 2a, b).

Fig. 1
figure 1

SEM images of nanoparticles (a) NiFe2O4, (b) CoFe2O4

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Fig. 2
figure 2

EDX spectrum of green synthesized Nanoparticles (a) NiFe2O4, (b) CoFe2O4

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Bashir et al. [31] synthesized NiFe2O4 using Persa Americano seeds as a reducing agent and observed that the particle size varied in the range of 15-20 nm. Makofane et al. [32] They synthesized nickel ferrite using the extract of Monsonia burkeana plant and used the NPs they produced for bacterial degradation. The size of the NPs produced in the study ranged between 35-87 nm. Kushwaha et al. [33] produced CoFe2O4 with a particle size of 23.85 nm using hibiscus extract and examined its humudity sensing properties. In addition, there are studies that combine green synthesis methods using aloe vera with methods such as the classical sol-gel method to synthesize cobalt ferrite NPs and produce CoFe2O4 NPs with particle sizes in the range of 50-65 nm [34]. Compared to the studies here, the particle size of the NPs produced in our study is higher. While studies generally focus on NP synthesis and characterization of produced NPs, the number of studies examining their potential for use in different fields is rare.

The typical IR spectra of the NiFe2O4 and CoFe2O4 nanoparticles in the range of 400–4000 cm-1 were given in Figure 3. In the FTIR spectrum of the NiFe2O4 sample, a prominent absorption peak at 510 cm−1 is attributed to the presence of Ni-O bonds [35]. The band associated with the bending vibrations of H2O molecules is situated at 1651 cm−1 on the spectrum [36]. A wide peak in the range of 3600–3000 cm−1 was identified as arising from the stretching vibrations of OH groups, found in both types of nanoparticles, possibly from hydroxyl or carboxyl groups. Furthermore, the presence of stretching vibrations in CH groups from either aromatic or aliphatic methyl groups was confirmed in both samples, as evidenced by the peak occurring at approximately 3000–2800 cm−1 [37]. The existence of aromatic compounds was indicated by the stretching vibration of the C=C group, which was observable in both samples with a peak occurring in the range of 1700–1500 cm−1. Additionally, within the spectrum, two primary absorption peaks were observed between 530-580 cm−1, corresponding to the stretching vibrations of metal-oxygen bonds [38]. The absorption peak observed at 510 cm−1 (NiFe2O4) and 596 cm−1 (CoFe2O4) was attributed to the inherent stretching vibrations of the metal ions [39]. We observed comparable peaks in both the nanoparticles (NPs) and the OLE sample, suggesting that the NPs preserved certain functional groups from the OLE. These retained functional groups likely played a role in stabilizing the synthesized nanoparticles.

Fig. 3
figure 3

FTIR images of nanoparticles (a) NiFe2O4, (b) CoFe2O4

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3.2 Cumulative bio-hydrogen production

Developments in biotechnological fields are increasing rapidly and the most popular studies in recent years are increasing the process efficiency by using nanomaterials in various bioenergy production studies [40, 41]. In this study, two types of NPs are used in fermentative bio-hydrogen generation experiments. In the studies performed, it was observed that the bio-hydrogen production potential increased with the use of both NiFe2O4 and CoFe2O4. Sets were established with 6 different NP concentrations. The agglomeration procedures frequently exert a substantial impact on the characteristics of particles [42]. Ligands and polymers can be used to prevent aggregation [43]. By employing plant extracts as reducing agents in eco-friendly synthesis techniques, a biopolymer-based coating is created around the nanoparticles (NPs) [44]. This research demonstrates that agglomeration was effectively minimized through the utilization of NPs derived from the eco-friendly synthesis process. A consistently mixed fermentation environment was established with a constant mixing speed throughout the fermentation process. Experimental data and model results are shown in Fig. 4.

Fig. 4
figure 4

Cumulative bio-hydrogen production (a) Nickel ferrite NPs addition (b) Cobalt ferrite NPs addition

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The highest hydrogen yield was obtained with the generation of 292.12±1.21 mL H2/g glucose and 265.74±1.89 mL H2/g glucose, with the usage of 200 mg/L NiFe2O4 and CoFe2O4, respectively. The maximum hydrogen production yield was 2.02 mol H2/mol glucose with the addition of NiFe2O4. Zhang et al. (2021b) conducted a research about the performance of NiFe2O4 NPs in the context of fermentation. Interestingly, their findings revealed that under mesophilic conditions, the optimal concentration of NiFe2O4 nanoparticles for achieving the highest hydrogen (H2) yield was 100 mg/L, resulting in an impressive yield of 222 mL H2/g glucose. However, when the experiment was conducted under thermophilic conditions, a different concentration, specifically 200 mg/L of NiFe2O4 nanoparticles, led to the highest H2 yield, which amounted to 130 mL H2/g glucose. In this study, higher hydrogen production efficiencies were obtained with NiFe2O4 NPs. This phenomenon could be caused by the NP production method and the characteristic properties of the obtained NPs. The NPs synthesized by Zhang et al. [45] had an average particle size of less than 50 nm. In this study, the average particle size was determined as 385 and 292. Higher performances are expected due to the increase in surface area at smaller nanoparticle sizes. But at the same time, the possibility of smaller nanoparticles passing into the cell and causing toxic effects increases. In this study, it was thought that achieving relatively higher bio-hydrogen production efficiencies may be due to the cells being exposed to less toxicity. Besides, NPs synthesized by green route are also known as microorganism friendly NPs which is the reason to get higher bio-hydrogen production yield [46].

It has been proven in many studies that the hydrogen production of iron-containing NPs is supported [47, 48]. Both NPs used in this study have iron content. Nanoparticles exhibit the capability to permeate the cellular membranes of fermentative bacteria, where they play a pivotal role in enhancing the rate of electron transfer within the cellular environment. This heightened electron transfer rate is integral to various cellular processes, thereby influencing the overall metabolic dynamics and performance of these microorganisms [49]. Iron addition increases the hydrogenase efficiency which indirectly increases hydrogen production. It has been observed how the NPs used in this study affect the hydrogen production mechanism as a result of the use of iron with cobalt and nickel. Obtaining higher efficiencies in nickel ferrite sets shows that nickel affects the electron transfer rate more positively than cobalt.

Cobalt (Co) assumes a pivotal role as the primary constituent of coenzyme B12 (C72H100CoN18O17P). Hence, judicious supplementation of cobalt significantly mitigates volatile fatty acid (VFA) accumulation during fermentation [50]. In a study by Zhang et al. [51], the utilization of nickel oxide nanoparticles (NiO NPs) derived from Eichhornia crassipes resulted in a notable 47.29% augmentation in dark fermentative hydrogen production rates. Conversely, [Ni–Fe] and [Fe–Fe] hydrogenase variants, distinguished by the central active metal, exhibit a higher prevalence in microbial organisms than the latter [52, 53]. The stimulatory influence of nickel on biohydrogen (bioH2) production is attributed to its metal cofactor [54]. Noteworthy is the observation that nanoscale metal oxides exert diminished toxicity towards microbes compared to their pure metallic counterparts [13]. The inclusion of nanoparticles containing nickel and cobalt expedites the synthesis of hydrogenases. Nickel further contributes to the formation of [Fe-Ni] hydrogenase and acetyl-CoA synthase. Consequently, it not only facilitates the catalytic reduction of protons to molecular hydrogen (H2) but also acts as a crucial intermediate in the generation of soluble microbial products (SMPs) by participating in acetyl CoA synthesis. Moreover, the liberation of Co and Ni ions from nanoparticles incorporating these elements optimizes bacterial community structure by supplying essential trace elements conducive to proliferation. These ions concurrently ameliorate the adverse impacts of inhibitory substances, such as humic acid.

The NiFe2O4 and CoFe2O4 boosted the hydrogen yield by 47% and 41%, respectively. For NPs concentrations higher than 200 mg/L, fermentation process was inhibited. This event was reported in another research paper [11]. This paper also reported that cell death occurs as a result of using large amounts of nanoparticles in dark fermentation. It was explained by Rittman and Herwig that the use of high amounts of iron and nickel negatively affects microbial metabolism [55]. However, some properties of metals, such as electrical conductivity, differ considerably at the nanoscale. For this reason, the effectiveness of metals in increasing the efficiency of microbial processes is frequently investigated. As in the micro dimension, NP additives above a certain concentration also created an inhibition effect at the nanoscale.

At the conclusion of the fermentation period, the control group achieved a peak hydrogen production rate, recording an impressive output of 155.28±2.71 mL of hydrogen per gram of glucose. This observation underscores the significant hydrogen-producing potential exhibited by the control conditions in our study. For the NiFe2O4 sets 231.74±1.41, 264.19±3.53, 292.12±1.21, 246.41±0.87, 236.5±1.34 and 186.89±3.76 mL H2/g glucose achieved for N1, N2, N3, N4, N5 and N6 reactors, respectively. For the CoFe2O4 sets, bio-hydrogen production yields were 194.17±1.43, 240.74±2.32, 265.74±1.89, 235.55±1.13, 212.69±1.67 and 153.08±1.21 mL/g glucose for the C1, C2, C3, C4, C5 and C6 reactors, respectively. Higher hydrogen yields were achieved with NiFe2O4 NPs containing reactors. Zhang et al. [56], using cobalt ferrate in their study and maximum hydrogen yield of 205.24 mL/g glucose was achieved with the 400 mg/L NPs addition. In another study conducted by Zhang et al. [45], the highest hydrogen production of 222 mL/g glucose was obtained with the 100 mg/L NiFe2O4 NPs. Compared to similar studies, higher hydrogen yields were achieved in this study. Taherdanak et al. [57] has also proven that higher hydrogen production (149.8 mL/g VS) potentials can be achieved using nickel NPs. In addition, dark fermentation was done with iron NPs in the same study. The use of both NPs has increased hydrogen production. It has been proven that higher hydrogen yields can be achieved by using NPs formed by the combination of nickel ferrite NP production and metals contributing to hydrogen production with this research.

Kinetic variables for fermentation sets were calculated with the Modified Gompertz Equation (Table 1). R2 values were calculated to validate the model data. According to kinetic variables and hydrogen yields, R2 values were ≥ 0.95. Additionally, the p-values for H2 and SMPs concentration calculated from the one-way ANOVA were not greater than 0.05 (p-H2: 0.042 and p-SMPs: 0.048), which indicated that the dosing of NPs at different levels had an obvious influence on the concentrations of hydrogen and metabolites. This showed that the experimental results could be modeled accurately with the Modified Gompertz Equation. The model data and the experimental results fit well. The usage of 200 mg/L of NiFe2O4 and CoFe2O4 resulted in peak hydrogen production rates of 17.17 and 16.18 mL/g/d, respectively. According to the results of the kinetic study, while the lag phase was 7.48 h in the control set, the lag phase was considerably shortened in the reactors containing NPs. Gadhe et al. [58] reported that in addition to the increase in hydrogen yield, the lag phase of fermentation shortened as a result of his work using nickel NPs. He stated that the reason for this development is that depending on the wide surface area of NPs, they improve the certain enzymes activities such as ferredoxin, hydrogenase and ferredoxin oxidoreductase proteins. In addition, the quantum size properties of NPs also provide some additional benefits.

Table 1 Modelling results for bio-hydrogen production sets
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Both nanoparticles achieved maximum biohydrogen production efficiency when applied at a concentration of 200 mg/L. Considering this positive effect of nanoparticles, their combined effect is also a matter of curiosity. However, it is clearly seen in Table 1 that a decrease in process efficiency begins at concentrations above 200 mg/L. If both NPs are used together, adverse effects will probably occur even if lower concentrations are applied. It was reported by Zhang et al. [56] that the release of trace metals such as Co, Ni and Fe in NPs into the fermentation liquid may create inhibitory effects. However, in lower concentration applications, significant increases in biohydrogen yield can be observed due to the combined effect. The present investigation determined that the cobalt and nickel nanoparticles (NPs) employed in this study exhibited inhibitory effects beyond a concentration threshold of 200 mg/L. In a parallel investigation [59] utilizing NiCo2O4 NPs, it was reported that an inhibitory effect manifested, impeding microbial activity at concentrations exceeding 400 mg/L, ultimately resulting in microbial mortality.

3.3 Effects of nickel ferrite and cobalt ferrite NPs on fermentation metabolism

During the experiments, the produced soluble microbial products are shown in Fig. 5. For control, the content of 1.10 g/L ethanol, 1.49 g/L acetic acid, 0.53 g/L propionic acid and 1.84 g/L butyric acid was determined. It was determined that the soluble metabolic products (SMPs) content of the N3 reactor, where the maximum hydrogen yield was obtained, was higher than the other reactors. Higher production of soluble metabolic products also results in higher hydrogen production. Total SMPS content was 4.9 g/L for control set. While the total SMPs amount was higher than the control for all reactors. NPs generally increase the substrate conversion rate according to results. Similar results were reported by researchers [45]. Within this context, it’s important to note that glucose can undergo divergent metabolic pathways leading to distinct end products. Specifically, glucose can be enzymatically transformed into acetic acid, as described by Equation (2), or alternatively, it can follow a separate enzymatic route resulting in the production of butyric acid, as outlined in Equation (3). This dual metabolic capability highlights the versatility of glucose utilization within the biochemical processes under consideration. When the appropriate amount of NPs is used, the formation of SMPs increases in the study. Simultaneously, the notable improvement in hydrogen production efficiencies indicates a substantial connection between the generation of SMPs and the production of hydrogen. This suggests a meaningful correlation between SMPs production and the enhanced hydrogen production observed.

$${\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6+2{\mathrm{H}}_2\mathrm{O}\rightarrow 2{\mathrm{C}\mathrm{H}}_3\mathrm{COOH}+2{\mathrm{C}\mathrm{O}}_2+4{\mathrm{H}}_2$$
(2)
$${\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6\rightarrow {\mathrm{C}}_3{\mathrm{H}}_7\mathrm{COOH}+2{\mathrm{C}\mathrm{O}}_2+2{\mathrm{H}}_2$$
(3)
Fig. 5
figure 5

The concentration of SMPs (a) NiFe2O4 containing reactors (b) CoFe2O4 containing reactors

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The use of NiFe2O4 NPs increased the production of SMPs more than CoFe2O4 NPs. SMPs results also reached its maximum in reactors where the highest hydrogen yield was obtained for both NPs. The maximum SMPs concentration for CoFe2O4 NP was 6.36 g/L (1.05 g/L ethanol, 1.82 g/L acetic acid, 0.47 g/L propionic acid and 3.03 g/L butyric acid) for the C3 reactor. In the N3 reactor, the SMP concentration was found to be 7.29 g/L (1.08 g/L ethanol, 1.92 g/L acetic acid, 0.79 g/L propionic acid and 3.50 g/L butyric acid). The reason why nanoparticles containing cobalt and ferrite show similar patterns is that both ions act to increase hydrogenase activity. Mishra et al. [60] reported that fermentative bacteria depend on nickel and cobalt ions for enzyme synthesis. Since both ions are necessary for enzyme synthesis and also play an active role in hydrogenase activity, adding NPs containing sufficient amounts of Ni and Co to the medium during fermentation will be beneficial in increasing fermentative hydrogen production efficiency. The superior performance of nickel ferrite compared to cobalt can be attributed to the direct integration of nickel ions within the hydrogenase structure. The inclusion of nickel in nickel ferrite enhances hydrogenase activity more significantly, leading to a more pronounced acceleration in catalytic performance when compared to cobalt-based counterparts.

Elevating the activity of hydrogenases directly contributes to the enhancement of hydrogen production [61, 62]. For this reason, the use of metallic substances containing Ni and Fe in the structure of hydrogenases in fermentation increases the hydrogenase efficiency. Metallic nanoparticles containing nickel and iron have been frequently used in studies in the literature [63,64,65]. According to SMP values, the use of metallic nanoparticles increases the ratio of butyric acid to acetic acid, allowing higher hydrogen production to be obtained. Similarly, an increase in the amount of butyric acid was observed in the study.

4 Conclusion

This study showed that olive leaf could be successfully used for nickel ferrite and cobalt ferrite nanoparticles production. Synthesized metalic nanoparticles by green route can increase the bio-hydrogen yield. It has been observed that the produced nanoparticles have an average size in the range of 200–400 nm. Both nanoparticles enhance the dark fermentation yield. Biohydrogen yield boosted 47% and 41% by nickel ferrite and cobalt ferrite nanoparticles, respectively. In prior researches, a variety of foliage has commonly been employed in the green synthesis approach. This research makes a valuable contribution to the existing body of literature by showcasing the potential for generating diverse nanoparticle types through the utilization of olive leaves, which could serve as a foundation for future investigations in this field.