pH-tailored monodispersed nanopolystyrene: environmental impact on PGPR and nitrogen cycling in agricultural soils

ABSTRACT

Emulsion polymerization was optimized to synthesize polystyrene nanoparticles (NPSt) with tailored sizes by adjusting pH and azobisisobutyronitrile (AIBN) content. An NPSt sample prepared with 20 mg AIBN at pH 8 showed the smallest size (88 nm) and narrowest distribution (24 nm peak width). XRD patterns indicated semi-crystalline nature for this optimized sample. The involvement of synthesized NPSt in the nitrogen (N) cycle enhanced our understanding of nitrogen transformations and the response of microbial populations to polystyrene nanoparticles in the environment. In vitro experiments revealed compatibility of NPSt with tested PGPR strains, with no inhibition zones observed. Furthermore, carrot growth in soil containing 400 mg/kg NPSt showed no significant changes in biomass or shoot height compared to control soil. This demonstrates the NPSt samples had no negative effects on PGPR or soil nitrogen transformations and bacterial communities. Overall, the study optimized the synthesis of NPSt, evaluated environmental impacts on soil nitrogen cycling and PGPR, and found no toxicity of NPSt on PGPR or carrot growth up to 400 mg/kg concentration. The insights contribute to understanding nanomaterial interactions in agricultural systems, highlighting the need to consider the environmental impacts of nanoparticles.

GRAPHICAL ABSTRACT

KEYWORDS:

• (AIBN)
• emulsion polymerization and agricultural soil

HIGHLIGHT

• Emulsion polymerization yielded nano polystyrene (NPSt).

• NPSt size is controlled by pH and AIBN content.

• In vitro study confirmed NPSt's safety for PGPR and soil nitrogen transformation.

1. Introduction

In recent decades, the field of nanopolymers has experienced tremendous growth and has emerged as a significant area of research and technological development. Nanopolymers, characterized by their nanoscale dimensions and unique properties, have captivated the attention of researchers and scientists from various disciplines. Their versatile nature has applications in various fields, including electronics, photonics, materials science, sensors, medicine, biotechnology, and environmental pollution prevention. Practically, the different synthesis methods, including evaporation of solvents, salting-out, dialysis, supercritical fluid method, micro- and mini-emulsions, surfactant-free emulsions, and interfacial polymerization, are indeed utilized in the synthesis of nanopolymers. These methods offer different approaches to achieve control over the size, structure, and properties of the resulting nanopolymers. Each method has its advantages and suitability for specific applications. Emulsion polymerization has emerged as a powerful green nanomaterials synthesis technique (1), enabled by developments like controlled/living radical polymerizations that provide precise control over polymer nanostructure and composition. Stimuli-responsive and inorganic–organic hybrid polymer nanoparticles can now be prepared via heterogeneous dispersion polymerization using bi-functional monomers, or encapsulation approaches (2). Microfluidic reactors and continuous flow methods offer scalable production of monodisperse particles. Bio-inspired dispersion polymerization within cell-derived vesicles or biopolymers allows biodegradable, non-toxic nanoparticles (3). Controlled polymerization, multicompartmental architectures, microfluidics, bio-inspiration and modeling have established dispersion polymerization as a versatile route for functional polymeric nanomaterials with applications in biomedicine, energy and environmental remediation (4).

Researchers choose the most appropriate method based on the desired nanoparticle characteristics, the nature of the polymer and monomers, and the targeted application. By employing these methods, scientists have created nanopolymers with tailored properties, opening up opportunities for various technological advancements (5–9). The chosen method depends on several variables, including temperature, nanoparticle size control, particle size distribution, and field of applications (10, 11). One of the simplest approaches is radiation polymerization because of its excellent penetrability and temperature independence (12–15). Certainly, chemical initiators play a crucial role in preparing monodisperse nanopolymer particles. Researchers can initiate and control the polymerization process by selecting appropriate initiators forming nanoparticles with uniform size and size distribution. Chemical initiators, such as azobisisobutyronitrile (AIBN) mentioned in your initial question, are commonly used to trigger polymerization reactions and produce nanopolymers with controlled characteristics (12, 16). Radiation is a co-friendly method with several benefits, such as an easy synthesis approach and a high-purity product (12, 17–23).

The emulsion polymerization process offers excellent control over particle size by manipulating various parameters, including the monomer concentration, surfactant/surfactant-free system, initiator concentration, and reaction conditions such as temperature and pH. By adjusting these parameters, researchers can achieve nanoparticles with a desired size range and narrow size distribution (24). Another method for nano-polystyrene Preparation is dispersion polymerization (25–27). In this study, we choose emulsion polymerization initiated by (AIBN) to produce nano-polystyrene with a well-defined monodispersed nanosphere. Using the water as a dispersed aqueous phase is environmentally sustainable and allows for excellent free radical production during polymerization. It may be graded as traditional or surfactant-free emulsion polymerization, depending on the use of a surfactant. Accounts for most emulsion polymerization processes the conventional emulsion polymerization is based on production worldwide (28, 29).

The parameters e.g. surfactant, pH and amount of (AIBN), play important roles in the size distribution of nanopolymer formation (30–32). The ingredients comprise water, non-polar monomers, aqueous soluble initiator materials, and a surfactant in the conventional heterogeneous system. High-energy radical transfer of energy or charge in one phase into the monomer molecule to transfer across the interface to an initiating radical at the water phase and form solid particles of nano polymer. These particles have a large surface-to-volume ratio, making absorbing active substances easier.

Soil microplastics are emerging as a growing agricultural issue globally. Research emphasizes the need to understand their impacts alone and combined with other stresses on crop productivity (33, 34). Microbially-assisted strategies show promise for more sustainable plastic-polluted agricultural systems (35). Soil microplastics can negatively impact crop growth by altering the soil environment and properties (36). Microplastic contamination influences soil enzymatic activity, nutrient availability and microbial community. Studies showed microplastics inhibited root growth, biomass accumulation and physiological processes in crops like wheat and rice (37). Effects were more severe under combined stresses like heavy metal toxicity or invasive plant presence (38). Microplastics can act as vectors, facilitating the spread and establishment of invasive alien plants by influencing root traits and competitiveness of native crops. This can advance sustainability goals. Future research should focus on elucidating molecular mechanisms of microplastic-crop interactions. Multi-stressor impacts under field conditions also need evaluation. Beneficial microbial associations could mitigate plastic effects (39).

This work aims to study the different preparation parameters, e.g. surfactant, pH and (AIBN) amount in the size distribution of nanopolymer formation. Here the surfactant is used as an emulsifier to polymerize the styrene monomer in the emulsion polymerization reaction initiated by (AIBN) and investigates the impact of pH and amount of (AIBN) on the size of NPSt nanoparticles formation by DLS and TEM techniques.

The resulting novelty in synthesized nanopolystyrene has been characterized in terms of environmental risk. The term ‘nanopolystyrene’ does not meet environmental sense and should be eliminated. Besides, some particles cause nitrogen transformation, so in this article, the effect of different-sized polystyrene nanoparticles was examined according to different kinds of growth-promoting rhizobacteria (PGPR) named Bacillus subtilis SBMP4, Achromobacter xylosoxidans NBRC15126 and Lysinibacillus fusiformis NBRC15717 were performed. This study demonstrates the successful synthesis of controllable NPSt through emulsion polymerization under ambient conditions. The pH-dependent size modulation provides a means to tailor NPSt properties. At the same time, evaluating their environmental impact on PGPR and nitrogen cycling highlights their potential for safe utilization in agricultural soil. These findings contribute to our understanding of nitrogen transformations and the microbial population responses to polystyrene nanoparticles in the environment. We hypothesize that the impacts of (NPSt) on red carrot development will depend on its effect on growth-promoting rhizobacteria (PGPR) named Bacillus subtilis SBMP4, Achromobacter xylosoxidans NBRC15126 and Lysinibacillus fusiformis NBRC15717. Thus, the effects of nanopolystyrene on the red-carrot plant growth were investigated.

2. Experimental

2.1. Materials

All the chemical compounds were used in high purity. Styrene monomer (99.5%) was supplied by (Acros Organics), Brussel, Belgium and kept at 4 ^oC. polyoxyethylene sorbitan monooleate (tween 80) Density: 1.064 g/cm³, purity is 99%, azobisisobutryonitrile (AIBN) and N, N-dimethylformamide (DMF) 99% were provided by (Sigma-Aldrich Co.), Germany. Sodium dodecyl sulfate (SDS) was of the grade for biochemical use (Merck Co.), Rahway, USA.

2.2. Preparation of nanoparticles polystyrene by emulsion polymerization using azobisisobutryonitrile (AIBN)

Scheme 1 shows the emulsion polymerization process for the preparation of nanoparticle polystyrene. The styrene monomer (4 ml, 3.624 g, 34.80 mmol) is taken and dispersed in a mixture of 18 ml deionized water and DMF (50:50 vol.%). The purpose of using a mixture of water and DMF is to control the reaction conditions and solubility of the monomer. The dispersion is typically carried out in a reaction flask. The monomer dispersion is bubbled with nitrogen gas for 2 min. Bubbling with nitrogen helps to remove oxygen from the system, which can interfere with the polymerization process. Sodium dodecyl sulfate (SDS) is added to the dispersion (1 g, 3.46 mmol). Tween 80 is added to the dispersion (1 ml, 1.07 g). Both surfactants, SDS and Tween 80, stabilize the monomer droplets and prevent coalescence during polymerization. The surfactants are absorbed on the surfaces of the styrene monomer droplets, creating a protective layer.

Scheme 1. The procedure of NPSt nanoparticle preparation by the emulsion polymerization process.

After the addition of surfactants, the mixture is subjected to sonication. Sonication helps further disperse and homogenize the monomer droplets in the aqueous phase, promoting the formation of a stable emulsion solution. The sonication process typically involves applying high-frequency sound waves to the mixture, breaking larger droplets into smaller, more uniform-sized droplets. As a result of the above steps, the mixture transforms into a white, stable emulsion solution. The emulsion solution is now ready for further processing and initiation of the polymerization reaction. Thus, in an emulsion polymerization system, the monomer droplets are reduced due to micelles’ formation and migrating monomers to the droplet inside. By reducing the size of the monomer droplets through the formation of micelles and facilitating the migration of monomers to the droplet interiors, the emulsion polymerization system ensures a higher surface area-to-volume ratio, which promotes efficient polymerization and the formation of nanoparticles with controlled size and morphology (40–42). The temperature was maintained at 20°C throughout the process. The pH of the emulsion solutions was adjusted using hydrochloric acid (HCl) solution (0.1M) to obtain a pH range from 1 to 8. The pH adjustment is important as it can influence the particle size, transparency, and stability of the resulting nanopolystyrene. After pH adjustment, the emulsion solutions were subjected to sonication to achieve a stable emulsion. Sonication helps break down larger droplets and promotes uniform dispersion, resulting in a stable emulsion. Each emulsion solution of styrene monomer was mixed with 20 mg of azobisisobutryonitrile (AIBN), a chemical initiator. The polymerization process took place at 77°C for 2 h, allowing the styrene monomers to polymerize and form nanopolystyrene particles.

X-ray diffraction (XRD) measurements were conducted by converting the suspension particles from the aqueous phase to the solid phase using spin coating. A thin layer of the nanopolystyrene sample was formed on a glass slide using a spin caster at a speed of 1200 RPM for 40 s. The samples were then dried in a desiccator under dry atmospheric conditions. The resulting dried samples on glass slides were subjected to XRD analysis to investigate their crystal structure and composition. Based on the observations, it was found that the transparency of the nanopolystyrene samples increased from pH 1 to pH 8. This indicates that the pH of the emulsion solution impacts the particle size and optical properties of the synthesized nanopolystyrene. It's important to note that the experimental details provided are specific to the described process, and adjustments can be made based on the desired outcomes and experimental requirements.

2.3. Nanoparticles polystyrene (NPSt) analysis

Several analytical techniques were employed to investigate the morphology and particle sizes of the synthesized nanopolystyrene (NPSt), for TEM analysis, such as the Transmission Electron Microscope (TEM) of the model FEI-CM30 electron microscope. TEM allows for high-resolution imaging of the nanopolystyrene particles, providing information about their morphology, size, and shape. The influence of pH and AIBN content on the particle sizes of NPSt was determined using TEM. The particle size distributions of the polystyrene nanoparticles (NPSt) were analyzed using the Malvern Zetasizer (DLS), ZS, ZEN3500 instrument from the UK. DLS is a widely used technique for measuring the hydrodynamic size and size distribution of particles in suspension. DLS provides information about the sample's average particle size and polydispersity by analyzing the scattering of light from the particles. XRD analysis was performed on the solid film of the NPSt suspension formed on glass slides. The XRD patterns were measured using a JEOL JDX-8030 X-ray diffractometer system. XRD helps determine the crystal structure, orientation, and phase composition of the nanopolystyrene particles. By analyzing the diffraction pattern, valuable information about the arrangement of atoms and the material's crystallinity can be obtained. The thermal stability and decomposition behavior of the nanopolystyrene structure were investigated using thermogravimetric analysis. TGA measurements were conducted using a TGA 550 instrument from TA Instruments. TGA provides information about the thermal stability, weight loss, and decomposition temperatures of the nanopolystyrene sample. It helps in understanding the thermal properties and behavior of the synthesized nanopolystyrene. These analytical techniques collectively provide comprehensive information about the morphology, particle size, crystallinity, and thermal properties of the synthesized nanopolystyrene. By combining the results from TEM, DLS, XRD, and TGA analyses, a thorough characterization of the NPSt samples can be achieved.

2.4. In vitro antagonistic experimental of (NPSt) toward the transformation of nitrogen and communities of rhizobacteria (PGPR) in an agricultural soil

To assess the interactions between the four tested samples of nanopolystyrene (NPSt) prepared at different pH levels (pH 1, 3, 5, and 8) and the rhizobacteria (PGPR), a dual-culture study was conducted in vitro. The specific strains of rhizobacteria used in the study were Bacillus subtilis SBMP4, Achromobacter xylosoxidans NBRC15126, and Lysinibacillus fusiformis NBRC15717, which were previously identified and reported in our published paper (43). Here is a summary of the experimental procedure. The source of Rhizobacteria from the soil in a plant area, serving as the primary source of the rhizosphere, was collected (15.0 g). A suspension was prepared by adding 100 mL of sterile distilled water to the soil. Serial dilution was performed using the suspension to obtain varying concentrations (10⁻²–10⁻⁶). Nutritional agar medium was used, and 0.1 mL of the diluted solution was cultivated on sterile Petri plates. The plates were incubated below 36°C for 24 h for bacterial growth. Each bacterial strain (Bacillus subtilis SBMP4, Achromobacter xylosoxidans NBRC15126, and Lysinibacillus fusiformis NBRC15717) was individually planted on Luria broth (LB) plates. A sterile scalpel was used to cut a portion (10 mm) from the culture plate, and sterile discs with a diameter of 6.0 mm were infected with the tested NPSt samples. Control plates without bacterial strains were also prepared. The plates were initially incubated for 16 min at temperatures below 4°C to allow for the distribution of the tested samples. Subsequently, the plates were incubated for 24 h at temperatures below 36°C to allow the tested PGPR strains to grow.

The antibacterial efficacy of the tested NPSt samples against the isolated PGPR strains was assessed through triplicate analysis. The antibacterial activity of amoxicillin/clavulanic acid (AMC), a standard antimicrobial, was also tested as a positive control. The dual-culture study aimed to investigate the antibacterial activity of the tested NPSt samples against the identified rhizobacteria strains. By comparing the growth and interactions observed on the plates, the antibacterial efficacy of the NPSt samples could be evaluated. The triplicate analysis ensured the reliability of the results obtained. It's worth noting that this study provides insights into the potential effects of NPSt on the tested rhizobacteria strains and their growth. The results obtained from this experiment contribute to our understanding of the interaction between nanopolystyrene and microbial populations in the soil environment.

3. Results and discussion

3.1. Effect of pH value on the particle size and activation energy of nano polystyrene

The activation energy of nanopolystyrene is influenced by the particle sizes, which are controlled by the pH of the solvent medium. The adsorption interaction between styrene monomer and surfactants is crucial in this process. Changes in the medium's pH significantly affect the size distribution of nanopolystyrene particles. Figure 1a-d illustrates the relationship between the particle size of NPSt and the pH values of the emulsion solution. The data shows that as the pH increases from 1 to 8, there is a corresponding increase in the particle size of NPSt. At pH 1, the particle size is 10 nm, while at pH 3, 5, and 8, the particle sizes are 45, 70, and 94 nm, respectively. This trend indicates that the pH of the emulsion solution influences the particle size of polystyrene nanoparticles. As the pH increases, there is a tendency for larger particles to form. This dependency can be attributed to the variations in the adsorption interaction between the styrene monomer and the surfactants in the emulsion system. The findings presented in Figure 1a-d provide valuable insights into controlling polystyrene particle size through pH adjustment in the emulsion polymerization process. By manipulating the system's pH, it is possible to tailor the particle size of NPSt, offering opportunities for precise control and optimization of nanopolystyrene synthesis. The dependence of polystyrene particle size on the pH of the emulsion solution can be explained by the interaction between the styrene monomer and the surfactants present in the system.

Figure 1. TEM images of NPSt were prepared using 20 mg (AIBN). and a pH of emulsion solutions was 1 (a), 3 (b), 5 (c) and 8 (d). and (e) SEM image of PS NPs of pH8.

At low pH values (acidic conditions), excess protons in the solution promote the formation of positive charges on the surface of the polystyrene particles. These positive charges facilitate the adsorption of precursor chains in the aqueous environment and the subsequent growth of a stable layer of surfactants on the surface of the styrene monomer. The resulting polystyrene particles acquire a negative charge due to the presence of the surfactants.

As the pH of the emulsion solution increases, the concentration of protons decreases, reducing the positive charge on the poly styrene particles’ surface. This decrease in positive charge weakens the interaction between the styrene monomer and the surfactants, reducing the binding between the surfactant and the precursor chains of polystyrene to give a bigger overall particle size. In other words, the pH of the emulsion solution affects the adsorption behavior of the surfactants on the styrene monomer and subsequently influences the growth and aggregation of the polystyrene particles. Higher pH values promote weaker adsorption and slower growth, resulting in larger particle sizes, while lower pH values enhance stronger adsorption and faster growth, leading to smaller particle sizes. Therefore, the particle size of NPSt can be controlled by adjusting the pH of the emulsion solution, providing a means to regulate and optimize the synthesis of nanopolystyrene particles with specific sizes for various applications.

Figure 2 a-d displays DLS curves and provides insight into the distribution's average particle diameter (dH) and peak width. The average particle diameter was measured as 88, 106, 121, and 133 nm for pH values 1, 3, 5, and 8, respectively. The corresponding peak widths were 24, 32, 35, and 38 nm. These results indicate that particle aggregation increases as the pH rises. This phenomenon can be attributed to the excess protons present at low pH, which promote the formation of positive charges on the surface of styrene particles. These positive charges facilitate the formation of precursor chains in the aqueous environment, stabilizing the two surfactants on the surface of the styrene monomer. It is worth noting that polystyrene carries a negative charge in this environment. The observed increase in aggregation with pH is consistent with the findings of Fuminori (41). Fuminori's research likely explored similar factors influencing the aggregation behavior of nanoparticles concerning pH variations. The increase in aggregation can be attributed to the changes in the surface charge and interparticle interactions as the pH of the emulsion solution increases. At lower pH values, the excess protons lead to a higher concentration of positive charges on the surface of the styrene particles. These positive charges can contribute to electrostatic repulsion between the particles, preventing significant aggregation. However, as the pH increases, the concentration of protons decreases, resulting in a reduced positive charge on the particle surfaces. This decrease in positive charge weakens the repulsive forces between the particles, making them more prone to aggregate. The weakened electrostatic repulsion allows for closer proximity and interparticle attraction, forming larger aggregates. The findings of Fuminori likely support this understanding of the relationship between pH and particle aggregation. Their research may have provided further experimental evidence or theoretical explanations for the observed phenomenon. The result, the increase in particle aggregation with increasing pH is a consistent observation. The interplay between the emulsion system's surface charge, electrostatic forces, and interparticle interactions can explain it.

Figure 2. The TGA thermogram of all polystyrene nanoparticles samples (a) Plot of $\ln [\ln (w_o-w_e\left)/\right(w_t-w_e\left)\right]$versus θ for polystyrene nanoparticles samples (b).

Figure 2e shows a low magnification scanning electron microscope (SEM) image of the NPSt sample slide at pH 8, revealing large-scale particles with beautiful and curly structures. These structures exhibit symmetrical arrangements, suggesting a well-organized and ordered assembly of NPSt particles. The observed symmetrical arrangement and well-dispersed nature of the NPSt structures indicate that the particles can align themselves in an ordered manner and self-assemble into cohesive structures. This self-assembly behavior indicates the nanoparticles’ ability to interact and organize themselves based on their inherent properties and the surrounding environment. The self-assembly of NPSt particles into well-dispersed structures is an intriguing phenomenon with potential applications in various fields, including materials science and nanotechnology. The ability to control and manipulate the self-assembly process of nanoparticles can lead to the development of advanced materials with tailored properties and functionalities. In summary, the size and morphology of nanopolystyrene particles are influenced by the pH of the emulsion solution. The particle size increases with increasing pH, and aggregation becomes more prominent. The observed self-assembled structures further confirm the monodispersed nature of the prepared NPSt samples.

The thermal stability of the obtained nanopolystyrene (NPSt) samples prepared at different pH values (1, 3, 5, and 8) was investigated using thermogravimetric analysis (TGA) and derivative thermogravimetric analysis (DTGA) techniques. TGA measures the weight changes of a sample as it is heated, providing information about its thermal decomposition behavior.

Figure 2a displays the TGA curves of NPSt samples over a temperature range from 30°C to 600°C. All the TGA curves exhibit a single degradation step, indicating that the thermal decomposition of NPSt occurs in a relatively narrow temperature range. Additionally, the absence of any residue after decomposition suggests that the NPSt samples are homogeneous and do not leave behind any significant by-products.

The characteristic temperatures of the thermal decomposition process, including the beginning of decomposition (T_i), the end of decomposition (T_f), the temperature at the maximum rate of decomposition (T_max), and the temperature at which 10% weight loss occurs (T₁₀), were determined from the TGA and DTGA data. T_i represents the threshold decomposition temperature and provides insight into the material's thermal stability. By analyzing Ti and T_f, as well as other parameters, it is possible to calculate kinetic coefficients related to the decomposition reactions, which can further elucidate the thermal behavior of nanopolystyrene during thermal pyrolysis. Table 1 summarizes the TGA data, including T_i, T_f, T_max, and T₁₀, for the NPSt samples prepared at different pH values. These data provide valuable information about the thermal stability and decomposition characteristics of the nanopolystyrene samples under the influence of heat. The TGA and DTGA analyses are important tools for assessing nanomaterials’ thermal stability and behavior, enabling researchers to understand their performance and potential applications in various fields, including materials science, polymer chemistry, and engineering.

Table 1. Temperature, weight loss characteristics and the energy of activation (Ea) of NPSt at different pH.

	Temperature [^oC]				Δw [%]	v [%/oC]	Ea [kJ mol−1]
	Ti	Tf	Tmax	T10
pH 1	392	446	422	385	85.74	1.59	138.2
pH 3	391	440	418	377	75.14	1.53	127.6
pH 5	386	436	420	360	74.61	1.49	123.4
pH 8	385	436	421	336	72.62	1.42	122.5

Where Δw % is the weight change between TS and TE, ΔT = Ti – Tf, $v={\displaystyle \frac{\mathrm{\Delta }w}{\mathrm{\Delta }T}}$ is the average specific decomposition rate in %/^oC (44). The higher the values of T₁₀ and T_max, the higher the thermal stability of the material such as nanopolystyrene (45,46). Based on the data in Table 1, it can be observed that the thermal stability of NPSt at pH 1 is higher compared to the samples at higher pH values. This trend indicates that as the pH increases, the thermal stability of NPSt decreases.

The higher thermal stability of NPSt at pH 1 can be attributed to the smaller particle size or dimension of NPSt. Smaller particles have a larger surface area-to-volume ratio, which can enhance their thermal stability. The increased surface area allows for better heat dissipation and reduced mass transfer limitations during decomposition. The improvement in specific surface area for smaller NPSt particles leads to increased interactions between the polymer chains and the surrounding medium, which can result in stronger intermolecular forces and higher thermal stability. Therefore, the smaller size of NPSt particles at pH 1 contributes to their higher thermal stability compared to the samples with larger particle sizes at higher pH values (18, 47). The enhancement of thermal stability for the smaller particle size of NPSt can be explained because of its very high surface areas (19, 48–50). Similar results were obtained for PS-based nanocomposites dispersed with various nanofillers (51–53).

Kinetic parameters of thermal degradation of nanopolystyrene are determined using Horowitz–Metzger's methods to investigate the degradation behavior for all samples. Horowitz–Metzger method was used to calculate the activation energy from the obtained TGA data (51).

The Horowitz–Metzger (HM) relation used to evaluate the degradation kinetics is: (1) $\ln [\ln (w_o-w_e\left)/\right(w_t-w_e\left)\right]=E_a\theta /R{T}_{max}^2$(1) where W_o, W_e and W_t are the initial, final and remaining weight of the nanopolystyrene sample, at a given temperature (T), respectively. E_a is the activation energy, θ = T – T_max and R is the gas constant (R = 8.314 J.K-1mol⁻¹). The plot of $\ln [\ln (w_o-w_e\left)/\right(w_t-w_e\left)\right]$versus θ for all samples is presented in Figure 2b which gives a straight line whose slope is equal to$E_a/R{T}_{max}^2$.

E_a was calculated for all samples and presented in Table 1. The obtained E_a values are 138.2, 127.6, 123.4 and 122.5 kJ/ mol for pH1, pH3, pH5 and pH8 StNP samples, respectively. The activation energy is higher for the smaller particle size samples and gradually decreases as the particle size increase.

The decrease in activation energy (E_a) as the particle size of NPSt increases can be attributed to the effect of particle size on the diffusion and mobility of reactant molecules during the thermal decomposition process.

The surface-to-volume ratio is higher than larger particles in smaller particle size samples. This means that a larger proportion of the polymer chains is exposed to the surface, where the decomposition reactions occur. The increased surface area facilitates the diffusion of reactant molecules and allows for a higher frequency of collisions between the molecules, leading to more efficient decomposition. Due to the smaller particle size, the reactant molecules have shorter diffusion distances to cover, which reduces the energy barrier for their movement and increases their mobility. As a result, the activation energy required for the reactant molecules to overcome the energy barrier and initiate the decomposition reactions is lower.

On the other hand, in larger particle-size samples, the diffusion distance for reactant molecules becomes longer, and their mobility is relatively reduced. This leads to a higher activation energy required for the reactant molecules to diffuse and reach the reaction sites on the particle surface, resulting in a higher E_a value. Therefore, the observed trend of decreasing activation energy as the particle size increases can be attributed to the enhanced diffusion and mobility of reactant molecules in smaller particle size samples, making the decomposition reactions more favorable and requiring less energy to initiate.

3.2. Effect of (AIBN) content on size distribution of nanopolystyrene (NPSt)

The results obtained from dynamic light scattering (DLS) measurements and TEM images indicate that the particle size of nanopolystyrene (NPSt) is influenced by the content of azobisisobutyronitrile (AIBN) used in the emulsion polymerization process. Figure 3(a, b, and c) shows the size distribution histograms of NPSt prepared with different AIBN contents (10, 20, and 30 mg). The average particle diameter (dH) obtained for each case was 170, 88, and 143 nm, respectively. For the case of 20 mg of AIBN, the NPSt particles were smaller than the samples prepared with 10 and 30 mg of AIBN. This can be attributed to the cross-linkage degree, which is the lowest for the sample prepared with 10 mg of AIBN. With a lower cross-linkage degree, the polymerization rate is slower, resulting in smaller particle sizes.

Figure 3. DLS and the TEM images of the NPSt were prepared at different content of (AIBN) of emulsion polymerization (a) 10 mg, (b) 20 mg and (c) 30 mg. prepared at pH of emulsion solution.

On the other hand, increasing the content of AIBN to 30 mg favored the generation of more radicals, which enhanced the polymerization rate compared to the 10 mg and 20 mg cases. This increased polymerization rate leads to larger particle sizes for the NPSt prepared with 30 mg of AIBN. Therefore, the content of AIBN in the emulsion polymerization process affects the cross-linkage degree and the rate of polymerization, which in turn influences the particle size of NPSt. A lower AIBN content results in smaller particle sizes, while a higher AIBN content leads to larger particle sizes. Also, the coinciding of the three distribution curves modes (intensity, number and volume) given by using Zetasizer software was observed to suggest that all the NPSt grew nearly homogeneously. The multimodal size distribution of suspension NPSt prepared by 20 mg is typical and coincides with the three distribution curves (intensity, number and volume) with a width 24 nm. Therefore, the mono distribution and very homogeneous spherical shape were formed at nearly 100% at 20 mg rather than 10 and 30 mg with width peaks 77 and 38 (nm), respectively. The results from TEM images further support the findings obtained from DLS measurements. In the case of NPSt prepared with 20 mg of AIBN, the TEM images reveal regularly shaped spherical particles with nearly uniform spherical forms. The average sizes of these particles range from 10 nm to 15 nm, indicating a narrow size distribution (Figure 3b). In contrast, for the emulsion polymerizations carried out with 10 and 30 mg of AIBN (Figure 3a and c), the average diameter of the nanopolystyrene particles was 200 nm and 150 nm, respectively. These particles also exhibited narrow size distributions with a relative standard deviation (RSD) below 10%. Overall, the TEM images confirm that 20 mg of AIBN to prepare NPSt resulted in particles with narrow size distribution and regular spherical morphology. In contrast, the use of 10 and 30 mg of AIBN led to slightly larger particle sizes but still maintained a narrow size distribution. These results highlight the importance of the AIBN content in controlling the size and distribution of NPSt particles, with the optimum conditions achieved at 20 mg of AIBN.

3.3. Effect of content of (AIBN) on crystallinity of nanopolystyrene (NPSt)

The crystallinity of the NPSt obtained after polymerization with 10, 20 and 30 mg content of (AIBN) are displayed in Figure 4 several information can be obtained. The presence of two diffraction peaks at 2Θ = 6.2˚ for the (110) plane and at 2Θ = 20.5˚ for the (211) plane indicates the crystalline state of syndiotactic polystyrene (sPS). In syndiotactic polystyrene, the polymer chains are arranged in a regular, alternating pattern of stereochemical isomers. This arrangement leads to the formation of crystalline regions within the polymer structure. The diffraction peaks observed in the X-ray diffraction (XRD) pattern correspond to the specific crystal planes of sPS.

Figure 4. XRD of NPSt is prepared by(a) 10 mg, (b) 20 mg, and (c) 30 mg of (AIBN).

The peak at 2Θ = 6.2˚ corresponds to the (110) crystal plane, associated with stacking polymer chains in the crystal lattice. The peak at 2Θ = 20.5˚ corresponds to the (211) crystal plane, which represents the arrangement of polymer chains in another direction within the crystal lattice. The presence of these diffraction peaks confirms the crystalline nature of syndiotactic polystyrene and provides information about the crystallographic structure and arrangement of polymer chains in the material (54, 55). The XRD analysis of the NPSt samples prepared with different contents of (AIBN)s reveals some changes in the diffraction patterns compared to the initial monomer. In Figure 4 (b, c, d), the peak at 2Θ = 6.2˚ corresponding to the (110) crystal plane is observed with low intensity, indicating a decrease in the crystallinity of the NPSt samples compared to the syndiotactic polystyrene (sPS) monomer. However, the positions of the diffraction peaks remain unchanged, suggesting that the material's crystal structure is still present. In addition, a significant broadening of the diffraction line at 2Θ = 20.5˚ corresponding to the (211) crystal plane is observed, and the extent of broadening varies with the content of (AIBN)s. This broadening indicates a decrease in the order and size of the crystalline regions within the NPSt samples as the (AIBN) content increases.

On the other hand, three strong diffraction peaks are observed in Figure 4b, where NPSt is prepared with 20 mg of (AIBN). The peak at 2Θ = 9.3˚ corresponds to the (210) crystal plane, the peak at 2Θ = 14.07˚ corresponds to the (220) crystal plane, and the peak at 2Θ = 16.38˚ corresponds to the (400) crystal plane. These diffraction peaks indicate the presence of a semi-crystalline state in the NPSt sample prepared with 20 mg of (AIBN), suggesting a higher degree of order and crystallinity than the other samples. The XRD results suggest that the crystallinity and degree of order in the NPSt samples are influenced by the content of (AIBN)s, with higher (AIBN) content leading to lower crystallinity and larger changes in the crystal structure (54, 55). The presence of large humps centered at 2Θ = 20.3˚ for the NPSt sample prepared with 10 mg of (AIBN) and at 2Θ = 20.11˚ for the sample prepared with 30 mg of (AIBN) indicates the presence of an amorphous phase in these samples. This peak is characteristic of the amorphous state of polystyrene, where the polymer chains are randomly arranged and lack long-range order. In contrast, the absence of such large humps in the NPSt sample prepared with 20 mg of (AIBN) suggests a higher degree of crystallinity in this sample. This could be attributed to the smaller particle size obtained for NPSt when using 20 mg of (AIBN). Smaller particle sizes often lead to a higher surface-to-volume ratio, promoting the formation of more ordered structures and enhancing crystallinity. Therefore, observing a more crystalline state in the NPSt sample prepared with 20 mg of (AIBN) compared to the samples prepared with 10 and 30 mg may be attributed to the smaller particle size obtained in the former case. Table 2 represents the particle size and crystal size of obtained StNPs in three contents of (AIBN) (10, 20 and 30 mg). The crystal size is calculated from XRD data using the Scherrer formula and the value of FWHM, peak position and x-ray wavelength used. $\mathrm{D}=\mathrm{k}\lambda /\text{β}\mathrm{c}\mathrm{o}\mathrm{s}\theta$where D denotes the crystallite size in nanometers; k denotes the form factor constant with a value of 0.89; β denotes the full width at half maximum in radian; λ is the wavelength of the X-ray that is 1.540598 nm for Cu-target Kα radiation, and θ is the angle of the Bragg diffraction.

Table 2. summarizes the particle size of NPSt obtained by three content of (AIBN) of 10, 20 and 30 mg.

NPSt/ (AIBN) mg	(dH) nm	Crystal size (nm)	Diameter Size (nm)
10	170	52	200
20	88	22	10
30	143	43	150

3.4. Impact of (NPSt) on the growth of red carrot plant

The environmental effect of nano polystyrene (NPSt) needs further investigation. whereas, soil and crops may be directly affected by the (NPSt) when used in different applications when accumulated in the soil. In this section, red carrot is grown in two pots with pristine soils as the control sample (Figure 5a) and soil amended with 400 mg/kg of a mixture of nano polystyrene (NPSt) (Figure 5b) until full-red carrot plant maturity. As shown in Figure 5 c and d the fresh leaf weight of red carrots remained unchanged in soil sample treatment concerning the control sample. In addition, Figure 5d shows no significant change in the high shoot of red carrots in soil sample treatment concerning the control sample. A possible explanation of the unremarkable change of red carrot leaf biomass by both soil samples this linked to the root nitrogen assimilation improvement. The micro-organisms responsible for fixed nitrogen in soil play important roles in plant growth and preservation, improving plant photosynthetic activity due to nitrification. Under aerobic conditions, nitrifying bacteria convert NH₄⁺-N to NO₃^--N, a process known as nitrification. The NO₃^--N content rise and the higher absolute abundances of the functional genes amoA and nxrA proved accelerated nitrification (56, 57).

Figure 5. Shows the growth of red carrot in (a) pristine soils as a control sample and (b) soil amended with 400 mg/kg of a mixture of nano polystyrene (NPSt), (c) leaf biomass (gm) and (d) plant height of red-carrot (cm) of the control soil sample and treated sample of (NPSt) 400 mg/kg.

3.5. Impact of (NPSt) toward transforming nitrogen and rhizobacteria (PGPR) bacterial communities in agricultural soil

Nitrogen is a critical nutrient for sustaining agricultural soil health and crop productivity. However, the information on the effects of nanopolystyren on soil nitrogen cycling and transformation is limited. This section demonstrated the effect of (NPSt) on nitrogen transformation and promoted nitrification. According to the relative abundances microorganisms in healthy agricultural soil, we choose three growth-promoting rhizobacteria (PGPR) three bacterial strains named Bacillus subtilis SBMP4, Lysinibacillus fusiformis NBRC15717, and Achromobacter xylosoxidans NBRC15126. The inhibitory influence of the tested (NPSt) samples (at four pHs = 1, 3, 5, and 8) on the identified three bacterial strains was tested following the addition of (NPSt) samples. The antagonistic action was estimated according to its capacity to hinder the extension of the bacterial colonies within the control plates (Figure 6 a–c). The test effects of in vitro antagonistic experimental of (NPSt) toward the transformation of nitrogen and communities of rhizobacteria (PGPR) in an agricultural soil indicated that there was no zone of inhibition (ZOI) throughout the four tested (NPSt) samples, thereby confirming that the incorporated sample at different pH, did not show any adverse effects on PGPR, demonstrating the safety of its use in agricultural settings. In both the below- and above-ground parts of the crops, creating plant origins by non-pathogenic bacteria could provide a definite wide-spectrum of defense response. Several advantages of Achromobacter species have also been mentioned. The two advantages are the creation of glutaryl-3-deacetoxy-7-aminocephalosporanic acid acylase, a key enzyme in manufacturing antibiotics like cephalosporin, and the stimulation of ionic transport to increase plant maturity.

Figure 6. Represents three bacterial strains named as (a) Bacillus subtilis SBMP4, (b) Lysinibacillus fusiformis NBRC15717, and (c) Achromobacter xylosoxidans NBRC15126.

4. Conclusions

The production of nanopolystyrene in a spherical shape was achieved using emulsion polymerization techniques. Preparation of nanopolystyrene using the emulsion polymerization method by applying different content of (AIBN) and pH values gives the different sizes of nanopolystyrene NPSts. The results indicate that with the increased pH value, the size of the produced particle also increases. While the content of (AIBN) for 20 mg is more suitable for producing spherical nanopolystyrene with a diameter of 10 nm compared with 200 and 150 nm at 10 and 30 mg, respectively. The obtained results from XRD, DLS and TEM analyses have shown the following. (i) Modifying the size and shape of the content of (AIBN) is successful. (ii) The crystallinity of nanopolystyrene in cases 20 mg is more than 10 and 30 mg. In addition, these emulsion phases were shown to be capable of solubilizing styrene monomers and could withstand the subsequent polymerization of styrene. Supported by X-ray measurements, remarkable crystalline particles were successfully obtained with crystal size of 52, 22 and 43 nm of StNPs at AIBN content of 10, 20 and 30 mg, respectively. DLS data shows polystyrene particle (NPSt) nucleation increased with pH. This is due to low pHs (acidic), excess protons promoted the positive charge formation on the surface of polystyrene particles, which aided the formation of precursor chains in the aqueous environment growing to the stable layer of the two surfactants on the surface of St monomer where polystyrene is negatively charged this confirmed the aggregation of NPSt was observed with pH rise. In addition, this study investigated the environmental effects of nanopolystyrene (NPSt) on soil nitrogen transformation and its compatibility with beneficial rhizobacteria in agricultural systems. The results demonstrated that NPSt promoted nitrification, which could impact soil nitrogen availability and nutrient cycling. However, the tested NPSt samples showed no inhibitory effects on the growth-promoting rhizobacterial strains, indicating their compatibility and safety in agriculture. This suggests that NPSt, when used at appropriate concentrations, may not pose significant risks to soil microbial communities crucial for plant growth and nutrient cycling. Furthermore, the study highlighted the potential benefits of Achromobacter species, including their involvement in antibiotic production and stimulation of ionic transport for improved plant growth. These findings contribute to our understanding of the environmental implications of NPSt and its safe application in agricultural practices. This study confirms the eco-friendly effect NPSt as proved by planting experimental red carrots. After tested NPSt against three bacterial strains named as (a) Bacillus subtilis SBMP4, (b) Lysinibacillus fusiformis NBRC15717, and (c) Achromobacter xylosoxidans NBRC15126. The obtained results show no visible inhibition zone was observed and NPSt not had significant effects on nitrogen transformation.

Authors’ contributions

Abeer S. Meganid: Conceptualization, Methodology, Investigation, Validation, Formal analysis, Writing – original draft, Writing – review & editing. Mohamed Mohamdy Ghobashy: Validatio . Dalal Mohamed Alshangiti: Methodology, Investigation, Writing – original draft. Sheikha A. Alkhursani: Methodology, Investigation, Writing – original draft. Samera Ali Al-Gahtany: Methodology, Investigation, Mohamed Madani, Writing – original draft, Writing – review & editing.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Disclosure statement

No potential conflict of interest was reported by the author(s).