A facile biodegradation of polystyrene microplastic by Bacillus subtilis

1 Introduction

Since its discovery in 1833 when Frenchman Henri Braconnot first stumbled upon this new material, plastic has emerged as a popular substitute for traditional materials like glass, metal, and wood [1,2,3]. Plastic pollutants disintegrate in the natural environment by physical, chemical, and biological processes, resulting in vast amounts of microplastics (MPs) (<5 mm in diameter) [1]. Following that, the MPs then disperse into the environment, where they can be transported into all environmental compartments. Humans can then be exposed to MPs through three different routes: skin contact, ingestion, or inhalation [4,5]. Furthermore, being regularly exposed to MPs even in a negligible amount is detrimental to human health. Moreover, when the MPs pervade through the skin from cleaners or foams, it may result in their bioaccumulation in the liver and digestive tract [6,7]. In addition, the MPs that are transmitted into the environment can enter the food chain, resulting in the consumption of contaminated food by humans [8].

Statistical exploration shows that MPs compose about 92.4 of plastic debris and are generally composed of polystyrene (PS), polyethylene (PE), and polypropylene [9]. PS plastic (often known as Styrofoam in its expanded form) is a major environmental concern due to its widespread use, persistence in the environment, and challenges in recycling. PS is a key component of environmental MPs, causing significant ecological and human health hazards at the cellular level. This substance, which is widely used in packaging, insulation, and consumer goods, fragments into smaller particles rather than entirely decomposing, resulting in a pervasive presence of PS MPs in all environmental compartments including the food chain. As a result, there is a pressing need for long-term methods to reduce PS pollution [10]. Currently, plastic in the environment can be degraded via three modes: oxidative photodegradation, thermo-oxidative declination, and biodegradation. Out of the three strategies, biodegradation is the most sustainable since it is both environmentally friendly and cost-effective. Photo-oxidation, which makes use of UV light and microbes, is the second-best option. Thermo-oxidative processing, on the other hand, is undesirable since it uses more energy than nature provides [11,12].

During bioremediation, microorganisms, especially bacteria, can alter the manifestation of MPs along with their functional group structures and other features [13,14]. There have been several studies [15,16,17] that have reported the biodeterioration of plastics. For example, Anabaena spiroides, Bacillus sp., Lysinibacillus sp., and Pseudomonas sp. were identified as ideal candidate strains [3]. Previous studies have also highlighted that Bacillus subtilis and Bacillus licheniformis, the most common bacteria in soil, can be potential candidates for microbial remediation of plastic contamination in the environment [3,18]. One of the most promising microbes used in MP degradation is B. subtilis. It has been effectively utilized in combination with other microbes in wastewater treatment attributed to its high degradative capacity for organic compounds including plastic. Biodegradation by B. subtilis used alone has been reported on PE films [18,19,20,21]. PS comprises a straight carbon backbone, and phenyl moieties are attached to alternate backbone atoms. There have been very few reports on the biodegradation of PS due to its distinct structure, which makes it difficult to break down. A comprehensive review by Ho et al. [22] reported findings of previous research on the biodegradability of various PS materials. PS materials employed in these various studies were pure PS, modified PS, or PS combined with other polymers. Pseudomonas spp. and Bacillus spp. have shown potential in degrading PS, particularly when pretreated, to make the material more accessible. The pretreatment approach is a crucial initial step before considering the biological strategy. Most prior studies involved pretreatments of MPs prior to biodegradation procedures. The pretreatment methods used range from heat treatment, γ-irradiation, and UV. Pretreatments are necessary primarily to reduce the hydrophobicity of the polymer, making them susceptible to microbial action [23]. Furthermore, the major research gap that needs to be addressed is the developing strategies focused on degrading PS micro- and nano-plastics in marine, freshwater, and terrestrial environments. Most of the previous studies have used PS plastic products or composites and have not focused much on the microplastic that adds up as a major environmental concern.

The concept served as a framework for the design of the present study to investigate the biodegradative potency of B. subtilis (ATCC 11774) on PS-MPs with and without pretreatment with heat and UV radiations. PS-MPs are frequently discovered as microplastic contaminant in ecosystems, making them an important material for environmental and biological impact research. The particle size (5 µm) used in the study is characteristic of MPs commonly detected in environmental samples. Colony-forming units (CFU) were used as the response parameter to identify the drivers of biodegradation after 30 days of testing. The ideal conditions for biodegradation of PS-MP by the designated bacterial strain were then ascertained using biodegradation tests.

2 Materials and methods

2.1 Materials

1. Commercially available, ready-to-use PS-MP beads (Alpha Nanotech Inc., Vancouver, Canada) 5 μm in size, colorless to pale white in color, and spherical in shape. PS-MPs were commercially obtained in wet form in solution at an initial concentration of 10 mg·ml⁻¹ and a density of 1.01–1.06 g·cm⁻³. PS-MPs were delivered in Milli-Q-Water medium and were used as received. Figure 1(a) and (b) shows the SEM micrographs and zeta distribution data of the PS-MPs prior to the study.

2. The bacterial strain, B. subtilis (ATCC 11774), used in the present study was obtained from the Department of Microbiology at Vision College, Riyadh, Saudi Arabia. The strain was transported within 30 min to the laboratory in a nutrient-rich medium such as Luria-Bertani (LB) broth in a cold storage bag containing ice to maintain a low temperature to ensure that the strain remained viable during transport. Bacterial cells were grown in LB broth, which contained sodium chloride (10 g·l⁻¹), tryptone (10 g·l⁻¹), and yeast extract (5 g·l⁻¹). NaOH was used to alter the pH of the LB broth to 7.4 (this is close to the optimal pH for several bacterial species, and many bacterial enzymes function optimally at or near physiological pH, which is 7.4). The mineral salt medium, Bushnell-Haas (BH) broth, was utilized in the experiment for the bacterial decomposition of PS-MPs. The BH broth was composed of K₂HPO₄ (1 g·l⁻¹), KH₂PO₄ (1 g·l⁻¹), NH₄NO₃ (1 g·l⁻¹), CaCl₂ (0.02 g·l⁻¹), MgSO₄ (0.20 g·l⁻¹), and FeCl₃ (0.05 g·l⁻¹); NaOH was used to adjust the pH to 7 [24]. The BH medium contains only essential inorganic salts and no carbon sources. This causes bacteria to use plastic as their only carbon source, allowing researchers to directly examine the microorganisms’ capacity to break down the polymer. In addition, it provides the necessary minerals and trace elements to support bacterial growth.

The strain was inoculated directly in nutrient broth media and left overnight at 37°C. Initially, the bacterial culture was transferred into the medium by adding a loopful of bacterial colonies (the loop used was a 10 µl in size, and the bacterial count was approximately 10⁷–10⁸ cells). Bacterial strains are ideally sub-cultured every 2–4 weeks to maintain viability. The plate count method and microscopic examination are used to assess the viability of the culture. The bacterial cells were kept stored at 4°C on a slant of LB agar. This form of storage can keep bacterial cells alive for up to 2 months.

Figure 1

(a) SEM micrograph and (b) zeta distribution data of the PS-MPs prior to the study.

3 Results

3.1 Bacterial growth

Bacterial growth was monitored through 4 weeks of the experimental period in all B. subtilis inoculated samples of PS-MPs with and without abiotic pretreatment by determining the CFU. Figure 2 shows the pattern of bacterial growth in all samples which was uniform and steady through the experimental period.

Figure 2

Bacterial growth curve showing log CFU·ml⁻¹ of the bacterium Bacillus subtilis in the optimized medium. Each value of the log (CFU·ml⁻¹) of the curve represents the mean ± SE of at least three replicates.

3.2 UV–Vis spectroscopy

Figure 3(a) and (b) shows the UV–Vis absorption spectra corresponding to the control (the pristine PS-MPs) and the groups with bacterial culture with and without pretreatment after 4 weeks that represent the beginning and end of the biodegradation experiment. A marked variation in the absorbance ratio of different inoculated samples (pretreated and untreated) is observed after 4 weeks in comparison to the control. The observable trends are as follows: Control (pristine PS-MPs, green curve) shows minimal absorbance changes owing to the intrinsic resistance of the pristine PS. Limited peaks indicate little or no structural change during the testing period. For the untreated inoculated group, PS + BS (orange curve): bacterial action induces chemical modifications, including oxidation and the introduction of chromophoric groups (e.g., carbonyls and hydroxyls), resulting in a moderate increase in absorbance in the shorter wavelength range (<300 nm). For the UV pretreatment group, PUV + BS (blue curve) UV pretreatment improves degradation by introducing oxidized functional groups before bacterial exposure, resulting in a higher initial absorbance. These groups absorb heavily in the UV range, indicating partial photo-oxidation. Bacterial growth increases deterioration following UV exposure, resulting in variations in absorbance across wavelengths. The temperature pretreated inoculated group, PT + BS (red curve) shows physical fragmentation increases surface area, facilitating microbial colonization and activity. The red curve shows distinct peaks and elevated absorbance compared to PS and PS + BS, suggesting intensified biodegradation.

Figure 3

UV–Vis spectra of PS-MP samples: (a) first week and (b) fourth week. PS (control, pristine), PS + BS (untreated inoculated with B. subtilis), PT + BS (temperature-treated PS and inoculated with B. subtilis), and PUV + BS (UV-treated PS and inoculated with B. subtilis).

In addition, a quantitative assessment of the degradation rate of inoculated PS-MPs (PS + BS, PT + BS, PUV + BS) was done on the basis of the reduction rate that was calculated by applying the first-order kinetic model shown under

(1) K = − 1 / t ln ⁡ C C 0

where K indicates the first-order rate constant for samples uptake per day, t indicates the time in days, C indicates the final concentration of samples, and C ₀ indicates the initial concentration of samples. Thus, the formula shown below was employed to compute the half-life of samples by integrating the value generated from the removal rate constant:

(2) t 1 / 2 = ln ⁡ ( 2 ) / K

Notably, UV pretreated PS-MPs (PUV + BS) exhibited a higher reduction rate (0.008037 day⁻¹) and a shorter half-life (86.2 days) followed by (PT + BS) and (PS + BS) (Table 1). However, the occurrence of biodegradation was further confirmed after analyzing the FTIR spectra.

Table 1

Biodegradation of inoculated PS-MP samples (treated and untreated) based on the reduction rate

Samples	Rate of constant (day−1)	Half-life (day)
PS + BS	0.00096	722.9
PT + BS	0.00118	587.3
PUV + BS	0.008037	86.2

3.3 FTIR analysis

The FTIR spectra were evaluated corresponding to the first week (Figure 4(a)–(d)) and the fourth week of the biodegradation experiment. It is in the region ranging between 4,000 and 250 cm⁻¹ of the spectrum that illustrates the most significant changes in different experimental setups. At the initial stage, all the inoculated groups share the same constituent functional groups as the control group (pristine PS-MPs). The prominent peaks between 3,800 and 3,200 cm⁻¹ correspond to the O–H stretching attributed to the alcohol group. The peaks at 2,422.59 and 2,376.30 cm⁻¹ correspond to the O═C═O stretching characterized by carbon dioxide. The bands at 1,635.64 and 840.96 cm⁻¹ correspond to C═C stretching followed by a band at 1,388.75 cm⁻¹ attributed to C–H bending. The bands between 1,111.00 and 925.83 cm⁻¹ correspond to C–O stretching. The band at 2,090.84 was observed only for the pristine PS-MPs that is attributed to the N═C═S group of isothiocyanates. Between the pristine PS-MPs and the inoculated groups (with and without thermal and UV pretreatment), a marked difference in the absorbance intensity was observed in the 3,100–3,441 cm⁻¹ and in the 1,388.75 cm⁻¹ region, which explains the alterations induced by the pretreatments. Figure 5(a)–(c) shows the FTIR spectra after 4 weeks of inoculation in both pretreated and untreated samples of PS-MPs. Inoculation without pretreatment (Figure 5a) showed marked changes in the peak intensities as well as the appearance of new bands 3,032.10 and 2,924.09 cm⁻¹ attributed to C–H stretching. Furthermore, additional peaks corresponding to C═O stretching at 1,728.22 cm⁻¹ followed by the appearance of a peak at 956.69 cm⁻¹ attributed to C–O stretching were observed for all three inoculated groups (Figure 4(a)–(c)) after 4 weeks. A closer comparison of the FTIR spectra of week 1 and week 4 for the inoculated sample that received thermal pretreatment (Figures 4(c) and 5(b)) illustrates that the most discernable changes that were observed after 4 weeks were the change in the intensity of peaks at 3,147.83 and 3,433.29 cm⁻¹ corresponding to O–H stretching and change in the intensity of the peaks at 1,643.35, 1,388.75, and 1,118.71 cm⁻¹. In addition, the FTIR spectra for week 1 and week 4 for inoculated UV-pretreated samples showed a conspicuous change in the peak intensity of bands at 3,402.43 and 3,147.83 cm⁻¹ corresponding to O–H stretching. Furthermore, observable change in the peak intensity was noted for the prominent peaks at 1,627.92, 1,388.75, and 1,111.00 cm⁻¹ corresponding to C═C stretching, C–H bending, and C–O stretching, respectively (Figures 4(d) and 5(c)). Furthermore, the extent of organic material degradation can be assessed based on the change in the FTIR absorption peaks, the evolution of oxygen-containing (e.g., hydroxyl, carbonyl, ester, etc.), or the disappearance of distinct functional groups (Figure 6). The oxygen evolution due to oxidative degradation can be monitored using one of the corresponding oxygen-containing functional groups like hydroxyl (OH), carbonyl (C═O), or ether (C–O–C), those typically observed at frequencies close to the ranges of 3,700–3,100 cm⁻¹ (OH stretching), 1,850–1,650 cm⁻¹ (C═O stretching), and 1,200–900 cm⁻¹ (C–O stretching). The OH and C–O bands are vulnerable to alteration by, e.g., moisture and overlapping with various other structural bands (e.g., C–C, C–N stretching, and C–H bending), respectively, and thus are less useful for reaction monitoring. The carbonyl band, on the other hand, is one typical product of oxidative degradation that has high molar absorptivity (more detectable) and thus being more acceptable for quantitative calculations. The CI can be calculated from the ratio between the area (or sometimes the intensity) under the C═O stretching band (1,650–1,850 cm⁻¹) and that of the methylene δ C–H scissoring peak (1,380–1,340 cm⁻¹) as given in the equation below:

(3) Carbonyl index ( CI ) = ( C ═ O ) peak area ( 1 , 650 – 1 , 850 cm − 1 ) ( C – H ) peak area ( 1 , 380 – 1 , 400 cm − 1 )

Figure 4

FTIR spectra of PS-MP samples in the first week (a) Control; pristine PS-MPs, (b) PS + BS (untreated inoculated with B. subtilis), (c) PT + BS (temperature treated PS and inoculated with B. subtilis), and (d) PUV + BS (UV-treated PS and inoculated with B. subtilis).

Figure 5

FTIR spectra of PS-MP samples in the fourth week (a) PS + BS (untreated inoculated with B. subtilis), (b) PT + BS (temperature-treated PS and inoculated with B. subtilis), and (c) PUV + BS (UV-treated PS and inoculated with B. subtilis).

Figure 6

FTIR spectra of plastic waste: PS pristine; plastic treated with temperature for a week (PT + BS-w1) and for 4 weeks (PT + BS-w4); treated with microorganism for a week (PS + BS-w1) and 4 weeks (PS + BS-w4); treated with light irradiation and microorganism for a week (PUV + BS-w1) and for 4 weeks (PUV + BS-w4). Spectral peaks were roughly categorized into six regions for simplicity.

To calculate the peak area, the FTIR spectra were set in the absorption mode. The data were drawn using Origin software; the baseline correction, derivatization, peak integration, and deconvolution were performed using the integrated functions.

Due to the overlapping of C═O peaks with C═C, a deconvolution was performed; an example of deconvolution results is demonstrated in Figure 7 for all groups (control and inoculated). Furthermore, as oxidative degradation typically results in various types of C═O containing compounds (e.g., aldehyde, ketone, acid, amid, etc.), multi-peaks were detected in this region; however, the complete region was treated as one during calculation while the peak center was selected at the highest intensity. As can be seen in Table 2, the CI values for the thermally pretreated inoculated group (PT + BS) were the highest compared to other samples, PS + BS and PUV + BS. The CI value of PT + BS samples was doubled after a week of conditioning compared to the pristine PS-MPs (i.e., increased from 0.053 to 0.106). After 4 weeks (PT + BS -w4), the CI has reached the value of 0.245 which is the highest detected CI value in the study. On the other hand, CI values of PS + BS did change moderately, while for PUV + BS there was no noticeable change observed, suggesting negligible oxidation. However, the extent of degradation has become considerable after 4 weeks of conditioning. Overall, the degree of degradation was in the order of PT + BS > PS + BS > PUV + BS.

Figure 7

Illustration of C═C and C═O peaks after deconvolution of the absorption band between, e.g., 1,550 and 1,850 cm⁻¹. PS-MPs, control pristine; PS + BS, plastic inoculated with B. subtilis; PT + BS, plastic pretreated with temperature and with B. subtilis and PUV + BS, plastic pretreated with light and with B. subtilis for 7 and 28 days.

Table 2

Carbonyl index for pristine plastic (PS-MPs) and its corresponding inoculated samples with and without abiotic pretreatment for week 1 and week 4

Peak center (cm−1)	Carbonyl index
	PS	PT + BS		PS + BS		PUV + BS
		Week 1	Week 4	Week 1	Week 4	Week 1	Week 4
1,725	0.053	0.106	0.245	0.049	0.144	0.042	0.093

3.4 Surface morphological and elemental analysis

Scanning electron microscopy (SEM) coupled with EDX was used to observe the surface morphological changes with the elemental analysis to further validate the degree of PS-MP biodeterioration. The pristine PS-MP control exhibited a smooth surface, as shown in Figure 8(a). However, thermal-pretreated and UV-pretreated PS-MP samples demonstrated various irregularities on their surface (Figure 8(b) and (d)). The SEM micrographs of the inoculated PS samples with prior UV and heat treatment showed the appearance of mechanical fatigue in week 4 characterized by the onset of the fracture/cracks occurring close to the surface (Figure 8(c) and (e). In addition, the micrographs after 4 weeks of bacterial inoculation without abiotic pretreatment also showed marked morphological changes, demonstrating the biodeterioration of the brittle PS-MP samples (Figure 8(f) and (g)).

Figure 8

SEM micrographs of PS-MP samples (a) PS (control, pristine), (b) PS + BS (untreated inoculated with B. subtilis) in the first week and (c) fourth week, (d) PT + BS (temperature treated PS and inoculated with B. subtilis) in the first week and (e) fourth week, (f) PUV + BS (UV treated PS and inoculated with B. subtilis) in the first week and (g) fourth week.

In addition, the samples were examined for PS-MP biodegradation by SEM/EDX analysis.

Based on their element footprints and surface modifications/properties. The detected elements included C, O, Na, and Si, which are frequently observed in samples of environmental water that contain microplastics (MPs). The elements detected were C, O, Na, and Si, which have been typically identified in environmental water samples containing MPs. Carbon and oxygen (C, O) were dominant, visibly illustrating the organic matrix of the PS-MPs (Figures 9–14). Table 3 shows the EDX analysis with the elemental map of each PS-MP sample (control, inoculated groups; pretreated and untreated). Variations in the surface atomic O/C ratio of all samples are visible, indicating that the scission reaction involves an oxidation process. The mean atomic O/C ratio for the control sample was 0.40 ± 0.01667, followed by a significant (p ≤ 0.05) observable increase in the ratio for the inoculated samples; 0.67 ± 0.00577 for untreated PS-MPs, 0.48 ± 0.00882 for UV-pretreated and 0.65 ± 0.00577 for heat/temperature treated (Table 4).

Figure 9

SEM and EDX analysis of the pristine PS-MP samples; control showing the elemental composition.

Figure 10

SEM and EDX analysis of the untreated inoculated PS-MP sample after 4 weeks showing the elemental composition.

Figure 11

SEM and EDX analysis of the temperature-treated inoculated PS-MP sample in the first week showing the elemental composition.

Figure 12

SEM and EDX analysis of the temperature-treated inoculated PS-MP sample after 4 weeks showing the elemental composition.

Figure 13

SEM and EDX analysis of the UV treated inoculated PS-MP sample in the first week showing the elemental composition.

Figure 14

SEM and EDX analysis of the UV treated inoculated PS-MP sample after 4 weeks showing the elemental composition.

Table 3

Spearman’s correlation (rho) between half-life (days) and CI within all experimental groups

			Half-life (days)	Carbonyl index
Spearman’s rho	Half-life (days)	Correlation coefficient	1.000	0.500
		Sig. (2-tailed)		0.667
		N	3	3
	Carbonyl index	Correlation coefficient	0.500	1.000
		Sig. (2-tailed)	0.667
		N	3	3

There is no statistically significant correlation at the significance level of (0.05) between half-life and CI.

Table 4

EDX analysis with the elemental composition of control and inoculated PS-MP samples with Bacillus subtilis showing the O/C ratio after 4 weeks

Elements	Control (pristine PS-MPs)		Untreated inoculated PS-MPs		UV-pretreated inoculated PS-MPs		Temperature-pretreated inoculated PS-MPs
	Weight%	Atomic%	Weight%	Atomic%	Weight%	Atomic%	Weight%	Atomic%
C K	64.48	70.85	52.84	59.88	54.38	64.65	44.16	56.11
O K	35.11	28.96	47.16	40.12	34.78	31.04	38.31	36.54
Other elements (Na, K, Ca)
Mean atomic O/C ratio	0.40 ± 0.01667a		0.67 ± 0.00577b		0.48 ± 0.00882c		0.65 ± 0.00577b

Values of the O/C ratio represent mean ± SE. Different superscripts are statistically significant (p ≤ 0.05).

4 Discussion

Research on the biodegradation of PS has demonstrated that this polymer may be broken down by specific microbes, including fungi and bacteria. According to Ho et al. [22] and Wen et al. [30], these organisms release enzymes that break down PS into smaller particles that can be further metabolized. The genera Bacillus, Pseudomonas, in addition to Paenibacillus, and Rhodococcus, are the most frequently utilized bacteria for PS breakdown [31,32,33].

The bacterial growth in all inoculated samples was stable through the experimental period of 4 weeks. The UV–Vis spectroscopy results showed that the absorbance ratios of the inoculated samples exhibited variation in comparison to the control (pristine PS-MPs) from week 1 to week 4. It has been suggested that biodegradation would happen if the sample absorbance ratio values do not remain constant [34]. The variations in absorbance ratios highlight the interplay of bacterial growth and pretreatment methods in facilitating PS degradation. Higher absorbance indicates the presence of chromophoric degradation intermediates (e.g., carbonyl-rich compounds) and reflects increased microbial metabolic activity, breaking down PS into oxidized by-products. Consequently, this variation implies that the polymer’s molecular structure was altered and that degradation might have occurred. UV-Vis spectroscopy is intrinsically confined to analyzing compounds containing chromophores, which are chemical structures that absorb ultraviolet or visible light. Thus, this limitation implies that the data acquired may not represent global changes in the overall plastic structure but rather specialized/localized changes in specific functional groups or additives. However, PS contains a benzene ring that absorbs significantly in the ultraviolet (200–280 nm) area. Thus, UV-Vis data will mostly reflect changes to these aromatic groups or any additions containing chromophores. Considering this, complementary analyses such as FTIR can detect changes in the entire molecular structure, including non-chromophoric functional groups and the aliphatic backbone [35].

Characteristic PS-MP peaks have been reported at 3,024, 2,847, 1,601, 1,027, and 694 cm⁻¹ in previous studies [33,36], which are close to the range of peaks corresponding to the results of the present study. Most of the characteristic peaks showed a decrease in their intensities after biodegradation by B. subtilis (with and without pretreatment). A similar decrement in intensities of characteristic FTIR peaks was reported after treatment by Pseudomonas alcaligenes (M2-23), as well. New peaks at 3,032.10 and 2,924.09 cm⁻¹, representing C−H stretching corresponding to aldehydes, and at 1,728.22 cm⁻¹ corresponding to C═O stretching attributed to the carbonyl group appeared in the spectra of inoculated PS_MP samples without pretreatment. The functional groups observed via FTIR spectra (–OH, C═O) are intermediates that can be metabolized by microorganisms and funneled into the TCA cycle. It has been postulated that during the process of oxidation, which is involved in biodegradation, functional groups such as hydroxyl or carbonyl groups could be formed via β-oxidation, which are known to be used in the citric acid (TCA) cycle or by the bacteria for energy metabolism, which subsequently enhances the hydrophilicity [33,37]. Thus, the integration of FTIR and metabolic pathways emphasizes the interplay between chemical analysis and biological processes in biodegradation research. In addition, the pretreated (thermal and UV) inoculated samples also showed the appearance of 1,728.22 cm⁻¹ corresponding to C═O stretching attributed to the carbonyl group validating the biodegradative process. The presence of the characteristic carbonyl band and its intensity directs the occurrence of oxidation reactions with chain scission [38]. These results are in line with the study by Miloloža et al. [33], where a similar characteristic weak peak at 1,715 cm⁻¹ was detected in the FTIR spectra of biodegraded PS by Bacillus cereus and P. alcaligenes, indicating the formation of carbonyl groups (−C═O). Furthermore, all inoculated groups showed the appearance of a band at 956 cm⁻¹ after 4 weeks. It has been previously reported that the band at 956 cm⁻¹ suggests its association with the C–O stretching mode of primary alcohols [39]. Consequently, the presence of more oxygen atoms on the plastic surface in regions where bacteria are growing is clear evidence of PS degradation [40]. Furthermore, based on previous literature, the carbonyl index (CI) has been applied for monitoring the physicochemical change and is one of the most acceptable among other methods used to probe the extent of oxidation [41]. Using the area under the band in FTIR spectroscopy is widely regarded as the most accurate and effective method for determining the CI [42]. The CI values calculated showed a notable change in the extent of degradation in all inoculated samples of PS-MPs after 4 weeks. Overall, the degree of biodegradation was in the order of PT + BS > PS + BS > PUV + BS. This increase in the CI value reflects the increase in carbonyl groups due to oxidative degradation, with the temperature pretreated sample being most pronounced. The CI values of degraded plastics have been reported in the range of 0–2 [41] but mostly less than 1 [43], which is in line with the obtained range in the present investigation (0–0.245).

These findings further corroborate the results of the SEM/EDX investigation, which showed a discernible increase in the atomic O/C ratio and was associated with the appearance of peaks in the FTIR spectra that were assigned to oxygen-containing functional groups (alcohol, aldehyde, and carbonyl). The SEM micrographs also demonstrated marked surface modifications of PS-MPs attributed to the abiotic pretreatment followed by the bacterial inoculation as well as the bacterial inoculation alone. Thus, the results clearly illustrated that bacterial inoculation of PS-MP samples with and without pretreatment modified the surface morphology after 4 weeks, as the presence of cracks/ridges indicates that the PS samples did become fragile [23].

It has been documented that PS biodegradation commences when microorganisms grow on its surface and release their enzymes, which break down the polymer into smaller molecular fragments such as oligomers and possibly monomeric units. Based on existing reports, proposed PS-biodegradation pathways and mechanisms involve the oxidation of styrene followed by its metabolic breakdown via the TCA cycle. Degradation might begin with either the cleavage of the main chain or the side chain, resulting in various degradation routes [44]. The breakage of the main chain can produce several aromatic monomers of PS, such as styrene and toluene, followed by two catabolic processes that use aromatic molecules as carbon sources. The first suggested process converts styrene into intermediate chemicals, which are then transformed into acetyl-CoA via the β-oxidation pathway. The citric acid (TCA) cycle leads to the core biosynthetic pathways. Another hypothesized mechanism begins with the cleavage of PS polymers’ side chains; monooxygenases or aromatic ring hydroxylases are viable candidates for breaking the aromatic ring of PS; however, the specific degradation pathway and enzymes involved have yet to be revealed [45]. Thus, certain microbes can utilize styrene as a carbon source for growth [22]. The results of the present study evidently support this explanation of PS biodegradation. The endpoints based on the biodegradability tests displayed an effective degradation of the PS-MPs in all inoculated groups with and without the abiotic pretreatment. The Bacillus strain alone was adequate to direct the biodeterioration process, which was observed in the larger magnitude of the modifications observed in the FTIR spectra, a moderately high CI, and the atomic O/C ratio of the untreated inoculated PS-MP sample. This is contrary to some previous studies that have reported that abiotic pretreatment (UV and thermal) enhances the biodegradative efficacy of microbial strains on polymers [23,38]. On the other hand, a study by Yao et al. [3] supported the notion that B. subtilis and B. licheniformis can effectively degrade PE without any abiotic pretreatment and could potentially be used as plastic-biodegrading microorganisms. Miloloža et al. [33] also investigated the biodegradation of MP-PS particles by the bacteria B. cereus and P. alcaligenes and reported that these bacterial strains offer a promising biodegradative potential for MPs in the environment.

When assessing the biodegradation of plastics, both half-life and carbonyl index are commonly used metrics, but they reflect different aspects of the degradation process and do not always correlate directly. This is in congruence with the quantitative data on half-life and CI in all inoculated groups in the current study. Spearman correlation coefficient, r _s , did not show a statistically significant (p ≤ 0.05) correlation between half-life and CI in the study. Half-life represents the overall rate of disintegration in terms of plastic mass or integrity, while the CI reveals chemical changes in the polymer; it mostly signifies oxidation and disintegration of the plastic’s molecular structure. UV-pretreated inoculated PSMPs exhibited the highest reduction rate of 0.008037 day⁻¹ and half-life of 86.2 days among all the inoculated groups, though the CI index was the least for this group. It has been reported that UV exposure causes the generation of free radicals in the polymer matrix, resulting in rapid chain scission without the formation of numerous carbonyl groups. This causes faster deterioration (shorter half-life) but lower CI. In addition, UV radiation might produce alternative degradation products (e.g., hydrocarbons, volatile organic compounds) instead of carbonyl-containing intermediates, lowering the measured CI [46,47]. On the other hand, the untreated inoculated group (PS + BS) showed a low reduction rate of 0.00096 day⁻¹ and half-life of 722.9 days even though the CI was moderately high along with the perceptible shift observed in the FTIR spectra. This explains that PS undergoes slow oxidative degradation, producing carbonyl-rich intermediates that accumulate because of the resistance of these materials to microbial degradation, extending the half-life [48].

Although several microbes with PS biodegradation potential have been identified, the mechanism and enzymes responsible for degradation have not been thoroughly investigated. However, researchers have proposed potential PS-degrading enzymes that include lipases, esterase, and oxidative enzymes [44]. Alkane hydroxylases can break PS main-chain C–C bonds under acidic or alkaline environments, as the stability of C–C bonds reduces [24]. In addition, depolymerizing enzymes that cleave the polymer backbone are required for reducing PS to monomers/oligomers such as styrene. Further degradation of styrene occurs via aromatic ring cleavage enzyme pathways, resulting in simpler molecules such as acetyl-CoA and succinyl-CoA that eventually enter the TCA cycle, which has been described above [44].

However, some study limitations should be acknowledged. The study could be planned over a longer period, elucidating the ability of B. subtilis to maintain its biodegradation efficiency over prolonged periods. There could be more analytical resolution and quantitative data. In addition, the environmental impact of by-products was not explored. These limitations could direct future research on investigating the ability of B. subtilis to degrade other types of plastics across different particle sizes (nano to larger fragments) and varied morphologies. In addition, using the bacterial strain in a natural consortium, optimization of abiotic treatments, and studies on understanding the enzymatic strategies involved in microbial degradation are imperative.

5 Conclusion

Taken together, the study highlights that B. subtilis, a commonly found bacteria in the soil, was effective in biodegrading the PS-MPs, which was evident from an integrated approach of using complementary techniques: UV–Vis, FTIR, SEM/EDX to provide a holistic view of both local and global degradation. On the basis of all the metrics assessed, pretreatment with temperature and untreated inoculated PS-MPs showed a perceptible oxidative chain scission. Thus, the bacterial strain was efficacious in biodegrading the PS-MPs with and without the abiotic pretreatment, which offers an alternative to abate energy-intensive processes, ensuring the credentials of green chemistry. Hence, the findings propose sustainable resolutions in waste management strategies, which are pivotal in the recent global scenario of plastic pollution.