Most researchers aim to determine the decomposition of plastic waste directly in the environment, such as soil or water, and this process is influenced by external physical and chemical factors in addition to microorganisms.
In our work, we therefore took a different approach and investigated the potential ability of so-called indigenous microorganisms isolated from the processing lines of various plastic products to decompose plastics. These indoor conditions are completely different from the natural environment, with specific external abiotic (UV radiation, pH values, humidity, extreme temperatures) and biotic factors, which include a wide range of different organisms. In the first part of the study, we determined the number and presence of individual groups of microorganisms in several stages of processing lines from different manufacturers and identified the most common bacterial and fungal species. Later, we focused mainly on the ability of the isolated bacterial strains to degrade PS material.
Aerobic mesophilic bacteria (TCC) (42.0% of the samples) were predominantly present on the examined surfaces, followed by moulds (30.0%), representatives of the genus Bacillus (28.3%), and staphylococci (19.9%) (Table S1).
No differences were found in the number of microorganisms between the individual sampling points of the processing lines, as many microorganisms were found on the raw materials, the machines in the various production stages and on the surfaces of the end products. As all three processing lines are not fully enclosed, microorganisms from the working environment in the production halls can enter the lines. Similarly, employees in two out of three companies do not wear protective clothing (gloves, hair protection, masks, gowns) and do not wash their hands thoroughly before handling the products, which could prevent the transmission of microorganisms from humans to the products.
As the production process required high temperatures at some sampling points, we incubated the samples at 37°C and 55°C to determine the presence of mesophilic and also thermophilic microorganisms. However, the latter were only present in very small numbers or not at all in most of the samples.
Most of the 96 isolated strains belonged to the Gram-positive genera Bacillus (45.6%) (B. licheniformis, B. cereus), Staphylococcus (39.6%) (S. epidermidis, S. hominis), and Micrococcus (11.5%) (M. luteus), as revealed by identification using MALDI-TOF MS. Staphylococci are part of the normal human skin microbiota, are easily transmitted and can survive on surfaces, especially on plastic and vinyl materials (Coughenour et al. 2011). Neely and Maley (2000) reported that common nosocomial pathogens such as staphylococci and enterococci survive longer on polyethylene and polyurethane than on cotton or cotton-polyester blended surfaces. It has been confirmed that Gram-positive bacteria survive longer on surfaces than Gram-negative bacteria (Lemmen et al. 2004). We were also able to clearly demonstrate that the contamination of plastic processing lines with Gram-positive bacteria was much more extensive than with Gram-negative bacteria. In our case, only about 3.1% of the strains belonged to the Gram-negative genera Burkholderia (B. fungorum) and Moraxella (M. nonliquefaciens) (Table S1). Surprisingly, no strains of Pseudomonas spp. were isolated from the samples, although members of this genus are thought to be commonly found on surfaces (De Abreu et al. 2014) and have been shown to have significant potential to form biofilms and degrade plastics (Muhonja et al. 2018; Amobonye et al. 2020).
The most common fungi in the samples, especially on the raw plastic pellets and the final products before sterilization (test tubes, Petri dishes), belonged to the genera Penicillium, Aspergillus, Chladosporium, and Fusarium. They could originate from the raw plastic material itself, presumably due to non-sterile processing and storage, or they could get onto the surfaces from the air of the production rooms (Haas et al. (2014) and Rejc et al. (2020). The ability of these fungi to survive and degrade plastic has been demonstrated in many studies (Muhonja et al. 2018; Srikanth et al. 2022).
The biodegradation of plastic polymers begins with the initial attachment of bacterial cells and the formation of a microbial biofilm, followed by a phase of biodegradation with the production and action of microbial exoenzymes on the mechanical, chemical and physical properties of the plastics. In the next phase of biofragmentation, the microorganisms secrete a variety of enzymes such as lipases, esterases and ureases, which convert polymers into oligomers, dimers or monomers that are transported into the microbial cytoplasm. The assimilated fragments are broken down by enzymes into smaller molecules (CO2, N2, CH4, H2O and H2S), which are further utilized by the microorganisms as available energy sources and finally returned to the atmosphere (Müller 2005; Shaji et al., 2024).
Kim et al. (2024) mentioned several analytical methods that can be used to study the degradability of plastics based on physical and chemical changes in polymer structure and/or monitoring of CO2/CH4 production and oxygen consumption. Of these, we selected clear zone, weight loss, scanning electron microscopy (SEM) and water contact angle measurements to determine the physical changes in PS, while Fourier transform infrared spectroscopy (FT-IR) was used to assess the chemical changes.
4.1 Clear zone determination
PS is one of the main polymer types in plastic waste and is known to be poorly biodegradable, resulting in PS waste remaining in the environment. Members of the genera Bacillus, Pseudomonas, Enterococcus, Listeria, Burkholderia, Enterobacteriaceae (Klebsiella spp., Cronobacter spp., Citrobacter spp.) and several others are PS-degrading microorganisms with known potential of genes in their genome for enzymes involved in this process (Mohan et al. 2016; Auta et al. 2017a; Hou and Majumder 2021).
The clear zones could be due to the hydrolysis of the polymer materials by the microbial extracellular enzymes that diffused through the agar and degraded the polymers to water-soluble materials. They may vary in clarity and number of days of appearance, suggesting differences in the metabolic states of the strains in the basal medium and under environmental conditions, as well as differences in the amount and activity of the excreted enzymes and their diffusion kinetics in the agar medium (Durairaju et al. 2024).
Only 3 (3.1%) of the 96 strains in our study showed a distinct clear zone around the colony on the culture medium with PS polymer, indicating their ability to degrade the plastic. All three strains belonged to the species B. licheniformis, and all were also obtained from raw materials for plastic products. Colony formation was observed in 64 (66.7%) strains, indicating that the bacterial strains nevertheless used the polymer in the medium as a carbon source, although we could not detect a clear zone around the colonies during the incubation period. Several studies have mentioned that Bacillus strains are among the most commonly isolated bacteria from the natural environment that can degrade plastic waste (Auta et al. 2017b; Zhang et al. 2018), which was confirmed in our research. The percentage of bacterial strains that can degrade PS is low in our study compared to other reports (Zhang et al. 2018; Joshi et al. 2022). However, it must be taken into account that in our case the isolated strains originate from the very limited environment of plastic processing plants and not from the natural environment where microbial biodiversity is much higher.
4.2 Weight loss measurement
Several studies have claimed that weight loss is the initial phase of depolymerization and biodegradation of plastics (Alamer et al. 2023). This method can also be used to determine the amounts of CO2 or CH4 produced during the degradation of plastics. The formation of CO2 is a viable indicator of polymer degradation, while CH4 is produced during the anaerobic degradation of plastics by microorganisms (Stegmann et al. 2022).
For the biodegradation test by measuring the mass loss of PS, 10 strains were selected that showed a transparent zone or pronounced colony growth on the polymer-containing culture medium. The selected strains were B. subtilis, B. licheniformis, B. cereus, M. luteus, B. fungorum and M. osloensis.
The average PS degradation rates of the isolated strains were between 0.21% and 2.15% after 60 days of incubation at 37°C. As in the clear zone assay, PS weight loss was highest for most Bacillus strains and lowest for the two Gram-negative isolates (Fig. 2). One of the possible reasons for this could be that some Bacillus strains are characterized by the formation of surfactants that can improve the efficiency of polymer degradation (Vimala and Mathew 2016). The weight loss of the polymer and changes in the pH of the media indicated that the isolated bacterial strains could degrade polymers. Biki et al. (2021) reported a decrease in pH of the medium with PE for Bacillus sp. strain SM1 from 7.12 to 7.03, showing that the bacterial strain consumes the polymer as a carbon source and becomes metabolically active during incubation. The weight loss of PE and the changes in the pH of the medium indicated that the isolated bacterial strain was able to degrade this polymer. In our study, the average pH changes in MSM bacterial cultures were up to 0.5, which could be an indicator of the metabolic activity of the bacterial cultures and their PS degradation in addition to the weight loss.
In some cases, our results on PS biodegradation were lower than in previous reports. For example, the strain M. luteus isolated from soil was responsible for weight loss of PS of up to 32% after 55 days (Sivasankari and Vinotha 2014). Auta et al. (2017) reported that Bacillus cereus and Bacillus gottheilii from mangrove sediments reduced the weight of PS granules in MSM by 7.4% and 5.8%, respectively, within 40 days, which was slightly higher than in our study. Similar results with a weight reduction of 1.52% and 1.45% by the soil isolates Acinetobacter johnsonii JNU01 and Pseudomonas lini JNU01 were obtained by Kim and coworkers (2021), while the weight reduction of PS after 60 days of treatment with the strain B. velezensis was 4.1% (Xiang et al. 2023). In the same range, the weight loss of PS after one month was 7.73% and 2.66%, degraded by the marine isolates Gordonia spp. and Novosphingobium spp. respectively (Liu et al. 2023b). In addition, the degradation of high-impact PS films using the Bacillus spp. strain, investigated by Mohan et al. (2016), showed a weight loss of up to 23% (w/w) in 30 days, while Yuan et al. (2022) reported a weight loss of 10.7% of PS, degraded by Bacillus cereus CH6 after 50 days of incubation.
However, the detailed biodegradation efficiency of PS cannot be compared in all cases, as the PS materials used had different structures (Kim et al. 2021) and originated from different sources. In addition, the degradation rate also depends on the presence of nutrients in the medium, temperature, pH, and incubation time, as well as agitation speed, concentration, and size of the PS particles (Miloloža et al. 2022). The aforementioned studies directly attempted to investigate the biodegradation of PS in environmental samples such as soils, mangroves, seawater, landfill sediment, activated sludge, waste disposal, and compost previously exposed to abiotic and biotic factors. We should keep in mind that the PS pellets in our study were pristine plastic, originating directly from the production lines and were exposed to UV sterilization for only 30 minutes after disinfection to avoid mechanical or chemical injury.
Asmita et al. (2015) reported the PS weight loss of 20.0% and 5.0%, caused by Bacillus subtilis and Pseudomonas aeruginosa cultured in NB, respectively. When bacteria were cultured in Bushnell-Hass broth without hydrocarbons, the weight loss of PS was higher for Bacillus cereus (58.8%), while no weight loss was observed for Pseudomonas aeruginosa. This suggests that providing a suitable culture medium for bacteria (Miloloža et al. 2022) significantly influences the rate of the biodegradation process. Our results are in agreement with these reports in that the rate of weight loss of PS, exposed to all strains tested, was significantly higher in MSM without hydrocarbons than in NB (Fig. 2). The presence or absence of nutrients namely affects the intensity of absorption of bacterial cells on surfaces (Shaikh et al. 2022). The more easily accessible nutrients in NB inhibit the bacterial production of enzymes to degrade the more difficult degradable PS.
4.3 The concentration, metabolic activity, and adhesion of bacterial cells
The final bacterial concentration of the tested strains averaged 5.6 log CFU mL− 1 after 60 days of incubation in MSM and was still relatively high compared to the initial concentration. Therefore, we can assume that the cells were still metabolically active, which was confirmed by the resazurin assay. This reaction is based on the reduction of oxidized non-fluorescent blue resazurin to a red fluorescent dye (resorufin) by the mitochondrial respiratory chain in living cells. The amount of resorufin produced is directly proportional to the number of living cells in constant average metabolic activity (Kuete et al. 2017).
Most Bacillus strains were found to be metabolically active after 30 and 60 days of incubation, while non-sporulating strains were weak or inactive after 60 days. The latter Burkholderia, Moraxella and some Micrococcus strains were generally characterized by a lower bacterial concentration in the suspension and low respiratory activity (Fig. 3). However, it should be emphasized that the activity was detected only after the addition of NB to the bacterial suspension in MSM and prolonged incubation for up to 3 hours to stimulate bacterial respiration, as it was otherwise undetectable. Both bacterial concentration and respiratory activity of most strains were quite high in NB compared to MSM. We assume that the Bacillus strains were present in nutrient-free MSM in the form of dormant spores, whereas in NB they were present as active vegetative cells. After spore activation at 80°C for 10 min, the resazurin assay also showed a positive reaction for the Bacillus strains incubated in MSM, while the non-spore formers were inactivated.
In general, the significant correlation between the concentration of cells in the suspension including their metabolic activity and their biofilm formation on coupons, and the degradation of PS could not be confirmed and was highly dependent on the strain tested (Fig. 2, 3, 4, Fig. S1, S2).
For example, strain No. 8 (B. licheniformis) was found to have relatively low PS degradation and high growth rate and metabolic activity, while strain No. 2 (B. subtilis) showed the opposite situation with the relatively low metabolic activity of cells in suspension and the highest PS degradation (Fig. 2, 3).
The attached bacterial cells were indirectly detected by staining with CV and then measuring the absorbance of the dye released from the cells after extraction in 96% ethanol. The absorbance of CV was significantly lower in all strains tested after 60 days of incubation than after 30 days. CV binds to peptidoglycan in the bacterial walls, but older cultures tend to lose peptidoglycan from cell walls. A higher amount of CV was released from cells of non-spore-forming strains incubated in MSM, while it was highest for most Bacillus strains in NB (Fig. 3). In MSM, Bacillus strains formed spores that were not stained with CV and showed little to no metabolic activity. Undamaged or dormant spores resist absorption of simple dyes, including CV, due to the impermeable cortex region, and therefore appear as non-staining units in Gram stain preparations (Kozuka and Tochikubo 1991; Todar 2020).
It has been reported that for the same type of plastic and strain, the higher weight loss is related to the higher absorbance of CV (Taghavi et al. 2021). In our study, this was confirmed for non-spore forming strains in MSM and NB, but not for Bacillus. When incubated in NB, there was also a correlation between the degradation of PS and biofilm formation in Bacillus strains, No. 6 and 8. The measured absorbance of CV as a function of the cells attached to the PS coupons shows that bacterial adhesion is highly dependent on the characteristics, metabolic stage, and type of bacteria, as well as environmental factors such as pH, nutrients, and temperature. The physical and chemical properties of the surfaces themselves, such as surface charge density, hydrophobicity, and roughness, also have a strong influence (Bohinc et al. 2014).
It should also be considered that CV staining cannot distinguish between living and dead cells in a biofilm. Therefore, we determined the metabolic activity of bacteria in suspension and biofilms using the resazurin assay and fluorescence microscopy, respectively.
4.4 Fluorescence microscopy
The presence and amount of living bacterial cells on the PS coupons were examined by fluorescence microscopy using two DNA dyes, SYTO9 and propidium iodide (PI).
The results of this study show that biofilm production and survival of bacterial cells after 60 days of incubation depend on the bacterial strain. The concentration of living cells on the surfaces was lowest for both Gram-negative strains as well as for a Micrococcus (No. 4) and a Bacillus strain (No. 3). The highest number of attached cells or spores was found in B. subtilis (No. 2), B. cereus (No. 6), and two M. luteus strains (No. 4, 5), which is consistent with the results of the SEM examination (Fig. 4, Fig. S2). For both Bacillus cultures (No. 2 and 8), we observed a high number of living cells or germinating/germinated spores and a low number of dead cells. However, for these strains, there was a characteristic negative correlation between the number of cells/spores in the biofilms and the absorbance of CV released from them (Fig. 3), probably due to the apparent sporulation after 60 days of incubation. Both Bacillus strains were also characterised by the highest degree of PS degradation, as determined by weight loss (Fig. 2), and ATR-FTIR measurements (Fig. 5, Fig S4). On the other hand, a weak but positive correlation was observed between the total number of living and dead cells and the results of CV staining in non-spore forming strains. In particular, strain M. luteus (No. 4) was characterised by the highest measured CV absorbance and showed the most efficient PS degradation, measured by weight loss, of all non-spore-forming strains (Fig. 2, 3, 5).
Bacterial endospores were thought to be impermeable to various intracellular dyes, but some studies confirmed the applicability of this method to detect endospores with flow cytometry (Comas-Riu and Vives-Rego 2002; Fan et al. 2019; Wei et al. 2023). They concluded that the membrane-permeable dye SYTO serves as an indicator of damage to the cortex, while the membrane-impermeable PI indicates damage to the inner membrane. It should be noted that the size of the molecules that can pass through the spore coat depends on the Bacillus species and is estimated to be between 2 and 8 kDa (Nicholson et al. 2000). LaFlamme et al. (2004) reported that germinating/germinated undamaged endospores stain brightly with SYTO 9 (a nucleic acid-binding dye) but do not take up PI. However, Cronin and Wilkinson (2007)
suggested that ungerminated or undamaged endospores only stain peripherally with these two dyes, while non-viable endospores and dead germinated endospores only stain with PI. To confirm the suitability of this method for the observation of Bacillus spores in our study, a commercially prepared B. subtilis spore suspension DSM 618 (110649, Merck, Germany) in MSM and NB with different incubation times was also used. All spores, detected by phase contrast microscopy (AE2000 Inverted Biological Microscope, Motic, USA), were stained green with the cell permeation agent SYTO 9 (data not shown in this article), as reported by Laflamme et al. (2004).
4.5 Assessment of bacterial biofilms by SEM
The SEM images show that individual bacterial cells or their aggregates were present on the surfaces of PS. The microorganisms colonised near and around the incisions and near rough surfaces, which was also demonstrated by Mohan et al. (2016). This is because the rate of attached bacteria increases with increasing surface roughness and hydrophobicity. The increased adhesion of bacteria on rougher surfaces is the interaction between the increasing effective surface area and the increasing number of defects on the surface (Bohinc et al. 2014). The deterioration of the surface and the formation of new cavities on PS were not clearly visible on the SEM images, as also reported by Taghavi et al. (2021). Based on the SEM analysis, we cannot prove that the attached bacteria degraded the PS and caused the formation of holes and cracks, because even the untreated surfaces were not completely smooth, so it is difficult to determine whether the observed nicks and holes on the inoculated surfaces were due to the microbial degradation. On the other hand, the weight loss and ATR-FTIR analysis results show minor changes in the weight or composition of the polymer that are difficult to detect by SEM observations of the surfaces (Fig. S2).
4.6 Changes in functional groups on the surface of PS pellets due to exposure to bacteria (ATR-FTIR)
It should be emphasized that in our study the PS coupons or pellets were treated with UV radiation for 30 min only for the purpose of their microbial decontamination. Cai et al. (2018) pointed out that abiotic factors, especially long-term UV radiation, contribute significantly to the success of polymer degradation. We believe that this is also one of the reasons for the relatively low level of PS degradation in our study, although some isolated bacterial strains from plastic production plants and products themselves show the potential for more intense degradation of PS materials. In contrast to the hydrolysable polymers (e.g., PET, PUR), which can be hydrolysed by enzymes to further degradable monomers, the non-hydrolysable polymers (PS, PE, etc.) are also much more resistant to hydrolysis and require oxidative cleavage of the backbone C-C bonds via enzymes with high redox potential (Amaral-Zettler et al. 2020). The degradation of the surface of PS has been confirmed in several studies by the formation of characteristic peaks for hydroxyl (-OH; at 3300 cm− 1), alkoxy (-C-O) or carbonyl groups (-C = O; at 1715–1740 cm− 1) in the ATR-FTIR spectra (Taghavi et al. 2021; Miloloža et al. 2022; Liu et al. 2023b). In our study, the oxidation of the surface of PS pellets after the decontamination process with UV was not observed (Fig. S3), therefore no degradation occurred, however, characteristic vibrational bonds for PS were determined as in previous studies (Ganesh Kumar et al. 2021; Taghavi et al. 2021; Miloloža et al. 2022; Wang et al. 2023).
Taghavi et al. (2021) reported that microbial enzymes and plastic samples cannot interact without the formation of hydroxyl, carbonyl and/or alkoxy bonds on the surface of plastic particles, which could impede the biodegradation process. Liu et al. (2023b), in addition to the appearance of oxygen bonds on the PS surface, also observed a decrease in the intensity of the characteristic ATR-FTIR peaks, especially those representing the aromatic ring of PS. In our study, a decrease in intensity and a lower occurrence of the aromatic ring bend but an increase in aromatic ring stretch was determined for the PS pellets exposed to the culture of B. licheniformis (No. 3 and No. 8) and B. cereus (No. 9). Due to the higher ratio 3024 cm− 1/696 cm− 1, representing aromatic ring stretch, in the samples of PS pellets exposed to other bacteria (strains No. 2, 4, 7, and 10) we cannot confirm ring cleavages and degradation of PS as reported by Kim et al. (2021) (Fig. 5, Fig. S3). Determination of minuscule changes on the surface of PS pellets might have been dimmed by the bulk properties of PS. Nevertheless, the results suggest that the Bacillus species influenced the chemical composition of the PS surface through the observed changes in aromatic ring vibrations and symmetric CH2 stretches.
4.7 Water contact angle
The primary mechanism for the biodegradation of plastics is oxidation or hydrolysis by enzymes to create functional groups that improve their hydrophilicity (Restrepo-Flórez et al. 2014). The size of the water contact angle between the surface of PS coupons and water is an expression of their hydrophobic and hydrophilic properties. In general, a water contact angle > 90° is considered a hydrophobic material, and a water contact angle < 90° is considered a hydrophilic material (Liu et al. 2023a). Hydrophobic bacteria prefer to adhere to hydrophobic surfaces, and hydrophilic bacteria prefer to adhere to hydrophilic surfaces (Koubali et al. 2021). Most, especially Gram-positive bacterial cells and spores are hydrophilic (WCA between 20 and 65°) due to the larger proportion of peptidoglycan in the cell wall (Ahimou et al. 2001; Dou et al. 2015; Eschlbeck and Kulozik 2017).
Oxidation is the crucial step in the biodegradation of plastics, transforming the hydrophobic plastic surface into a hydrophilic surface, which can be qualitatively confirmed by the change in contact angles between water droplets and the plastic surface. The oxidation of the surface makes it more susceptible to microbial attack, which increases the degradation of plastics (Ray et al. 2023).
Our results indicate that the contact angle of the PS surfaces decreased by seven (70%) isolates and increased slightly by only three of them. The average WCA of the biologically treated samples was 73.64 ± 0.45° and was 1.07° to 22.48° smaller than that of the control sample (80.55 ± 0.39°), depending on the strain (Fig. 6). The correlation between the decrease in WCA values and the weight loss results was negative but not significant.
Similar results were obtained by Kim et al. (2020), with the reduction of WCA of PS surfaces from 91.56° to 79.39°, caused by strain Pseudomonas sp. DSM 50071, confirming the increased binding energy on the PS surface and the conversion of C–C bonds to C═O bonds on the PS surface during degradation by Pseudomonas sp.
Zhang et al. (2023) reported, that the WCA of PS decreased from 76.3° to 66.9° after 180 days of incubation in the presence of nitrogen-metabolizing flora in microcosm wetlands. The FTIR peaks of the exposed surfaces increased in comparison to untreated plastics. Additionally, new peaks were observed in the wavenumber range of 1000–1500 cm− 1, representing vibrational regions of hydroxyl, carboxyl, and carbonyl functional groups. Despite the observed decrease in the WCA of PS after 60 days of incubation with bacterial strains, ageing or degradation in terms of chemical structural changes cannot be confirmed due to the absence of FTIR peaks at the wavenumbers, characteristic of oxygen-containing functional groups. Therefore, the decrease in the WCA was presumably due to the formation of the biofilm on the surface of PS. The hydrophobicity of the surface is not the main factor that determines the adhesion of bacteria to surfaces, but is also influenced by the roughness and electrostatic charge of the surface, the type of bacteria, the temperature and pH of the medium, etc. (Eschlbeck and Kulozik 2017; Abram et al. 2021).