Biodegradation of microplastics by Armadillidium vulgare and microbial isolates from an aged landfill

Plastics, the most used product that exhibits unparalleled attributes, have become a persistent cause of pollution, spiraling in every nook and corner of the earth^1,2,3,4. Microplastics, defined as plastic particles less than 5 mm in diameter, have emerged as a pervasive environmental pollutant, infiltrating both terrestrial and aquatic ecosystems^5,6,7. These tiny plastic fragments originate from a variety of sources, including the breakdown of larger plastic debris, synthetic textiles, and personal care products. Due to their small size and widespread distribution, microplastics pose a great challenge to environmental health, particularly in soil ecosystems where they can affect soil structure, nutrient cycling, and the health of soil organisms^8,9,10.

The persistence of microplastics in the environment is a growing concern. Unlike organic pollutants, microplastics do not readily degrade and can persist in the soil for extended periods, leading to long-term ecological impacts^11,12. Traditional remediation methods, such as physical removal and chemical treatments, often prove inadequate for addressing microplastic pollution due to the microscopic size of the particles and their extensive dispersion in the environment. These methods can also be labor-intensive, costly, and potentially harmful to the environment.

Considering these challenges, there is an urgent need for innovative and sustainable approaches to mitigate microplastic pollution^2,13,14,15. Bioremediation, the use of living organisms to degrade or remove pollutants, has gained attention as a promising solution for microplastic contamination. This approach leverages the natural metabolic processes of organisms to break down pollutants into less harmful substances, offering an eco-friendly alternative to conventional remediation methods^16,17,18,19.

Among the various bioremediation agents, terrestrial isopods, commonly known as pill bugs (Armadillidium vulgare), have shown potential due to their natural ability to ingest and fragment organic matter^20,21,22,23. Armadillidium vulgare are detritivores, meaning they feed on decaying organic material, and their digestive processes can break down complex substances into simpler compounds. This study hypothesizes that Armadillidium vulgare can similarly process microplastics, leading to their fragmentation and potential biodegradation.

As plastic is a relatively new product and the biodegradation of it is not so popular among bacteria, only^24,25,26,27. Researchers tried to use marine bacteria for the bioremediation of microplastics and can reduce the content; however, this method can not be used in the non-saline environment, for example, vegetable gardens^28,29,30,31. The earthworms were tried to clean up the microplastic contaminated soil, but they can not survive on the surface of the soil due to the sunlight and heat^32,33,34. Proteobacteria can degrade the biofilms of microplastics within 20–60 days, which was reported by researchers for the PE and PP. However, the days of degradation should be reduced more effectively^35,36,37. Numerous studies have explored methods for removing microplastics from water bodies, such as photocatalytic degradation, microbial decomposition, and ultra-high-temperature composting (HTC) technology^38,39,40. Researchers haven’t paid much attention to soil contaminated with microplastics because they didn’t understand its effects on humans and animals, and the relation between the contamination of soil by microplastics.

This research aims to investigate the feasibility of using Armadillidium vulgare for the bioremediation of microplastics in soil, along with microbes isolated from the old landfill site. The effectiveness of bioremediation of PE and PP will be monitored by using Armadillidium vulgare only and to compare it with the Armadillidium vulgare and microbes simultaneously in Pyrolysis Gas Chromatography Mass Spectrometry and SEM analysis.

Variation of PE in bioremediation experiment by Py-GC-MS analysis while using Armadillidium vulgare and microbes

Figure 1 illustrates the variation in polyethylene (PE) content within microplastic-contaminated soil following bioremediation using both microbes and Armadillidium vulgare. This analysis was conducted using Pyrolysis gas chromatography-mass spectrometry (Py-GC-MS), where the PE peak was observed at a retention time of 10–11 min. All samples were analyzed in triplicate for this paper. The results indicated a meaningful reduction in PE content, with a decrease of ~28.4% after bioremediation compared to the initial levels before treatment. This reduction was achieved over a period of three weeks through the combined action of Armadillidium vulgare and microbes sourced from an old landfill site. The observed decrease in PE content underscores the efficacy of the bioremediation process, highlighting the potential of using biological agents to mitigate microplastic pollution in soil. The bioremediation process leverages the natural degradation capabilities of microbes and the physical and digestive activities of pill bugs. Microbes break down the complex polymer chains of PE into simpler compounds, which can then be further degraded or assimilated. Armadillidium vulgare contributes by ingesting soil particles containing microplastics, which are then fragmented and excreted in a more biodegradable form. This synergistic interaction between microbes and pill bugs enhances the overall degradation rate of PE in the soil. Previous studies have demonstrated that earthworms can also reduce microplastic levels in soil. However, this study highlights the potential of pill bugs to similarly decrease microplastic contamination. Comparative analysis suggests that while earthworms are effective, Armadillidium vulgare offers an alternative or complementary approach to bioremediation. The ability of Armadillidium vulgare to thrive in various soil conditions and their natural behavior of burrowing and ingesting soil particles make them suitable candidates for large-scale bioremediation projects. The findings of this study have substantial implications for environmental remediation strategies. The ability to reduce microplastics in soil enhances the prospects for employing natural bioremediation methods to address soil contamination. This approach offers a sustainable and eco-friendly alternative to conventional methods, which often involve chemical treatments or physical removal processes that can be costly and environmentally damaging.

Variation of PP in bioremediation experiment by Py-GC-MS analysis while using Armadillidium vulgare and microbes

A similar trend has been observed in Fig. 2 for the case of polypropylene (PP) microplastics contaminated soil. The amount of PP reduced greatly after the bioremediation. The peak appears at the retention time of 10–11 min in GCMS for the PP, which is similar to the PE. It was observed that the amount of PE reduced after the bioremediation by almost 26% by comparing it without before bioremediation. This PP value was reduced because of bioremediation after 3 weeks with the combination of Armadillidium vulgare and the microbe collected from the old landfill site.

Variation of PE in bioremediation experiment by Py-GC-MS analysis while using Armadillidium vulgare only

Figure 3 illustrates the reduction of polyethylene (PE) in contaminated soil after the application of Armadillidium vulgare, as analyzed by Py-GC-MS. The data reveal a reduction of approximately 16%, which is notably about 14% lower than the reduction observed when both microbes and Armadillidium vulgare were used together, as depicted in Fig. 1. This finding provides compelling evidence of the Armadillidium vulgare’s capability to biodegrade microplastics independently. However, the comparative analysis underscores that the combined use of microbes and Armadillidium vulgare results in a greatly higher reduction of PE. This suggests a synergistic effect when both agents are employed simultaneously, leading to enhanced biodegradation efficiency. Therefore, for achieving optimal reduction of microplastics in contaminated environments, a combined approach utilizing both microbes and pill bugs is recommended. Similar results have been observed for bioremediation of microplastics by marine bacteria^2,41,42.

Variation of PP in bioremediation experiment by Py-GC-MS analysis while using Armadillidium vulgare only

Figure 4 illustrates the reduction of polypropylene (PP) in contaminated soil following the application of Armadillidium vulgare, as analyzed by Pyrolysis-Gas Chromatography-Mass Spectrometry (GC-MS). The data indicatea reduction of ~20%, which is about 10% lower than the reduction achieved when both microbes and Armadillidium vulgare were used together, as shown in Fig. 2. This finding provides clear evidence of the Armadillidium vulgare’s ability to biodegrade microplastics independently. However, the comparative analysis highlights that the combined use of microbes and Armadillidium vulgare results in a notably higher reduction of PP. This suggests a synergistic effect when both agents are employed simultaneously, leading to enhanced biodegradation efficiency. Moreover, a fascinating observation is that Armadillidium vulgare exhibits a greater capacity to biodegrade PP compared to polyethylene (PE) in the soil. This differential biodegradation potential underscores the importance of selecting appropriate biological agents based on the specific type of microplastic contamination.

Percentage reduction of PE and PP in soil by using various earth creatures and microbes

Figure 5 illustrates the percentage reduction of polyethylene (PE) and polypropylene (PP) in soil using various earth creatures and microbes. The data reveals several key insights into the effectiveness of different bioremediation agents. Firstly, the combination of microbes and Armadillidium vulgare is shown to accelerate the reduction of microplastics in the soil. This synergistic effect results in a faster decrease in microplastic content compared to when earthworms or pill bugs are used individually. The collaborative action of microbes and Armadillidium vulgare enhances the breakdown and assimilation of microplastics, making this combination particularly effective.

Secondly, the reduction rate of polypropylene (PP) is more pronounced than that of polyethylene (PE) when Armadillidium vulgare is employed⁴³. This indicates that Armadillidium vulgare has a higher efficacy in degrading PP, possibly due to differences in the chemical structure and physical properties of the two types of plastics. The Armadillidium vulgare’s digestive processes and microbial interactions may be better suited to breaking down PP, leading to a more substantial reduction.

However, when it comes to polyethylene (PE), earthworms demonstrate greater ability in bioremediation. Earthworms’ burrowing activities and gut microbiota contribute to the breakdown of PE, making them more effective for PE reduction in contaminated soil. This suggests that earthworms have unique mechanisms or microbial partnerships that enhance their ability to degrade PE. The findings depicted in Fig. 5 highlight the importance of selecting appropriate bioremediation agents based on the type of microplastic contamination. The combination of microbes and Armadillidium vulgare is highly effective for overall microplastic reduction, while specific creatures like earthworms and Armadillidium vulgare show varying degrees of effectiveness depending on the type of plastic.

SEM analysis of PP contaminated soil before and after bioremediation by Armadillidium vulgare

Figure 6 presents scanning electron microscopic (SEM) images of polypropylene (PP) contaminated soil before and after bioremediation by Armadillidium vulgare. The top image, representing the soil before bioremediation, shows a higher concentration of visible PP particles. In contrast, the lower image, taken after bioremediation, reveals a meaningful reduction in the number of PP particles. This visual evidence strongly supports the effectiveness of Armadillidium vulgare in biodegrading PP particles. The pill bugs utilize the carbon components of the PP for their growth through natural metabolic processes. This observation underscores the potential of Armadillidium vulgare as a viable biological agent for the bioremediation of PP-contaminated environments. The SEM images provide a clear, visual confirmation of the biodegradation process, highlighting the transformation of the soil’s microstructure. This transformation is indicative of the pill bugs’ ability to break down PP particles and integrate the carbon into their metabolic activities. In conclusion, the SEM analysis not only demonstrates the Armadillidium vulgare’s capacity for biodegradation but also emphasizes the importance of employing biological agents in environmental cleanup efforts. The reduction in PP particles post-bioremediation showcases the practical application of Armadillidium vulgare in mitigating plastic pollution and promoting a healthier ecosystem.

The survival rate of the Armadillidium vulgare was around 95% after the experiment and then resealed in the natural garden.

This study has demonstrated the potential of Armadillidium vulgare as a viable bioremediation agent for microplastic (both PE and PP) pollution in soil. The experimental results indicate that Armadillidium vulgare can ingest and fragment microplastics, suggesting a novel and eco-friendly approach to mitigating this pervasive environmental issue up to 16 to 30% revealed by GC-MS. However, the synergistic bioremediation of Armadillidium vulgare and landfill-derived bacteria for microplastic degradation in soil is more prominent than Armadillidium vulgare only. The innovative use of Armadillidium vulgare for the bioremediation of microplastics presents a promising avenue for sustainable environmental management. This approach not only addresses the pressing issue of microplastic pollution but also aligns with broader efforts to develop eco-friendly and cost-effective remediation strategies. After 3 weeks, the Armadillidium vulgare were released in nature. The mechanism of the bioremediation of microplastics by using microbes and earth creatures should be studied in the future including the bioinformatic analysis of metabolic pathways and a detailed kinetic assessment.

Preparation of artificially contaminated soil by microplastics

The preparation of artificially contaminated soil with microplastics involves a meticulous process to ensure consistency and reliability in experimental conditions. The soil contamination was achieved by incorporating two types of microplastics: polyethylene (PE) and polypropylene (PP). Both PE and PP were finely powdered to a particle size of 13㎛ and precisely measured to 0.03 g each. These microplastics were then thoroughly mixed with 3 g of soil to achieve a uniform distribution. The concentration of microplastics in the natural soil varies from agricultural field, urban areas, and natural forest areas. So, in this study, the concentration of MPs has been selected for a higher range, which is commonly found in agricultural and urban soil. Before the addition of microplastics, the soil underwent autoclaving to eliminate any existing microorganisms, thereby preventing any interference with the experimental outcomes. The autoclaving process ensures a sterile environment, allowing for accurate assessment of the biodegradation capabilities of Armadillidium vulgare. The physicochemical properties of the soil were carefully monitored and adjusted to create optimal conditions for Armadillidium vulgare. The water content was maintained at 12%, the pH was adjusted to 7.2, and the electrical conductivity was set at 0.0034 mS/cm. These parameters are crucial for sustaining the biological activity of Armadillidium vulgare and ensuring its survival throughout the experiment.

To further support the living conditions of the Armadillidium vulgare, the organic content of the soil was standardized at 8% across all samples. This was achieved by incorporating leaf molds, which provide a natural source of organic matter and nutrients necessary for the pill bugs’ metabolic processes.

Isolation of bacteria from an old landfill site

The isolation of bacteria was conducted at an old landfill site in Japan, focusing on soil samples collected from areas containing partially degraded plastic waste. These samples were meticulously processed to isolate the present bacteria. The degraded plastics were found in the aged landfill and soil was collected from that area to isolate the bacteria. Figure 7 highlights the prominent bacterial species identified in the soil. Among these, Cytobacillus hornekea was successfully isolated and subsequently cultured in the laboratory, following protocols established in previous studies^41,44. Once cultured, Cytobacillus hornekea was applied to soil samples contaminated with microplastics, specifically polyethylene (PE) and polypropylene (PP) 10 ml. This application aimed to evaluate the bacteria’s potential in biodegrading these common types of microplastics. The application of Cytobacillus hornekea to PE and PP-contaminated soil provides valuable insights into the biodegradation capabilities of this bacterial strain, paving the way for more effective bioremediation strategies in the future.

Collection of Armadillidium vulgare

The collection of Armadillidium vulgare, was conducted within the garden of the Yamaguchi University campus, a common habitat for these creatures in Japan. To ensure consistency in experimental conditions, Armadillidium vulgare of uniform size were selected. Armadillidium vulgare, roly-polies, or woodlice, typically measure between 8.5 mm and 18 mm in length, with an average weight of ~37.96 mg. These small crustaceans are characterized by their distinctive oval shape, being longer than they are wide. They thrive in moist environments and are frequently found under rocks, logs, and leaf litter. As detritivores, they play a crucial role in ecosystems by feeding on decaying plant material, thereby contributing to nutrient recycling. One of the most fascinating behaviors is their ability to roll into a tight ball when threatened. This defense mechanism, known as conglobation, protects their vulnerable underbelly from predators. This unique adaptation not only serves as a protective measure but also highlights the evolutionary success of Armadillidium vulgare in various environments. In summary, the collection and study of Armadillidium vulgare provide valuable insights into their ecological role and potential applications in bioremediation. By understanding their behavior, physiology, and environmental interactions, we can harness the natural capabilities of Armadillidium vulgare to address environmental challenges, such as the biodegradation of microplastics. The pill bugs were fed dried leaves collected from the garden every day and checked the survival rate of it throughout the experiment (3 weeks).

Experimental set up

A 15 cm long and 7 cm wide cylindrical container was used for the experiment. The microorganisms were applied in liquid form, around 5 ml, to the soil and mixed thoroughly. To avoid escaping the pill bugs from the container, the height of the container was chosen in that way. Five Armadillidium vulgare of the same size were used for each set of experiments which were placed on the surface of the soil. Then the container was placed in an incubator at a constant temperature of 25 degrees Celsius and 50% humidity, as shown in Fig. 8.

The experiment was conducted for 3 weeks. To keep the moisture of the soil, distilled water (5 ml) was sprinkled over the soil surface at regular intervals (once/3days).

Pyrolysis gas chromatography mass spectrometry

To investigate the bioremediation of the soil, Pyrolysis Gas Chromatography Mass Spectrometry (Py-GC-MS) by Simidzu (GCMS-QP2050) was used. Pyrolysis–GC/MS was used to characterize and quantify polymeric microplastics extracted from the samples. Polymer quantification was performed using certified reference standards of PE and PP prepared at 0.1–50 µg and analyzed under identical conditions to generate calibration curves based on characteristic pyrolysis products (e.g., n‑alkenes for PE, 2,4‑dimethyl‑1‑heptene for PP), all exhibiting linearity with. Samples (5–20 µg) were pyrolyzed in a micro‑furnace pyrolyzer at 600 °C with a heating rate of 20 °C ms⁻¹ and a 10 s hold, using helium carrier gas at 1.0 mL min⁻¹ and an interface temperature of 300 °C. Pyrolysis products were separated on a DB‑5MS column (30 m × 0.25 mm × 0.25 µm) using a GC oven program of 40 °C (2 min), ramping at 20 °C min⁻¹ to 320 °C, and holding for 10 min; the injector operated in split mode (50:1) with a 280 °C transfer line. Mass spectra were acquired under EI at 70 eV in full‑scan mode (m/z 35–500), and polymer identity was confirmed by comparison with NIST library spectra and reference standards. All samples were analyzed in triplicate.

Statistical analysis was conducted using Microsoft Excel, a versatile tool for data management and analysis. Raw data was collected from the experimental results, including the percentage reduction of polyethylene (PE) and polypropylene (PP) in soil samples treated with different earth creatures and microbes. Basic descriptive statistics, including mean, median, standard deviation, and range, were calculated to summarize the data and provide an initial understanding of the distribution and central tendencies. Comparative analysis was performed to evaluate the effectiveness of different bioremediation agents. T-tests and ANOVA were used to determine the statistical significance of differences observed between groups.