Biodegradation of polyethylene by indigenous fungi from waste recycling site, South West, Nigeria

Background: Indiscriminate disposal of polyethylene materials has become a regular practice among developing nations of Africa, especially in Nigeria. This has resulted in environmental pollution; hence, this study investigates the microbial degradation of polyethylene obtained from a polyethylene dumpsite in South West, Nigeria, under static in vitro condition. Soil samples were analysed for mineral composition and physicochemical characteristics. The fungal isolates were screened for polyethylene degradation using minimal salt medium containing polyethylene as sole source of carbon and nitrogen for their ability to degrade polyethylene. Gravimetric analysis and Fourier-transform infrared spectroscopy (FTIR) were used to monitor the biodegradation of the polyethylene. Results: Aspergillus flavus , A. nidulans , Penicillium chrysogenum , Mucor mucedo , Eurotium repens , A. fumigatus and Rhizopus stolonifer were enumerated. Mean microbial count ranged from 1.37 × 10 7 to 8.2 × 10 8 SFU/g. Individual weight loss was observed in the polyethylene strip cultured with P. chrysogenum (1%), E. repens (1%) and A. nidulans (2%). The changes observed in the FTIR spectra especially the polyethylene sample inoculated with A. nidulans con-firm the significant role of fungi in polyethylene degradation. Hence, its usage in the treatment of polyethylene in the environment is a cheap eco-friendly alternative. Conclusion: Aspergillus nidulans , E. repens and P. notatum play significant roles in the biodegradation of polyethylene which necessitates incorporating in polyethylene products waste management to foster a cleaner environment.


Background
Polyethylene (PE) constitutes 64% of the total synthetic plastic as it is being used in huge quantity for the manufacture of bottles, carrier bags, disposable articles, garbage containers, margarine tubs, milk jugs and water pipes (Alabi et al. 2019). Annually, 0.5 to 1 trillion PE bags are used all over the world (Biki et al. 2021). The durability, light weightiness and process ability of these polymers cause them to linger in nature for centuries and end up in landfills and natural water resources creating a severe threat to the environment and its ecosystems (Dawoud et al. 2021).
Polyethylene littering of the environment is a common problem in most urban centres in Africa, because majority of its wastes are often not recycled (Gwada et al. 2019). PE bags have been reported to cause the death of terrestrial animals such as cow (Venkatesh et al. 2021). The degradation of most synthetic plastics in nature is a very slow process lasting thousands of years and involves synergistic action between environmental factors and microorganisms (Mohanan et al. 2020). Microorganisms such as bacteria, fungi and algae are involved in the biodegradation of materials (Sarmah and Rout 2020).
Microorganisms such as bacteria and fungi have been reported to be associated with biodegradation of PE (Yuan et al. 2020). Biodegradation involves a consortium of microorganisms, some of which breakdown the polymer into smaller constituents, while others utilize the monomers and release less toxic and recalcitrant by-products that serve as energy source for other microbial groups (Moharir and Kumar 2019). Fungi have hydrolytic enzyme system which produces hydrolases that help to degrade polysaccharides (Srivastava et al. 2021). Hence, this study was carried out to isolate and identify fungi from a polyethylene dumpsite in Akure, South West, Nigeria, in an effort to devise an environmentally friendly strategy for safe treatment of PE wastes and mitigation of environmental pollution.

Study design
This is an experimental study conducted between January and June 2016.

Description of study location
Ondo State lies between longitudes 4°30′ and 6° East of the Greenwich meridian 5°45′ and 8°15′ North of the Equator. The State lies entirely in the tropics. Akure is the capital of Ondo State, and it is located in South-Western Nigeria. Akure lies between latitudes 7.25′ N and longitude 5.195′ E (Fig. 1). The study location was accessed through experimental design. These areas are moderately populated with moderate standard of living. Residents living in these areas are majorly farmers, teachers, traders, civil servants, legal and medical practitioners.

Collection of samples
Polyethylene and soil samples (depth of approximately 10 cm) were collected from the Ondo State Integrated Wastes Recycling and Treatment Project (OSIWRTP) located along Igbatoro Road, Akure in Ondo State, Nigeria.

Isolation and Identification of Fungi
A measure of 1 g of soil sample, crushed and slightly heated, was diluted in 9 ml of sterile distilled water, followed by serial dilution. Serial diluents were aseptically inoculated into different Petri dishes, and sterile medium after cooling to 45 °C was poured unto the inoculums. Sub-culturing was done until distinct colonies were obtained. Microscopic and macroscopic examinations including staining (with lactophenol cotton blue) for morphological characteristics were carried out on fungal isolates, and identification was done based on the characteristics (Adesemoye et al. 2006).

Screening for polyethylene-degrading fungal isolates from soil samples
Standard dilution plating technique on potato dextrose agar (PDA) (Oxoid, Basingstoke, UK) at 28 ± 2 °C for 72 h was carried out. Purification of fungi was done by sub-culturing distinct colonies on freshly prepared PDA media. Confirmation of fungal isolates for degrading capability was done by growing isolated colonies on plates containing aseptically prepared polyethylene Mineral Salt Medium (PMSM). All the plates were incubated at 30 °C and were observed between 3 and 7 days for growth (Alshehrei, 2017).

Purification of polyethylene-degrading fungi
Isolates that grew on the compounded mineral salt medium with polyethylene powder as the sole carbon source were sub-cultured into already prepared malt extract agar and incubated for 7 days at 30 °C. The pure fungal isolates were preserved in cryo vials aseptically prepared with malt extract agar at ambient temperature.

Biodegradation of polyethylene strips via polymer over-layer method under static condition
This was carried out according to the method of Vimala and Mathew (2016) with slight modification. Prior to medium preparation, polyethylene strips were subjected to thermal ageing in the oven at 70 ºC. Then, polyethylene strips were treated with 70% ethanol for 30 min to remove any plasticizers and colouring agents, and then, they were air-dried for 15 min. Mineral salt medium was prepared and aseptically poured into the Petri dish with the polyethylene strip gently placed with the aid of a sterile glass rod on the already solidified agar. The polyethylene mineral salt medium was inoculated with purified fungal isolates and incubated at 30 °C for 72 h and observed for growth.

Spectroscopic method for biodegradation of polyethylene using Fourier-transform infrared (FTIR) spectroscopy
Spectroscopic analysis was carried out to detect degradation which detects changes in the structural units of the polyethylene. Fourier-transform infrared spectroscopy is both quantitative and qualitative analytics with quantification parameters comprising peak, height, peak area and derivatives. The PE strips recovered after the static biodegradation experiment were thoroughly washed with sterile distilled water, dried for 24 h in the oven at 50 ºC and packed for analysis (Dierkes et al. 2021). The FTIR analysis was carried out using Agilent FTIR exoscan 4100 (Texas, Florida, USA). Sample interface was cleaned with methanol. Background spectrum was taken after which the polyethylene sample was placed on the interface and run automatically.

Physical/chemical analysis of soil samples
The physicochemical analyses of the soil samples were determined using the methods the Association of Official Analytical Chemists (AOAC 1990).

Statistical analysis of data
Data obtained were subjected to one-way analysis of variance (ANOVA), and treatment means were separated using Duncan's new multiple range test (DNMRT) at p ≤ 0.05 level of significance with the aid of Statistical Package for Social Sciences (SPSS) version 23. Table 1 shows counts from three different dilutions (10 −5 , 10 −6 and 10 −7 ). Fungal colonies decrease with increasing dilution. The number of fungal colonies is least in 10 −7 and highest in 10 −5 for all plates. Hence, the number of fungal colonies decreases with increasing concentration. Macroscopic and microscopic characteristics of fungal isolates including their probable identities are shown in Table 2. Pigments/colouration observed include green and whitish spores with septate and non-septate hypha was observed in fungal isolates in Fig. 2a-d. Table 3 shows the occurrence and percentage occurrence of the fungal isolates. Spore counts are shown with varying degree of percentage occurrence. Yellow-greenish fungal spores have the highest percentage occurrence (25%), while the light-greenish have the least (6%).

Results
The fungal isolates obtained were screened for their ability to utilize PE as carbon and nitrogen sources. All the fungal isolates were able to utilize PE as both carbon and nitrogen source, although the growth pattern differed. Four out of the seven fungal isolates had rapid growth with sporulation on the minimal salt medium (MSM) in which PE was added as both carbon and nitrogen source, while the remaining three showed slow growth with no sporulation and hence were not used for the biodegradation study. The four fungal isolates, with rapid sporulation, were further purified and used for the biodegradation study.
Weight loss during biodegradation process by fungal isolates of test polythene strip is shown in Table 4. The table revealed percentage weight loss from the first week (week 1) through the last week (week 6) when biodegradation was terminated. There was no percentage weight loss in polyethylene (PE) sample inoculated with Aspergillus flavus. PE sample inoculated with Aspergillus nidulans showed a 1% and 2% weight loss in weeks 5 and 6, respectively, while the Penicillium chrysogenum and Eurotium repens samples showed a 1% weight loss in week 6.
The Fourier-transform infrared spectroscopy (FTIR) analysis was used to monitor changes in the functional groups of the polyethylene strips during the biodegradation experiment. The FTIR analysis carried out on the experimental and control PE sample strips showed little/no changes in wavenumbers in Figs. 3, 4, and 5. This implied that there was an insignificant level of degradation of polyethylene samples. Figure 6 shows significant changes in the functional group of the PE strip as there was formation of new peaks in the experimental sample.
A comparative physical/chemical analyses and mineral composition of polyethylene and non-polyethylene dumpsite soils are shown in Tables 5 and 6, respectively. Table 5 shows the physical properties of the sampled soils. These included soil colour, soil texture, particle size, consistency and soil odour. The table shows a significant difference in soil properties such as colour, particle size and odour, while properties such as texture and consistency show little difference for both sampled soils (polyethylene and non-polyethylene dump soils). Table 6 shows the chemical properties and mineral composition of the sampled soils. These include pH, moisture content, available phosphorus, organic carbon, organic matter, total organic nitrogen, cation exchange capacity, sodium, potassium, calcium, magnesium, copper, iron, manganese, nickel, cobalt, lead, cadmium and chromium. The table shows differences in the chemical characteristics and mineral composition of both the polyethylene dump soil and non-polyethylene dump soil samples. All parameters analysed for both polyethylene and non-polythene dump soils were significantly different from each other with the exception of chromium.

Discussion
Three hundred and forty-two fungi (Table 1) were isolated from the sampled soils using standard plate count technique. This fungal population may be as a result of the high organic carbon and organic content of the polyethylene dump site soils. This may also be as a result of the increased availability of biodegradable organic and inorganic substrates from the variety of municipal wastes continuously being dumped at these sites. This is similar to the findings of Tanunchai et al. (2021) who reported the presence, in large number, of fungi in soils harbouring plastic films. The high fungal count of 8.2 × 10 8 SFU/ ml (Table 1) (Douglas et al. 2020). The fungal isolates' polyethylene degrading ability could be as a result of the ability of these fungi to survive better in static-solid medium; this is similar to the findings of Khruengsai et al. (2021). The seven fungal isolates were subjected to identification after their macroscopic and microscopic characterization. Four out of the seven fungal isolates were selected and used for the biodegradation process (   Mishurov et al. (2020) who isolated Penicillium and Aspergillus from polymer while working on using polymeric materials as substrates for micromycetes. Gravimetric method used to determine the percentage weight loss of polyethylene sample (Table 4) was similar to the method used by Yu et al. (2019) in their findings of degradation of polyethylene by fungal consortium. Table 4 Percentage weight loss of the polyethylene strips during six weeks of degradation in static condition

Sample Week 1 (%) Week 2 (%) Week 3 (%) Week 4 (%)
Week 5   Polymer breakdown by fungi prior to biodegradation is aided by the action of pre-treatment with high temperature. Fungi such as Penicillium chrysogenum, Eurotium repens and Aspergillus nidulans showed ability to degrade the polyethylene samples and were subjected to thermal pre-treatment. This is similar to the findings of Peterson et al. (2019) who reported that temperature is a crucial factor in determining the rate of thermo-oxidation. It was noted that Penicillium pinophilum, Aspergillus niger and Phanerochaete chrysosporium showed biodegradative capability on thermally treated low density polyethylene.
FTIR spectra from this research showed little alteration in the structural composition of the sampled polyethylene (Figs. 3,4,5,6). This is similar to the findings of Janczak et al. (2020) who studied degradation of polyethylene using organisms isolated from compost soil. They studied degradation by inoculating isolated microorganisms into mineral salt medium containing one gram of polyethylene film and reported that carbonyl band corresponds to the ketone and ester functional groups, which is a typical product of thermal-oxidation degradation of polyethylene. A significant structural change was observed in the 3000 cm −1 band of the asymmetric stretching in the FTIR spectra of the sample polythene strip cultured with Aspergillus nidulans (Fig. 6), which indicates bending deformations around bands 1500-2500 cm −1 and 1600-1300 cm −1 . This is similar to the findings of Kelkar et al. (2019) who observed structural changes after investigating the effect of thermal ageing in sulphuric acid solution of high-density polythene (HDPE).
The result from the FITR analysis of polyethylene subjected to microbial attack indicated little or no significant alteration in functional group bonding of the polythene strip (Figs. 3, 4, 5), but the displacement of the CH group by OH at band 3000 cm −1 and a slight decrease in the carbonyl group at band 1600-1300 cm −1 showed considerable alteration (Fig. 6). Some Aspergillus species have been reported to produce aflatoxin, a toxic metabolite with OH as one of its functional group. This functional group was observed after six weeks of the biodegradation process. The formation of intermediate product such as polymeric OH bond can easily be converted to water and degraded by the fungus. This is similar to the findings of Zhang et al. (2020) who reported that some new intermediate products were observed after polymer breakdown by Aspergillus flavus.
From the study, soil sampled was both gritty and slightly coarse (sandy in nature) (Table 5). This observation agrees with the findings of Akinbile et al. (2021), who observed the sandy nature of top soils sourced from several municipal dump sites in Akure, Western Nigeria. However, this trend is in contrast with the findings of Daniel et al. (2021), who observed that majority of soil samples collected from waste dump sites in Port Harcourt, Nigeria, were silty in nature. This contrasting observation about soils textural class could be as a result of differences in geographical locations of soil sites and is plagued by different weather conditions. Minerals and the physicochemical parameters of both sampled soils have the same compositions with little variations in quantity (Table 6). This accounts for the similarity in the microbial type present in both soils. Heavy metals were in trace amount, while the pH of test sampled soils was alkaline with a substantial amount of total organic carbon (TOC) and small particle size. This is in agreement with the findings of Khadhar et al. (2020) who observed that pH, total organic carbon and particle size distribution are among several components of soil that affect its availability, retention and mobility of metals. Also, the alkaline nature and presence of trace amount of heavy metals in sampled soils agreed with the findings of Yuan et al. (2021) who demonstrated that soils with acidic pH levels tend to have an increased heavy metal concentration and vice versa.