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/lp/springer-journals/isolation-and-screening-of-low-density-polyethylene-ldpe-bags-L1e1qhoTDy
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- Springer Journals
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- Copyright © The Author(s) 2023
- ISSN
- 1590-4261
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- 1869-2044
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- 10.1186/s13213-023-01711-0
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Abstract
Purpose This study aims to screen bacterial isolates from the Addis Ababa municipal solid waste dumping site (Koshe) for the biodegradation of low-density polyethylene bags and analyzes their efficiency in degrading plastic bags. Methods In this study, low-density polyethylene bag-degrading bacteria were isolated from the Koshe municipal solid waste disposal area in Addis Ababa, Ethiopia. Screening of isolates for low-density polyethylene bag biodegrada- tion was carried out using a clear zone method. Additionally, the efficiency of the isolates for low-density polyethyl- ene biodegradation was evaluated using the weight loss method, scanning electron microscopy analysis, and Fourier transform infrared analysis. Finally, molecular identification of potential low-density polyethylene degrader bacterial isolates was done by 16S rDNA sequencing. Results Isolates KS35, KS14, and KS119 resulted in significant weight loss of low-density polyethylene film (42.87 ± 1.91%, 37.2 ± 3.06%, and 23.87 ± 0.11% weight loss, respectively). These isolates were selected for further bio- degradation study using scanning electron microscopy and Fourier transform infrared analysis. Scanning electron microscopy analysis shows the formation of pores, pits, and distortion of the plastic surface. Fourier transform infrared analysis indicates the appearance of new peaks at the surface of low-density polyethylene films. Phylogenetic analysis of the three potential bacterial isolates was also carried out, and the result indicates that the sequence of isolate KS35 had 99% similarity with sequences of Methylobacterium radiotolerans MN525302. Isolate KS119 had 100% similarity with Methylobacterium fujisawaense KT720189, and the sequence of isolate KS14 had 99% similarity with species of Lysinibacillus fusiformis. Conclusions Weight loss, scanning electron microscopy analysis, and Fourier transform infrared analysis results show that isolates KS35, KS14, and KS119 have high potential in degrading low-density polyethylene bags. Keywords Bacterial isolates, LDPE, Scanning electron microscopy, Fourier transform infrared spectroscopy, Biodegradation *Correspondence: School of Chemical and Bioengineering, Addis Ababa Institute Mesfin Tafesse Gemeda of Technology, Addis Ababa University, Addis Ababa, Ethiopia mesfin.tafesse@aastu.edu.et Biotechnology and Bioprocess Center of Excellence, Addis Ababa Department of Biotechnology, College of Biological and Chemical Science and Technology University, Addis Ababa, Ethiopia Engineering, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Nademo et al. Annals of Microbiology (2023) 73:6 Page 2 of 11 Aspergillus niger, Aspergillus flavus (Deepika and Jaya Background 2015), and Aspergillus versicolor (Gajendiran et al. 2016). Polyethylene is one of the most abundant commercially Biodegradation of LDPE film using Bacillus amylolique - produced synthetic plastic materials. It is a polymer of faciens has been reported by Das and Kumar (2015). ethylene, CH2-CH2, having the formula (−CH2-CH2-) They incubated LDPE film with two strains of Bacil - n, where “n” is the number of carbon atoms (Sandhu and lus amyloliquefaciens for 60 days and obtained a 16% Shakya 2019). Among the polyethylene family, LDPE weight loss of film. The incubation of polyethylene film (low-density polyethylene) accounts for 60% of the total with Pseudomonas species, A. niger, A. flavus, and Strep - production of plastic bags, and it is the major component tomyces species for 6 months revealed the reduction in of municipal solid waste (Gajendiran et al. 2017). molecular weight of LDPE film by 24.22 ± 0.01%, 26.17 ± Due to its high molecular weight, long carbon chain 0.05%, 16.45 ± 0.01%, and 46.7 ± 0.01%, respectively (Lee backbone, three-dimensional structures, hydrophobic et al. 1991). Gajendiran et al. (2016) identified Aspergil - nature, and lack of functional groups recognizable by lus clavatus as polyethylene-degrading fungi with 35% microbial enzyme systems, LDPE is very resistant to bio- weight loss of films after 90 days of incubation. degradation (Chiellini et al. 2003). Under normal condi- Even though many researchers have reported bacterial tions, the mineralization of LDPE takes more than ten and fungal degradation of LDPE, significant degrada - decades (Otake et al. 1995). The extensive usage of LDPE tion of LDPE wastes for environmental applications has is a severe environmental threat to terrestrial and marine not yet been achieved (Montazer et al. 2020). This study ecosystems. Plastic waste disposed of into the environ- aimed to screen indigenous bacterial isolates that have ment entangles the animal or bird’s body, thereby caus- the potential to degrade LDPE from plastic harboring ing the mortality of the organism (Kumar and Raut 2015). municipal solid waste soil samples. It also blocks the sewage system and creates a breeding ground for mosquitoes. Improper disposal of plastic Materials and methods waste can result in lost revenue from tourism by deterio- Sample collection and substrate preparation rating the natural beauty of the environment (Muthuku- Soil samples were collected from Koshe solid waste dis- mar and Veerappapillai 2015). Plastic materials dumped posal area in Addis Ababa, Ethiopia. The site is located into the earth prevent the production and mobilization in the Kolfe Keraniyo sub-city southeastern part of Addis of nutrients in the soil (Gharahi and Zamani-Ahmad- Ababa (Fig. 1). This area serves as a solid waste disposal mahmoodi 2022). Some plastic products cause human site for over 50 years. A total of 15 samples were collected health problems by causing immune and enzyme disor- from different sites randomly using closed sterile con - ders, hormonal disruption, and even infertility and are tainers and transported to the laboratory in the ice box. carcinogenic (Koteswararao et al. 2014). The samples were homogenized and stored at 4 °C until Different countries have adopted a range of approaches used. LDPE granules were collected from Ethiopia Plastic to discourage the use of plastic bags. In Ethiopia, Envi- Factory and used for enriching LDPE-degrader bacterial ronmental Protection Authority has cited a proclamation isolates after being prepared in powder form. The powder prohibiting granting permits for the company manufac- was prepared by immersing LDPE granules in xylene and turing or importing non-biodegradable plastic bags with boiling them for 15 min (Bhatia et al. 2014). The powder a thickness of less than 0.03 mm (Gazeta 2007). However, was washed with 95% ethanol, dried overnight in a hot due to poor awareness of society, and the lack of a strong air oven at 50 °C, and stored at room temperature for regulation system, different types of plastic bags are pro - further use. Low-density polyethylene films required for duced in large quantities and improperly disposed of. the biodegradation study were purchased from a local Different methods are practiced for the management market and prepared by cutting into 1.5 cm × 1.5 cm of plastic waste. These include recycling, incineration, size pieces. biodegradation, and dumping in a landfill. Moreover, bio - degradation is an environmentally sound full and cost- Culture enrichment and isolation of LDPE‑degrading effective method of plastic waste management (Kumar bacteria and Raut 2015). Recent reports on discovering certain Culture enrichment was performed to isolate bacteria fungi and bacteria that degrade synthetic plastics have that use LDPE as the sole source of carbon. The culture received scientific attention. Bacterial species associ - was enriched by suspending 1 g of the soil sample in 50 ated with the degradation of LDPE bags include Bacil- ml of sterile saline water and incubating it in a rotary lus cereus (Raut et al. 2015), Pseudomonas knackmussii, shaker at 120 rpm for 4 h. Then, 5 ml of soil suspension Pseudomonas aeruginosa (Hou et al. 2022), and Strep- was transferred into a 250 ml Erlenmeyer flask contain - tococcus species (Das and Kumar 2015). Polyethylene ing 100 ml of sterile mineral salt broth (1g/L K HPO , bags could be degraded by some fungal species such as 2 4 Nademo et al. Annals of Microbiology (2023) 73:6 Page 3 of 11 Fig. 1 Location and map of Koshe dumping site 0.2 g/L KH PO , 1 g/L (NH ) SO , 0.5 g/L MgSO .7H O, 5 ml of the enriched culture was transferred into 100 ml 2 4 4 2 4 4 2 1 g/L NaCl, 0.01 g/L FeSO .7H O, 0.002 g/L CaCl .2H O, of freshly prepared mineral salt medium supplemented 4 2 2 2 0.001 g/L MnSO .7H O, 0.001 g/L CuSO .5H O, 0.001 with 0.2% (w/v) LDPE powder. The third and fourth 4 2 4 2 g/L ZnSO .7H O and pH 7.0) and 0.2% (w/v) LDPE pow- transfers were done successively under similar condi- 4 2 der. All Erlenmeyer flasks were incubated in a shaker tions. After four cycles of enrichment, 0.1 ml of seri- incubator at 35 °C and 120 rpm. After 1 week of growth, ally diluted sample was spread on nutrient agar plates. Nademo et al. Annals of Microbiology (2023) 73:6 Page 4 of 11 Determination of dry weight of the residual polymer Isolated pure bacterial colonies were transferred into the The percentage of weight loss was determined after 60 nutrient broth and used for further study. days of incubation on a rotary shaker (120 rpm) at 35 °C. To determine weight loss, residual LDPE films were col - Screening of isolates for biodegradation of LDPE lected and mixed with 2% (w/v) of aqueous sodium dode- Screening of bacterial isolates for LDPE degradation cyl sulfate (SDS). The mixture was incubated in a shaker was carried out by the clear zone method. The synthetic incubator (120 rpm) for 4 h and then rinsed with distilled medium required to determine clear zone formation water to remove microbial film and residual medium. around the colony was prepared by mixing polyethylene Finally, residual LDPE samples were collected on filter glycol (the soluble form of polyethylene) with a mineral paper and dried overnight at 60 °C before being weighed. salt medium at a concentration of 0.2% (w/v) and 15% The weight loss was calculated and compared based on (w/v) agar (Rosario and Baburaj 2017). The media was the following formula (Montazer et al. 2019): autoclaved at 121 °C, 15 lbs pressure for 15 min, and allowed to cool to 45 °C, and poured into sterile Petri (Initial weight − Final weight) Weight loss (%) = × 100 plates. Once solidified, the isolated colonies grown Initial weight on nutrient agar were inoculated using an inocula- tion loop and then incubated at 30 °C for 2 weeks. After 2 weeks of incubation, plates were stained with Analysis of surface topography 0.1% Coomassie Brilliant Blue solution and destained The surface morphology of the LDPE film was analyzed to visualize a clear zone around the colony. Coomassie after 60 days of incubation at 35 °C and 120 rpm through Brilliant Blue solution was prepared by dissolving scanning electron microscopy (SEM) to check for any 0.1% (w/v) of Coomassie Blue into 40% (v/v) methanol structural changes made by the activities of bacteria on and 10% (v/v) acetic acid. The destaining solution LDPE film. The film was subjected to SEM analysis after was prepared by adding 40% (v/v) methanol into 10% washing with 2% (w/v) of aqueous sodium dodecyl sul- (v/v) acetic acid. Agar plates were flooded with 0.1% fate and distilled water repeatedly through mild shaking solution of Coomassie Blue R-250 for 20 min. The for a few minutes. Additionally, the film was flushed with solution of Coomassie Blue was poured off, and the 70% ethanol to get a maximum surface to be exposed plates were flooded with a destaining solution for 20 for visualization. A piece of air-dried film was placed min. The bacteria producing a clear zone in a blue on the sample holder, coated using a master coater, and background are considered polyethylene degraders then analyzed under a high-resolution scanning electron (Gupta et al. 2016). microscope (Gajendiran et al. 2016). Biodegradation studies Fourier transform infrared (FT‑IR) analysis In the present study, untreated LDPE was used to The structural change in the LDPE surface was investigated analyze the biodegradation efficiency of bacterial using the Fouriiier transform infrared (FT-IR) spectrom- isolates, while most early studies of microbial degra- eter. FT-IR analysis detects any change in the functional dation of LDPE used pretreated films. A biodegra- groups. Spectrum was recorded at 450–4000 wave numbers −1 dation test was performed using 0.2% (w/v) of LDPE cm for all LDPE samples (Gajendiran et al. 2016). films (1.5 × 1.5 cm) that had been dried overnight at 60 °C, weighed, disinfected (30 min in 70% etha- Sequencing and phylogenetic analysis nol), and air-dried for 15 min in laminar air flow Total genomic DNA was isolated from the bacterial cul- chamber. The films (0.2 g) were aseptically added to tures grown for 24 h using the Bacterial Genomic DNA Erlenmeyer flasks containing 100 ml of sterile min- Purification Kit (GeneMark) following the manufactur - eral salt medium supplemented with 0.01% (w/v) of er’s instructions. The 16S rRNA gene was amplified using yeast extract. Each flask was inoculated with 1 ml of universal primers (forward primer (27F 5′-AGA GTT 24-h old culture grown in a nutrient broth medium. TGA TCC TGG CTC AG-3′) and reverse primer (1492R Then, cultures were incubated on a rotary shaker at 5′-GGT TAC CTT GTT ACG ACT T-3′)) (Olukunle 2019). 35 °C and 120 rpm for 60 days. Flask without inocula- The PCR was performed on a Prime thermal cycler, UK, tion served as a sterile control. The extent of biodeg- using Taq DNA polymerase. A total of 30 cycles of ampli- radation of LDPE film was determined after 60 days fication were performed with template DNA. PCR reac - of incubation using the weight loss method, scanning tion was performed as follows: denaturation at 94 °C for electron microscope, and Fourier transforms infrared 4min, primer annealing at 56 °C for 1min, primer exten- (FT-IR) analysis. sion at 72 °C for 1 min, and final extension at 72 °C for Nademo et al. Annals of Microbiology (2023) 73:6 Page 5 of 11 8 min. Finally, the PCR product was visualized through their colony (Fig. 2). Bacterial isolates were selected based electrophoresis on a 1% agarose gel and sequenced using on the diameter of the clear zone around their colony. the Sanger sequencing technology (Applied Biosystems, India). Sequence analysis was done using the NCBI Biodegradation studies blast tool, and the best-matched organisms having valid Determination of dry weight of residual LDPE names were recovered. The phylogenetic tree was con - We calculated the weight loss of the polythene strips. structed using the neighbor-joining method (MEGA Out of fourteen bacterial isolates that form a clear zone version 11) after multiple sequence alignments with a around their colony, ten resulted in weight loss of LDPE bootstrap value of 1000 replicates. The 16S rRNA gene films (Table 1). In our study, some isolates which form partial sequences are deposited in the NCBI database Table 1 Percentage of weight loss of LDPE films (values are under accession numbers OK336096, OL315394, and duplicates and expressed as mean ± standard deviation) OK465137. Isolates code Initial Final weight (g) Percentage of weight loss weight Statistical analysis (g) Data were subjected to one-way ANOVA to observe the variation in weight loss among the bacterial isolates after KS119 0.2 0.1615 23.87 ± 0.11 60-day incubation. Post hoc (Duncan) test (p < 0.05) was KS114 0.2 0.197 1.25 ± 1.06 performed to determine the significance of the difference KS16 0.2 0.1999 0.025 ± 0.03536 between bacterial isolates in reducing LDPE film weight KS17 0.2 0.1998 0.05 ± 0.07071 after 60 days of incubation. Statistical analysis was done KS19 0.2 0.196 2.05 ± 1.48 using the software SPSS version 16 (Awasthi et al. 2017). KS35 0.2 0.1405 42.87 ± 1.91 KS116 0.2 0.176 13.31 ± 0.46 Results KS110 0.2 0.194 2.88 ± 3.36 Isolation and screening of LDPE‑degrading bacteria KS117 0.2 0.199 0.35 ± 0.21 Isolation of bacteria for biodegradation of LDPE was KS118 0.2 0.1999 0.025 ± 0.03536 made after successive enrichment of culture using min- KS15 0.2 0.198 0.75 ± 0.35 eral salt broth supplemented with 0.2% of LDPE powder KS25 0.2 0.196 1.91± 2.42 as a sole carbon source. Isolation of the bacteria was car- KS26 0.2 0.1999 0.025 ± 0.03536 ried out on a nutrient agar medium, and a total of sixty KS14 0.2 0.1465 37.2 ± 3.06 bacterial isolates were obtained. Screening of bacterial Control 0.2 0.2 0 isolates for LDPE degradation was carried out by the Differences in means are indicated with lowercase letters. Means with same clear zone method. Out of 60 bacterial isolates obtained, superscript letters are not significantly different, while means with different superscript letters are significantly different (P < 0.05) fourteen isolates formed detectable clear zone around Fig. 2 Clear zone formed by isolates after 2 weeks incubation in mineral salt medium supplemented with 0.2% polyethylene glycol (“A” growth of bacteria on medium before staining with 0.1% Coomassie Brilliant Blue solution. “B” and “C” Clear zone around bacterial colony after staining with 0.1% Coomassie Brilliant Blue solution) Nademo et al. Annals of Microbiology (2023) 73:6 Page 6 of 11 a clear zone around their colony did not show a signifi - and irregularities on the LDPE film. The control film cant change in the final weight of LDPE film. The maxi - appeared with a smooth surface (Fig. 3). mum degradation was achieved by isolate KS35, followed by KS14 and KS119 (42.87 ± 1.91%, 37.12 ± 3.06%, and Fourier transform infrared spectroscopy analysis 23.87 ± 0.11%, respectively) after 60 days of incubation. Oxidation or hydrolysis of LDPE by bacterial enzymes Furthermore, strain KS35 and KS14 showed significant creates functional groups that improve the polymer (p < 0.005) weight loss of LDPE film compared to other hydrophilicity and degradability by microorganisms. In isolates. Three of them resulted in maximum weight this study, the LDPE film biodegradation potential of the loss (KS14, KS119, and KS35) were selected as potential isolates was confirmed by FT-IR analysis. FT-IR analysis LDPE degraders for further SEM and FT-IR analysis. was used to detect the change in concentration of exist- ing functional groups or the formation of new func- Scanning electron microscopic (SEM) analysis tional groups. FT-IR spectra of LDPE films after 60 days Scanning electron microscopic analysis was carried out of incubation with selected bacterial isolates are shown for three potential bacterial isolates (KS14, KS119, and in Fig. 4. The result shows that incubation of LDPE film KS35), which showed better activity during the biodeg- with bacterial isolates has resulted in the generation of radation study using the weight loss method. Scanning new functional groups, changes in the concentrations electron micrograph showed various holes, cracks, pits, of existing functional groups, and disappearance of a Fig. 3 Scanning electron micrograph of LDPE films after 60-day incubation with bacterial isolates showing surface disruption, holes, and wrinkles on the surface. A Control, B LDPE film after treatment with KS14, C LDPE film after treatment with KS119, and D LDPE film after treatment with KS35 Nademo et al. Annals of Microbiology (2023) 73:6 Page 7 of 11 −1 Fig. 4 FT-IR spectra of polyethylene sheet treated with bacterial inoculum and the control incubated at 35 °C for 60 days. Peak at 1643.78 cm −1 which represent C–C=C stretch of alkenes disappeared in all bacterial-treated LDPE film except KS 119-treated film. New peaks at 1250.9 cm and 1246.44 cm−1 attribute to C–O stretch of ester’s group few functional groups at the surface of LDPE film either stretching of esters groups. The most prominent struc - because of their consumption or production. tural change was observed in the LDPE film treated with The FTIR spectrum of LDPE after treatment with KS14 KS35 bacterial isolate. and KS119 showed a decrease in wavelength number for O–H stretching of alcohol, and the band shifted from Sequencing and phylogenetic analysis −1 3391.53 to 3401.04 cm (O–H stretching of alcohol). PCR amplification of 16S rRNA gene was carried out, −1 −1 The peak at 3391.53 cm , 1473 c m (C–H bend stretch- and the PCR products were sequenced using Sanger ing vibration of alkanes), and 1643.40 (C=C stretching of sequencing technology (Fig. 5). Sequences of the three alkenes) in KS35-treated LDPE film disappeared. Addi - bacterial isolates were compared against the sequences −1 tionally, the peak at 1643.40 cm (C=C stretching of available in the NCBI, nr database using BLASTn. −1 alkenes) in KS14-treated film and peak at 1473 cm in The sequence of isolate KS35 had 99% similarity with KS119-treated LDPE film disappeared due to the effec - Methylobacterium radiotolerans MN525302. The sequences tive degradation of polyethylene film by bacterial isolates. of isolate KS119 had 100% similarity with Methylobacterium A new peak appeared in KS35- and KS14-treated LDPE fujisawaense KT720189, and isolate KS14 had 99% −1 −1 film at 1250.33 cm and 1246.44 cm wavelength num- similarity with species of Lysinibacillus fusiformis bers, respectively. Both peaks correspond to the C–O (Fig. 6). Nademo et al. Annals of Microbiology (2023) 73:6 Page 8 of 11 Discussion Low-density polyethylene could degrade by different fun - gal and bacterial species isolated from various sources. Several researchers explored microbial populations inhabiting landfills (Muhonja et al. 2018), rhizosphere soil of mangroves (Sangale 2012), and marine water (Ambika 2014) for their polyethylene-degrading potential. Simi- larly, in this study, the Koshe solid waste disposal area in Addis Ababa, Ethiopia, was selected for isolation and screening of LDPE-degrading bacteria. In many LDPE biodegradation studies, the weight loss method was used to determine microbial consumption of polymers (Das and Kumar (2015), Jamil et al. (2017), and Gyung Yoon et al. (2012)). In our study, the percentage of LDPE weight loss was calculated, and the highest value was recorded by isolates KS35 and KS14 (42.87 ± 1.91% and 37.2 ± 3.06%, respec- tively) (Table 1). This shows better degrading ability than the previously reported work by Kalia and Dhanya (2022), Fig. 5 PCR product analysis of 16S rRNA (1500bp) gene from in which they have documented 4.38% (untreated) and LDPE-degrading bacteria, lane 1, 1kb DNA ladder; lane 2, KS 35; lane 12.09% (xylene treated) LDPE films weight loss after 30 3, KS 119; and lane 4, KS14 days of incubation with Lysinibacillus fusiformis. Maroof et al. (2021) also reported a comparatively low percent- age of thermo-oxidized and UV-treated LDPE films weight loss (8.46 ± 0.3%) after 90 days of incubation with B. siamensis. Montazer et al. (2019) observed a percent Fig. 6 Maximum likelihood phylogenetic tree of isolates based on 16S rRNA sequencing. The newly sequenced isolates are highlighted in bold. NCBI accession numbers of the respective sequences are noted behind the species names Nademo et al. Annals of Microbiology (2023) 73:6 Page 9 of 11 decrease in LDPE (untreated and without additives) mass film (Fig. 4). A similar pattern of LDPE film spectra was by 33.7% ± 1.2% for C. necator H16, which is comparable reported by Gajendiran et al. (2017), where new peaks −1 with our result. Gajendiran et al. (2017) also reported a were observed at 1263.37, 1078.21 cm (C–O stretch −1 35% weight loss of LDPE films after 90 days of incubation of ethers), and 987.55 cm (=CH2 stretch of alkenes) with the fungi Aspergillus clavatus. A maximum decrease after 90-day incubation of film with fungi Aspergillus. in weight loss (48.40%) of pre-treated LDPE films after Muhonja et al. (2018) also analyzed the biodegradability 90 days of incubation with C. lunata SG1 in T-80 added of untreated LDPE using FT-IR and observed the forma- −1 medium was reported by Raut et al. (2015). tion of new peaks at 1700–1650 c m and 1000–1100 −1 While weight loss provides solid evidence of polymer cm . The shifting, addition, and deletion of peaks indi - degradation, SEM analysis confirms the biodegradation cate structural changes made by microbial activity (Bha- ability of isolates by elucidating the change of the surface tia et al. 2014). of LDPE films. Scanning electron microscopy analysis Phylogenetic analysis of the three potential LDPE showed the deformation of the LDPE film and the forma - degrader bacterial isolates was performed using MEGA tion of pits and holes after incubation of the film for 60 11 software, and the result showed that they are closely days with selected bacterial isolates (Fig. 3). In agreement related to Methylobacterium radiotolerans (KS35), with the present study, Das and Kumar (2015) reported Lysinibacillus fusiformis (KS14), and Methylobacterium that several cracks developed on the surface of LDPE film fujisawaense (KS119). Previously, these bacterial spe- treated with the bacterial isolate Bacillus amylolique- cies were reported to involve in the biodegradation of faciens BSM-1 after 60 days of incubation, whereas the LDPE and other hydrocarbons. Montazer et al. (2021) control sample had an appearance of a smooth surface. have reported biodegradation of low-density poly- Esmaeili et al. (2013) have also noticed surface erosion ethylene by Lysinibacillus fusiformis species isolated and the formation of pits and cavities on the surface of from larvae of the greater wax moth, Galleria mel- the LDPE samples after bacterial treatment. In a study lonella. Lysinibacillus species isolated from dumpsites by Yoon et al. (2012), SEM evidence confirmed that the were also identified as effective polyethylene degrad - smooth surface of the LDPE sheet became eroded as ers (Muhonja et al. 2018). Kalia and Dhanya (2022) a result of the biodegradation of the polymer by Pseu- reported that Lysinibacillus fusiformis had xylenes domonas sp. E4. Incubation of LDPE films with Asper - treated and untreated LDPE degradation potential. gillus clavatus strain JASK1 for 90 days also resulted in Photolo et al. (2021) isolated M. radiotolerans that can surface erosion, cracks, folding, and fungal colonization detoxify heavy metals and promote plant growth from (Gajendiran et al. 2016). municipal solid waste. Nzila et al. (2016) also reported In our present study, Fourier transform infrared spec- that M. radiotolerans isolated from soil contaminated tral analysis was carried out to check the chemical deg- with petroleum products was able to use naphthalene radation of polyethylene. The spectrum of LDPE films, as the sole source of carbon, and this bacterial strain incubated with the selected bacterial isolates, showed the grows efficiently in the presence of ethanol. Degrada - appearance of new bands and the disappearance of exist- tion of LDPE by microorganisms had known for several ing bands due to bacterial activity. Analysis of the poly- years, and there is no report on the biodegradation of ethylene spectral figures indicates the formation of new LDPE by Methylobacterium radiotolerans and Methylo- −1 −1 peaks at 1250.33 cm and 1246.44 cm , which corre- bacterium fujisawaense so far. This is the first experi - sponds to the C–O stretching of esters groups in KS35 mental report on LDPE utilization as a carbon source and KS14, treated LDPE film, respectively. Functional under laboratory conditions by showing the effective groups such as an ester group, a carbonyl group, or an ability of Methylobacterium radiotolerans and Methylo- ether group are formed when a hydrogen atom on a long bacterium fujisawaense. carbon-carbon bond is replaced by an oxygen atom (Ren The present work indicates that soil bacteria from et al. 2019). Alkane hydroxylases are the key enzymes solid waste dump sites show great efficacy in degrad - mediating aerobic alkane degradation by hydroxylation ing virgin polyethylene. Previously, many researchers of carbon–carbon bonds and the formation of primary or evaluated the biodegradability of LDPE after abiotic secondary alcohols (Montazer et al. 2020). In our study, pretreatment. Abiotic pretreatment such as UV irra- −1 the peak intensity of the band 1045.72 c m , which cor- diation, chemical oxidation, and thermal treatment responds to C–O of the ether group, increased. Ether is was employed to facilitate the microbial degradation formed during the biodegradation of LDPE as a result of of the polymer (Yoon et al. 2012). In the present study, the epoxidation of alkenes by microbial alkene monooxy- untreated LDPE film was used for the biodegradation genase (Hou et al. 1979). A new peak was also observed study, and promising results were obtained. A further at 888.07 (=C–H stretch of alkenes) in KS14-treated effort to improve this degrading capacity through the Nademo et al. Annals of Microbiology (2023) 73:6 Page 10 of 11 Received: 19 September 2022 Accepted: 4 January 2023 assessment of optimum conditions for microbial activ- ity is necessary. 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Journal
Annals of Microbiology – Springer Journals
Published: Jan 19, 2023
Keywords: Bacterial isolates; LDPE; Scanning electron microscopy; Fourier transform infrared spectroscopy; Biodegradation