Chemicals
All chemicals used in this study were of high purity and analytical grade
Isolation and screening of microorganism
Soil samples were collected in sterile sample bags at the depth of 5–15 cm, from transformer oil spillage site in Ile-Ife, Osun State, Nigeria, and taken immediately to the laboratory for microbial analysis. A stock solution was prepared by introducing 1 g of the soil sample in 10 mL distilled water, thereafter, serial dilutions were carried out to a concentration of 10− 6. A concentration of 0.1 mL of 10− 4 concentration was plated on nutrient agar and incubated at 37\(℃\) for 18–24 hrs. Colonies obtained were sub-cultured to obtain pure isolates which were used for downstream analysis.
Pretreatment and preparation of LDPE films
The LDPE films used in this study were food grade low density polythene (Ziploc bags) of about 18µ thickness, cut to an approximate size of 3 x 3 cm and a weight of 0.1000 g using an analytical balance were used. The polythene films were washed with deionized water and sterilized for about 3 hours using 70% (v/v) ethanol (HiMEDIA Laboratories, India) and air dried at 60\(℃\). These were then used as carbon source for the aforementioned microorganisms in biodegradation experiment.
Inoculum preparation for biodegradation
Bacterial isolates were standardized to uniform inoculum size, by inoculating them into nutrient broth. They were agitated, incubated at 30℃ for 24 hours and spun in a centrifuge at 3000 rpm for 15 minutes. Thereafter, adjusted to 0.5 Mcfarland Standard at 590 nm wavelength, and used as the starting culture for other analyses [12].
LDPE biodegradation experiment
The sterilized LDPE films were introduced into 100 mL of mineral salt medium (1 g K2HPO4, 1 g KH2PO4, 1 g NH4NO3, 0.02 g CaCl2, 0.05 g FeCl3, 0.2 g MgSO4 and 1 g glucose) sterilized at 121\(℃\) for 15 min. An overnight broth culture of the isolate (15 ) was inoculated into the flasks. The flasks were incubated in an incubator shaker at 200 rpm for 78 days at 30 \(℃\) alongside with a control containing no bacteria isolate. Each sample with a volume of 2 mL were analyzed at intervals of 3 days at a wavelength of 600 nm for degradation using an ultraviolet spectrophotometer.
At the end of 78 days, the LDPE films were removed from the incubator shaker and washed with deionized water, and immersed in 2% (v/v) sodium dodecyl solution for one hour to free the LDPE film from the metabolic protein of the cultured media. Thereafter, the LDPE films were rinsed with distilled water, oven dried overnight at 60\(℃\), and weighed to determine weight loss. They were then stored in sterile clean air-tight container for further analysis [13].
Percentage LDPE weight reduction
After 78 days’ incubation period, the LDPE films were dried and weighed using an analytical balance. The percentage weight reduction of the LDPE films after degradation was calculated Eq. 1 according to Samanta et al. [8]:
% weight reduction = [(Wi – Wf)/Wi] x 100 (1)
Where: Wi is the initial weight of plastics before degradation; Wf is the final weight of the plastics after the degradation period.
Samples with high percentage LDPE weight reduction were then subjected to chemical analysis.
Chemical characterization of degraded LDPE films
The surface morphology characterization and elemental composition of the unincubated LDPE film (control), incubated LDPE film without bacteria isolate (control NG) and incubated LDPE films with bacteria isolate was carried out using Field Emission Scanning electron microscope (FE-SEM) (JEOL JSM- 7600F) coupled with an Energy Dispersive X-ray Spectroscopy (EDX).
16S rDNA identification of the isolates
The bacteria with good activity were cultured in a nutrient broth at \(37℃\) for 24 hours, and centrifuged at 4,722 rpm for 5 min. The pellet was washed with phosphate buffered saline (PBS) and resuspended in PBS for DNA extraction. Genomic DNA of the isolates were extracted using the ZymoBIOMICS™ DNA MiniPrep kit (Zymo Research Corp., USA) according to the manufacturer’s instructions, as previously used by Onajobi et al. [14] and prepared in a 96 well plate for cycle sequencing. PCR amplification of the 16S rDNA gene was carried out using the universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1525R (AAGGAGGTGWTCCARCCGCA). The reaction consists of 2.5 uL of 10x PCR buffer, 1 uL of 25 mM MgCl2, 1 uL each of forward primer and reverse primer, 1 uL of DMSO, 2 uL of 2.5 mM DNTPs, 0.1 uL of 5 u/uL Taq DNA polymerase, and 3 uL of 10 ng/uL DNA. The total reaction volume was made up to 25 uL using 13.4 uL Nuclease free water. The PCR cycling was carried out at 94\(℃\) for 5 minutes for initial denaturation, annealing at 56\(℃\) for 30 secs, elongation at 72\(℃\) for 45 seconds for 36 cycles with final elongation at 72\(℃\) for 7 minutes. The amplicons were then visualized on ethidium bromide stained 1.5% agarose gel. The PCR amplicons were purified using Ethanol/ EDTA precipitation method and sequenced using ABI-PRISM 3500 genetic analyzer (Applied Biosystems, USA).
Bioedit software was used for opening and editing the sequences, the sequence identification was carried out using GenBank’s Basic Local Alignment Search Tool (BLAST) algorithm on National Centre for Biotechnology and Information (NCBI) database for the isolate identification.