Screening and Optimisation of the Biodegradation Potential for Low Density Polyethylene (LDPE) Films by Fusarium Equiseti and Brevibacillus Parabrevis

The accumulation of low density polyethylene, used extensively in packaging for industrial and agricultural applications, in the ecosystem is a great threat. This study focuses on the isolation of micro-biota from the plastic polluted sites to screen and optimise their potential for low density polyethylene (LDPE) film biodegradation. Firstly, the plastic samples from soil dumping plastic debris and plastic polluted water were collected; then fungi and bacteria were isolated using potato dextrose agar media and nutrient agar media, respectively, while screening low density polyethylene film biodegradation performed on mineral salt media (MSM) using the isolated micro-biota. The measurement of the potential biodegradation was assessed by visual observation. The most microbial colonization for low density polyethylene films was identifying molecular which was then utilized for optimisation of the biodegradation processes with different parameters such as media type, inoculum size, shaking speed, different incubation temperature and pH at different incubation time. Then the weight loss in the LDPE films percentage was calculated measuring dry mycelium weight and bacterial absorbance. The results revealed that, among the isolated micro-biota fifteenth, the most colonization was Fusarium equiseti and Brevibacillus parabrevis depending on the scanning electron microsope (SEM) and Fourier transform infrared (FTIR) analysis, in addition to optimum media, inoculum size, shaking speed, incubation temperature, pH, MSM, 2 disks and 2 ml, 30° C and 35°C, pH5 and pH7 for 30:20 days for F.equiseti and B.parabrevis, respectively. The overall results confirmed that F.equiseti and B.parabrevis from the plastic polluted sites play an essential role in low density polyethylene films biodegradation.

Plastic are polymeric compounds, nowadays synthetic plastic apply at many different fields due to their advantages such as durability and tenability 1 . The plastic waste disposal into the environment and land pollution problems has led to concern about plastics waste management 2 .
The plastic waste accumulation, caused a serious threat to the environment. Furthermore, swallowing the waste plastic debris by animals affect animal health 3 . Because of the disadvantage of conventional plastic degradation methods, the biological biodegradation methods become more demanding to be apply 4 . Polyethylene is a synthetic plastic commercially produced, while chemically it is petroleum-derived terephthalate acid (TPA) and ethylene glycol. Annually low density polyethylene production reached 60%. Due to the absence of appropriate disposal methods, polyethylene wastes are usually burnt in open areas which cause environmental pollution 1 .
Biodegradation is eco-friendly process relies on living organisms to degrade the polymeric compounds 5 . There are different factors affecting the plastic biodegradation process such as surface area, organism type, polymer nature, temperature, pH, and the addition of nutrients. The degradation process carried out at different steps. Firstly, the plastic is converted to its simple form, and then are mineralized 6 . Recently, using of microorganisms to degrade the plastic wastes becomes more attractive rather than the chemical and physical disposal methods. The most commonly used methods for the evaluation of plastic biodegradation process are (SEM), and (FTIR) 7 . Moreover, the mass loss of test specimens widely applied degradation tests to measure weight loss or determine residue polymer. The main distinguishing objectives of this study are: screening and optimization the biodegradation potential for low density polyethylene films using F.equiseti and B.parabrevis as novel microbial strains, and local eco-friendly methods.

Materials
A. Low density polyethylene (LDPE) used in this study was obtained in the form of LDPE films from plastic bags of market, Cairo, Egypt.

B. Media
Media for microbial isolation were added in g/L. Rose Bengal was added to inhibit bacterial growth; pH was adjusted to pH7 using either (0. Water samples were collected from surface and depth 10 cm through sterile glass bottle (Screw Cap bottles) and stored until used at 4ÚC. isolation of micro-biota isolation from soil The collected soil was sieved through mesh with a 2mm pore size sieve; then, two media for fungal and bacterial isolation were used by the isolation method (serial dilution 10 ). One gram of soil was added in 9 ml of sterile distilled water, and this suspension vortex was left to settle down.
Next, a series of 10-fold dilution was used to isolate fungi. One ml of each dilution was cultured on potato dextrose agar (PDA); meanwhile, in case of bacteria, nutrient agar media was used.

isolation from water
Water samples were prepared for fungal and bacterial isolation by serial dilution methods, and the plates were prepared through pouring plate techniques using PDA and Nutrient Agar media. Pure cultures were sub-cultured and then preserved until used.
Screening of the isolated microbiota biodegradation potential for low density polyethylene films Basal media were prepared as previously described by Esmaeili

Identification of the most microbial colonization of LDPE films Molecular identification of F.equiseti
The fungal strain was grown using Czapek's yeast extract agar (CYA) for Penicillim species and V8 Juice for Alternaria species followed by incubation for 7 days at 28ÚC 13 . The growing culture was prepared for DNA extraction using Patho-gene-spin DNA/RNA extraction kit provided by the Intron Biotechnology Company, Korea.
The fungal DNA was then sent to SolGent Company, Daejeon, South Korea for polymerase chain reaction (PCR) and rRNA gene sequencing. PCR was performed using ITS1 (forward) and ITS4 (reverse) primers which were incorporated in the reaction mixture.
The obtained sequences were analyzed using Basic Local Alignment Search Tool (BLAST) from the National Center of Biotechnology Information (NCBI) website. Phylogenetic analysis of sequences was implemented with the help of Meg Align (DNA Star) software version 5.05.

Molecular identification of B. parabrevis
Bacterial strain was cultured in sterile test tubes containing 10 ml of nutrient broth medium 9 . Culture was incubated at 28ºC for 48 hours.
The purified PCR products (amplicons) were reconfirmed using a size nucleotide marker (100 base pairs) by electrophoresis on 1% agarose gel. Purified amplicons were sequenced in the sense and antisense directions using 27F and 1492R primers with the incorporation of dideoxynucleotides (dd NTPs) in the reaction mixture 14 . Sequences were further analyzed using Basic Local Alignment Search Tool (BLAST) from the National Center of Biotechnology Information (NCBI) website. Phylogenetic analysis of sequences was carried out using Meg Align (DNA Star) software version 5.05. Measurement of biodegradation I. Visual observation (microbial attack the plastic surface bio-film or hyphae penetration formation). II. SEM in order to check for any changes in surface morphology. III. FTIR analysis to detect the degradation on the basis of changes in the functional groups.

(seM) analysis
The positive control and the treatment were prepared for (SEM) analysis (washing with70% ethanol). The samples were pasted onto the (SEM) sample stub using a carbon tape and the sample was coated with gold and analyzed under high-resolution scanning electron microscope (Quanta FEG 250, FEl, USA) at Desert Research Center, Cairo, Egypt.

(Ftir) analysis
Fourier transform infrared spectroscopic analysis was performed for the control and the treatment. The analysis was performed using Perkin-Elmer Spectrum version 10.5.4 at Central lab, Faculty of Science, Helwan University, Cairo, Egypt. IV. Weight loss measurement (determine of residue polymer) The weight loss in LDPE film percentage calculated using the equation 1: Weight loss in (LDPE) (%) = (Initial Weight -Final Weight) / Initial Weight) x 100 15 . Where: Initial weight= before treatment. Final weight= after treatment with F.equiseti and B.parabrevis.

optimisation of the biodegradation potential of LDPE films by F.equiseti and B.parabrevis
The growth conditions effect such as media types, inoculum size, shaking speed, incubation temperature, pH at different incubation time were studied.

Effect of media types Plastic film
There are two types of broth media used, namely, Czapek-Dox broth and mineral salt media (MSM). One disk (8 mm in diameter) of F.equiseti was inoculated into each of three sets of autoclaved flasks (250 ml) containing 50 ml of broth supplemented with sterilized LDPE films (1.5 cm x 1.5 cm), then it was incubated at 30ÚC for 21 days. After incubation, the dry mycelium weight was determined through a fungal mycelium filtration by using vacuum filtration, then dried mycelia were weighed by using digital balance, and weight loss in the (LDPE) films percentage was also calculated. On the other hand, in the case of B.parabrevis, one ml (bacterial suspension) was inoculated into each of the three sets of autoclaved mineral salt media, separately supplemented with sterilized (LDPE) films (1.5 cm x 1.5 cm) as shown in Pictures (4A & 4B), then incubated at 35ÚC for 21 days. After incubation, the bacterial growth was determined at 600 nm spectrophotometrically and weight loss in the LDPE film (%) was calculated. Positive control was media + plastic film (LDPE), while the negative control was media+ inoculum (B.parabrevis or F.equiseti).

Effect of inoculum size changes
Different inoculum size of F.equiseti (1, 2, 3 disks with 8 mm in diameter) and B. parabrevis (1, 2, 3 ml bacterial suspension) in three sets of 250 ml contained 50 ml (MSM) were supplemented with sterilized (LDPE) films (1.5 cm x 1.5 cm), then incubated at 30ÚC and 35ÚC for F.equiseti and B.parabrevis, respectively, for 21 days. After incubation, weight loss in (LDPE) films percentage was calculated.

Effect of shaking speed and static
Mineral salt media were supplemented with sterilized films and dispensed in three sets of 250 ml flasks containing 50 ml, and then the flasks were inoculated with two ml B.parabrevis. F. equiseti inoculated with two disks (8 mm in diameter) and incubated at both static and shaking speed (150 rpm) for 21 days at 30ÚC and 35ÚC for F. equiseti and B.parabrevis, respectively. After the end of the incubation, weight loss in the (LDPE) films percentage was calculated.

Effect of incubation temperature at different incubation times
Fifty ml of liquid (MSM) with one piece

results and discussions isolation of micro-biota
A total of fifteen, micro-biota were isolated from the soil dumping of plastic debris and water polluted of plastic. The isolates were labelled as 1S through 15 S. PDA was used for fungal isolation and nutrient agar for bacterial isolation as shown in Table (1). The first seven, micro-biota was isolated from the water polluted of plastic, and the other eight, micro-biota were isolated from the soil dumping of the plastic debris. screening of the isolated micro-biota biodegradation potential for low density polyethylene films Plastic film Screening micro-biota biodegradation potential was determined by visible observation   (1, 2)  s a m p l e F u s a r i u m s p 1 : F u s a r i u m  e q u i s e t i a u M c 1 5 1

Measurement of biodegradation seM analysis
Picture (6) shows SEM of LDPE film surface before and after 21 days of incubation with F.equiseti and B.parabrevis. Before, the sample had a smooth surface with no pits, cracks or any attached on the surface (Pic. 6A, 6B). However, after incubation with F.equiseti and B.parabrevis, surface with defects and changes was observed (Pic. 6C, 6D, 6E, 6F, 6G, 6H & 6I). For both F.equiseti and B.parabrevis, the different places on the surface of LDPE film colonized forming biofilm; this proved its strong adhering capabilities as well as LDPE utilization capacities. These results are similar to those obtained by Merina and Santosh 16 . In the case of F.equiseti, it was found the hyphal growth over LDPE film surface and hyphal penetration (Pic. 6C, 6D & 6E) while in the case of B.parabrevis, several cracks and cavities on the surface developed in addition to the biofilm formation were observed (Pic. 6F, 6G, 6H & 6I). Our results are agreed with 11 .

Ftir analysis
There was a variation in the intensity of bands after incubation with F.equiseti and B.parabrevis) compared with control as shown (Fig. 3A, 3B  show that the weight loss in the (LDPE) films % was achieved using (MSM) whereas the optimum absorbance for B.parabrevis and the optimum dry mycelium weight for F.equiseti were achieved using Dox's media, compared to positive and negative control.
Therefore, (MSM) is chosen to optimize the weight loss in the (LDPE) films percentage. This observation may go back to MSM's defect to carbon source while Dox's media contain carbon source which retards (LDPE) film as a source of carbon.

Effect of inoculum size changes
The results in Figure (6A) reveal that the best inoculum size at which the highest weight loss in (LDPE) films percentage for B.parabrevis and F.equiseti was achieved was two ml and two disks, respectively.
In addition, there was not a significant difference between two and three ml/disks. Thus, two ml/disks were chosen for optimizing the weight loss in (LDPE) films.

Effect of shaking speed and static
The results in Figure (6B) show that the optimum weight loss in LDPE was achieved at static condition compared to shaking conditions after incubation for 21 days using MSM media with the following values: 40% and 45% for B.parabrevis and F.equiseti, respectively. These may be due to static conditions allowing the (LDPE) colonization by B.parabrevis and F.equiseti incontast shaking conditions.

Effect of incubation temperature at different incubation times
The results presented in Figures (7A, 7B) show that the optimum weight loss in (LDPE) film percentage was observed at 35ÚC for B.parabrevis after incubating for 20 days while in the case of F.equiseti, it was at 30ÚC for 30 days.