## 3 Results
### Detection of LDPE Biodegradation Assay
#### 3.2.4 Gs-Ms Analysis  
A wide range of substances, such as volatile and semi-volatile alkanes, aromatic hydrocarbons, esters, saturated fatty acids, and unsaturated fatty acids, were identified in the LDPE-treated and untreated samples using GC-MS analysis (Figures 5 and 6). Additionally, a number of unidentified compounds were obtained. The analysis revealed the media treated with APCK5 had 42 compounds, while the media treated with APCZ14 had 54 compounds; 30 of these compounds were present in both treatments. The analysis revealed 42 and 54 compounds in the media treated with APCK5 and APCZ14, respectively, 30 compounds of which existed in both treatments. There were 30 similar compounds out of 54, whereas three of the compounds were unidentified in PACK5, as well as 20 compounds in APCZ14. Three and 20 unidentified compounds were reported in the APCK5 and APCZ14, respectively. However, 39 compounds were identified in the control sample, two of which had unknown chemical structures. As for the control (Figure 5a), 36 were found that were similar to APCK5, as were 31 similar to APCZ14. Three dominant products (butylated hydroxytoluene, heneicosane, and 2,4-di-tert-butylphenol) are found in APCK5 (Figure 5b) and APCZ14 (Figure 5c). Figure 4: FTIR spectra of LDPE powder after 180-day incubation with APCK5, APCZ14 and control. (a) double bonds (1640 cm\({}^{-1}\)), (b) acids (1715 cm\({}^{-1}\)), and (c) ketones (1740 cm\({}^{-1}\)). were obtained from a water sample at Al-Ahsa Plastic Products Company. Both isolates exhibited potential LDPE degradation, as evidenced by the change in pH values of the media, the reduction of the LDPE weight, and the FTIR and GC-MS analyses. Based on 16S rRNA gene similarities, APCZ14 and APCK5 were determined to be most closely related to the genus _Brucella_. APCZ14 exhibited a 99.48% homology with _Brucella cytisi_ ESC1 (accession number AY776289), whereas APCK5 showed a 99.33% similarity level to _Brucella tritici_ SCII24 AJ242584. It is generally established that the 16S ribosomal RNA gene is a powerful tool for identifying bacteria at the genus and species levels and is utilized for phylogenetic inferences across prokaryotes [51]. It was revealed, on the basis of similarities in their 16S rRNA gene sequences, that APCZ14 and APCK5 are most closely related to the genus _Brucella_. APCK5 displayed a similarity level of 99.33% to _Brucella tritici_ SCII24 (accession number AY242584), whereas APCZ14 showed a homology of 99.48% with _Brucella cytisi_ ESC1 (accession number AY776289). Similarly, phylogenetic analyses using 16S rRNA gene sequences revealed that the APCZ14 and APCK5 strains were clearly clustered within the _Brucella_ clade, providing a credible identity for the strains APCZ14 and APCK5. These findings provide further evidence for the efficiency of the 16S rRNA for the identification of bacterial species. The reduction in pH of the SM inoculated with APCK5 and APCZ14 demonstrated that both isolates have the potential to consume LDPE as a carbon source and become metabolically active during incubation. Similar results have been obtained by [52], who showed that the pH of the media containing LDPE was reduced from 7.12 to 6.67 for _Ralstonia_ sp. and from 7.12 to 7.03 for _Bacillus_ sp. after 180 days of incubation. It can be speculated that the biodegradation of LDPE due to the bacterial action (APCK5 and APCZ14) resulted in the secretion of certain intermediates, including acids, which could lower the pH value of the medium. It has been suggested that the reduction in pH indicates the presence of extracellular enzymes and the metabolic activity of the microorganisms to utilize LDPE as a source of carbon. Additionally, the depolymerization and fragmentation of LDPE would lead to the formation of products such as fatty acids and organic acids, which could result in a decrease in pH [53; 54]. A large body of evidence indicates that the biodegradability of LDPE is accompanied by a decrease in weight.