## 4 Discussion  
Biodegradation is an effective biological process to clean up polluted environments through the isolation of efficacious halo-organic compound biodegraders. Notably, the BLASTn search on the 16S rRNA gene sequence revealed that MH2 has the 98% identity match with _Lysinibacillus boronitolerans_ (accession number: KC59351.1). Hence, to identify the capability of MH2 to degrade the sample pollutants, this study grew the MH2 bacterial isolate in TCA-enriched minimal media as the sole carbon source (pollutants). The initial composition of the culture medium and the concentration of the substrate (TCA) are two important parameters that can affect the degrading ability of the bacteria. Using artificial seawater or sterilized seawater as a medium for cultivation of sea-isolated bacterium was refrained from due to the potential interference of natural chloride ions in seawater with those liberated during the growth experiment. Minimal medium contained basal salts solution, trace metals solution, and distilled water was used in this study to provide the required growth culture for the bacteria. The existing nitrilotriacetic acid (N(CH\({}_{2}\)COOH)\({}_{3}\)) in the trace metals solution is low enough not to interfere significantly with the results of the experiment and to be consistent with the hypothesis of utilizing TCA as the sole carbon and energy source in the bacteria growth process. The optimal concentration of the TCA pollutant sample was selected 10 mm in the study of the growth profile and the ion liberation experiments. Using TCA in concentrations higher than 20 mm can stifle the cell growth due to toxic effects while the lower concentration of pollutants was not enough to observe the induced catalytic reaction of the dehalogenase-producing bacteria. Growth was strictly monitored by measuring the cells' turbidity and the amount of chloride ions released at appropriate time intervals. An uninoculated flask treated in the same way was used as a control. This is important to make sure the chloride measured in the growth medium was due to the cells using the TCA rather than the auto-degradation of the substrate in the growth medium. MH2 strain was capable of degrading hydrocarbon compounds. The nature and type of carbon sources are among the most important factor to determine bacterial growth. Haloacetates (i.e., MCA, DCA, and TCA) are common classes of water chlorination by-products. Several bacteria are available that can grow on MCA. _Burkholeria_ sp. DehCL1 [62], _Bacillus_ sp. TW1 [63], _Xantobacter autotrophic  
Figure 5: Neighbor-joining tree of strains M2 based on 16S rRNA gene sequences. Scale Bar 0.001 indicates sequence divergence. GJ10 [64; 65], and _Pseudomonas_ sp. R1 [66] are examples of bacteria that can degrade MCA haloacetates. Additionally, [PERSON] and [PERSON] [67] described _Xanthobacter autotrophics_ GJ10 that can degrade DCA. To the best of the authors' knowledge, this is the first report on _Lysinibacillus_ sp. isolated from seawater adapted in metabolizing TCA. The results showed that the highest growth in _L. boronitolerans_ MH2 bacterium was achieved at 96 h. Low absorbance was observed for the lag phase due to the low intensity and slow growth of the bacterial biomass produced. In broth, the doubling time of the _L. boronitolerans_ MH2 was 32 h. Chloride ion concentration during the biodegradation process was monitored by a chloride ion assay [61; 68]. Based on the standard curve of the NaCl, the released chloride ion by _L. boronitolerans_ MH2 strain after 96 h was \(0.33\times 10^{-3}\) mol\(\cdot\)L\({}^{-1}\). The study of the growth curve shows that the _L. boronitolerans_ MH2 was capable of growing in a minimal medium having TCA as the sole source of carbon and energy. Growth on TCA was further analyzed by preparing the cell-free extracts from growth on TCA as described in Figure 1 and Section 3.2. The presence of dehalogenase was detected by measuring the enzyme-specific activity. The _L.