Today, the environmental pollution by cyclic aromatic substances has led to serious problems due to the use of these substances in different sectors. Phenanthrene and pyrene are belonging to this group of substances that cause abnormal effects on the environment and the health of organisms. The high cost and technical problems of the usual methods to remove these cyclic toxins have led to the use of biological methods by engineered bacteria as an economical and environmentally friendly option. In order to perform this test, 36 bioreactors were built with plastic media bed with inoculation broth lactose culture medium solution, 18 bioreactors had genetically engineered bacteria and 18 bioreactors had non-genetically engineered bacteria. Then pyrene and phenanthrene aromatic substances were added with different concentrations. (10, 50, and 100 ppm) theat were added to the bioreactors. there was during the addition of the above solutions, the pH of each bioreactor was adjusted to normal with the help of sodium hydroxide and 0.1 hydrochloric acid, so that the bioreactors had 3 different pH 3, 7, and 9. Finally, the residual concentrations of pyrene and phenanthrene aromatic substances were measured by liquid chromatography (HPLC). There are some well result . The results of this research show that there is a significant difference between the efficiency of removing aromatic substances by two groups of engineered and non-engineered Pseudomonas putida bacteria, and the biodegradation of phenanthrene and pyrene by the bioreactor containing the genetically engineered Pseudomonas putida bacteria is much higher than the non-engineered Pseudomonas putida bacteria. Therefore, this bacterium can be used in the biological degradation of different concentrations of the above pollutants and cleaning the environment.Introduction



Purification and purification of water contaminated with hydrocarbon substances such as phenanthrene and pyrene is very important due to its dependence on the food cycle. So far, various methods have been used to remove these types of pollutants, each of which has several strengths and weaknesses. The presence of such aromatic compounds in drinking water should be considered in terms of pathogenicity, taste, smell and effect on the appearance of water. Phenanthrene and pyrene are cyclic organic compounds that cause negative and adverse effects on the ecosystem in such a way that even in low concentrations they can be harmful to human health and most of these compounds are toxic, genotoxic and carcinogenic. Therefore, these compounds should be removed from water. Removing organic pollutants from water is an important issue in the industrial field. Considering the importance and high efficiency of bioreactors for the removal of cyclic organic compounds in this research, we have investigated the removal of cyclic organic compounds by constructing an innovative bioreactor, genetically engineered Pseudomonas putida biofilm media from polluted water.

Materials and methods

In this research, to investigate the biofilm performance of two genetically engineered Pseudomonas putida producing deoxygenase enzyme and non-genetically engineered Pseudomonas putida in removing aromatic substances phenanthrene and pyrene, first 36 bioreactors were constructed with plastic media substrate inoculated with lactose broth culture medium solution. 18 bioreactors had genetically engineered bacteria and 18 bioreactors had non-genetically engineered bacteria. Then pyrene and phenanthrene aromatic substances were made with different concentrations (10, 50, and 100 ppm) and added to the bioreactors. In other words, a total of 72 samples were tested. During the addition of the above solutions, the pH of each bioreactor was adjusted to 0.1 normal with the help of sodium hydroxide and hydrochloric acid, so that the bioreactors had 3 different pH values of 3, 7, and 9. Then, the residual concentrations of pyrene and phenanthrene aromatic substances were measured by liquid chromatography (HPLC). Finally, the obtained results were analyzed by statistical studies and the mean and standard deviation values were obtained. Also, independent t-tests, analysis of covariance, one-way and two-way analysis of variance, analysis of repeated measures were used in version 25 of SPSS software.

Discussion

After the cloning of the NahH gene in the PUC18 vector, which was confirmed by the BamHI and EcoRI restriction enzymes, the recombinant plasmids extracted from E. Coli were examined using the PCR method with the help of the HindIII enzyme. The transfer was well to P. putida and then the presence of NahH gene of 924 bp in P. putida was confirmed by PCR. It was observed that the bioreactor containing genetically engineered bacteria, after 8 weeks, produced phenanthrene and pyrene in three concentrations of 50, 10 and 100 mg/liter, on average, in the amount of 94.417, 65.99, and 72/417 respectively. 33 and 45.93, 62.93, 33.54 percent were destroyed. This is while these values were 93.41, 57.72, 17.44 and 92.992, 57.89, 17.75% for the non-engineered bacterial biofilm of Pseudomonas putida, respectively (Tables 8 and 9). In Table 10 and 11, the data related to the comparison of pyrene removal efficiency in engineered and non-engineered bacteria by different concentrations, comparison of removal efficiency between two bacteria by different concentration and pH are given. the percentage of phenanthrene and pyrene removal efficiency is shown in concentrations. The removal efficiency at the concentration of 10, 50 and 100 is different between two bacteria and a significant difference is observed between the removal efficiency of the groups. Therefore, investigating the transformed forms of the recombinant vector (PUC18-nahH), P. Putida with genetic engineering produced through the biosynthesis of catechol 2,3-dioxygenase enzyme for the biological degradation of phenanthrene and pyrene aromatic substances inoculated in the aqueous environment with different acidic, basic and alkaline pH and with three different concentrations of these pollutants in comparison with the non-engineered strain of this bacterium,

Conclusion

Investigating the amount of degradation and removal of phenanthrene and pyrene pollutants by the modified forms of the recombinant vector (PUC18-nahH), P. Putida with genetic engineering show that a significant difference is observed between the efficiency of removing aromatic substances by two groups of engineered and non-engineered Pseudomonas putida bacteria. Therefore, with proper planning in various industries containing these pollutants, the studied engineered forms can be used in the destruction and removal of various concentrations of the mentioned pollutants in order to have a healthier environment.