Molecular study on thermotollerant bacteria that degraded nitroarmatic compoundsr

The main aim of this study was isolating and identifying thermophilic bacteria capable of utilizing aromatic hydrocarbon that form part of petroleum components. For this purpose (95) hydrocarboncontaminated
soil samples were collected from different places in Basrah and Baghdad. Fourty eight bacterial isolates that utilize crude oil were isolated. Thirty eight mesophilic bacterial isolates showed good ability to grow on
crude oil as the only carbon and energy source. These isolates were characterized as: (20) Pseudomonas, (3) Staphylococcus, (3) Acinetobacter, (2) Alcaligenes, (2) Aeromonas, (2) Flavobacterium, and (6) Bacillus. In addition to these genera, ten thermophilic bacterial isolates showed an excellent ability to utilize crude oil as the sole source of carbon, nitrogen and energy. Utilization of aromatic compounds (Naphthalene, Carbazole, Nitrobenzene, and ρ-Nitrophenol) of these ten isolates was studied. Results showed a diverge utilization ability. These ten isolates cannot be distinguished by ordinary morphological, physiological and biochemical tests; therefore, they (for four selected isolates) were subjected to molecular identification through PCR amplification of 16S rDNA by using 7 primers (fd1, fd2, fd3, fd4, rd1, rp1 and rp2) which represent primers for the PCR amplification of eubacterial 16S rDNA, and followed by sequencing. The nucleotide sequence data was compared with 16S rDNA sequences of other culture on BLAST of the National Center of Biotechnology Information database (NCBI database). According to the results of molecular identification, these isolates characterized as, 2G (Ir1) (Geobacillus thermoleovorans), 3A (Ir2) and 4A (Ir3) (Anoxybacillus rupiensis), and were deposited in the National Genebank database under the accession number, JQ912239, JQ912240, and JQ912241 respectively. Anoxybacillus rupiensis strain Ir3 (JQ912241) was selected for its ability to utilize all the aromatic compounds efficiently and used throughout this study. The plasmid profile of the selected isolate A. rupiensis strain Ir43 (JQ912241) showed that this bacterium contain large and small plasmid DNA bands. In order to determine the role of plasmid in utilization of aromatic compounds (carbazole for example), many attempts were made to cure A. rupiensis strain Ir3 (JQ912241) plasmid (s) using Sodium dedocyl sulphate (SDS) and Ethidium Bromide (ET Br). Results indicated that no cured colonies (lost their ability to utilize carbazole at 70◦C) were obtained. Plasmid isolated from some of these colonies after treated with Et Br, indicated that these colonies are still harboring the large plasmid, which is difficult to cure, and the trait might be located on it. The optimum conditions for growth of A. rupiensis strain Ir3 (JQ912241) were determined. The results showed that growing in LB
(Luria-Broth) medium (pH 7) containing 0.5 to 1% of NaCl, and incubated with shaking (150 rpm) at 55 -65◦C for 24h. It was also found that this bacterium was able to withstand 80◦C for 90 min. The optimum
conditions for growth of A. rupiensis strain Ir3 (JQ912241) on minimal medium (CDM) were adjusted, pH to 7 and incubated at 55-65◦C and the bacterial growth was increased with carbazole concentration increasing.
This means that bacterial growth with Carbazole (CAR) as an N-source was concentration-dependent.
To confirm the ability of A. rupiensis strain Ir3 (JQ912241) to utilize the aromatic compounds, analytical experiments include HPLC (High performance liquid chromatography) and FTIR (Fourier transform infrared) were used. The results of quantitative analysis (HPLC) indicated that this bacterium showed as much as 99.62% consumption of carbazole, 99.4% of ρ-nitrophenole, 97.73% of nitrobenzene and 98.89% of naphthalene. Qualitative analysis of FTIR spectra showed that A. rupiensis strain Ir3 (JQ912241) has the ability to convert carbazole to anthranilic acid, indicating the presence of meta cleavage enzyme, this
also confirmed by using 2, 3-dihydroxybiphenyl through converting the colonies color on Luria-Bertani (LB) and minimal agar plates to brown.