الفهرس 
Aromatic and chloroaromatic compounds occurrence and persistence in theOnly 14 pages are availabe for public view
 |  | from | 132 |  |  |
| | | from | 132 | | |
Abstract
Aromatic compounds are considered as priority pollutants in the environment due to its hazardous effects both on humans and on the whole ecosystem. Besides those aromatic compounds are hard to be biodegraded in normal environments, it is harder to be degraded in extreme environments either in hyper-salinity and hyper-alkalinity.
In this study; a novel bacterium isolated for the first time on the basis of its ability to utilize benzene, toluene and chlorobenzene from the extremely saline and alkaline lakes of Wadi El Natrun. Chlorocatechol 1, 2-dioxygenase gene was amplified from the identified bacterial strain Alcanivorax HA03.This gene was also cloned in E.coli JM109. Heterologous expression of chlorocatechol 1, 2-dioxygenase from Alcanivorax sp. HA03 was also confirmed.
Thus, the isolation of bacterium Alcanivorax sp. HA03 is of great concern. The ability of Alcanivoax sp. HA03 to grow on benzene, toluene and chlorobenzene had been tested. It showed its ability to grow on these compounds as a sole carbon source. Its growth took rationally long time on these compounds only as a sole carbon source but it showed a rapid growth in case of presence of precursors to its growth like glucose, salicylate, and pyruvate beside benzene, toluene or chlorobenzene. The role of these precursors may be the initiation of growth till the bacterium adapted to the new conditions which enable it to use benzene, toluene and chlorobenzene in its metabolic system.
On the other hand Alcanivorax sp. HA03 showed no growth in case of addition of lactate as precursor for growth beside the presence of benzene, toluene and chlorobenzene. This may denote to the ability of lactate to inhibit its growth or the lack mechanism in its metabolic system to grow on it. The degradation was also increased in the presence of glucose compared to salicylate or pyruvate. The findings proclaimed the ability of the isolated strain Alcanivorax sp. HA03 to be used in the future as an in situ- bioremediation processes. Alcanovorax sp. HA03 showed its ability to grow on different salt concentrations beside its ability to degrade benzene, toluene and chlorobenzene. As it has the ability to grow on in salinity ranging from 3% to 15%. Both growth rate and degradation rate differed according to salt concentration. As in case of salt concentration 3%, the growth rate was high and also the biodegradation rate in only two weeks. Alcanivorax sp. HA03 has the ability to degrade the added content of benzene, toluene and chlorobenzene to the media in 2 weeks at a rate of 2.85, 2.15 and 0.75 μmol/flask/day respectively. With increasing salt concentration to 7% , the bacterium showed slower growth rate and also biodegradation rate, as benzene, toluene and content was consumed in four weeks with rate of 1.45 and 1.25 μmol/flask/day respectively while chlorobeneze degradation did not detected. Only benzene was degraded in salt concentration 10%-15% in a rate of (1 to 0.75 μmol/flask/day), while no growth or biodegradation detected for both toluene and chlorobenzene.
Also, it is for the first time to prove the ability of genus Alcanivorax represented in Alcanivorax sp. HA03 isolated from soda lakes in Wadi E1Natrun to degrade low molecular weight hydrocarbons over a broad range of salinity. Alcanivorax sp. HA03 also showed a regioselectivity of attack on chlorobenzene not previously reported.As 3- chlorocatechol is the bottle neck of the degradation chlorobenzene we attempted to isolate gene(s) responsible for its degradation which is initiated by 1, 2 chlorocatechol dioxygenase. A degenerative primer fw-cldioxmtand rev-cldioxmt was used to amplify fragments of the gene responsible for 1, 2 chlorocatechol dioxygenase. This primer set targeted the highly conserved region localized on chlorocatechol 1, 2-dioxygenase involved in the degradation of chlorobenzene. The obtained fragment was as expected amplification product of ~280 bp. The resultant PCR products was ligated into pGEM®-T Easy Vector and transformed into E. coli JM109 after heat shock. Then Sequence analysis performed to the PCR products which revealed the similarity between the obtained fragment of Alcanivorax sp. HA03 and that of 1, 2 chlorocatechol enzyme in Delftia acidovorans. This was confirmed by amplification and sequencing of the fragment with vector-specific M13–forward and M13-reverse primers
A specific primer set CC1, 2HF and CC1, 2HR was used to isolate the complete gene 1, 2 chlorocatechol from Alcanivorax sp. HA03 and the expected size was 760bp. The resultant PCR products was ligated into pGEM®-T Easy Vector and transformed into E. coli JM109 after heat shock. Then Sequence analysis performed to the PCR products. The transformed colonies can grow on 3-chlorocatechol and this is confirmed by HPLC analysis as presence of 2-chloromuconate was detected. Analysis of tfd gene was detected on SDS-PAGE and it was on 27.5 kDa and also was overexpressed even with diluted sample.