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Abstract
The present study was aimed to study the effect of some heavy metals and the herbicide 2,4-D on growth of Azotobacter sp. isolated from agriculture soil irrigated with wastewater (Site A) and ground water (Site B) in Sohag governorate. In addition, the most resistant isolates were identified and its ability for fixing nitrogen under stress of two heavy metals and the herbicide 2,4-D were assayed. Finally, the growth pattern and the behavioral response of protein profiles of the most resistant isolate that had the highest nitrogen-fixing ability in the presence of different concentrations of the two heavy metals and the herbicide 2,4-D were studied.
The results obtained from this investigation are summarized in the following points:
1. Determination of heavy metal contents of soil samples indicated that soil irrigated with wastewater (Site A) were contaminated with high levels of a variety of heavy metals as compared to soil irrigated with ground water (Site B).
2. The CFU determination indicated that the bacterial total counts in site (A) is lower than the bacterial total counts in site (B).
3. The higher the heavy metal content of the sample, the lower the bacterial total counts.
4. Examination of the heavy metal resistance of Azotobacter isolates revealed a high incidence of heavy metals resistance among the isolated strains from site (A) compared to these isolates from of site (B) as ~ 42%, 3%, 16%, 5%, 16%, 19%, 17% and 16% of the isolates recovered from site (A) exhibited a maximum MIC of 100 μg/ml for Hg2+, 400 μg/ml for Cd2+, 800 μg/ml for Cu2+, Cr3+, Co2+, Ni2+, Zn2+ and 1200 μg/ml for Pb2+, respectively. On the other hand, 0%, 0%, 4%, 2%, 5%, 4%, 7% and 0% of the isolates recovered from site (B) exhibited the same MIC of the previous metals, respectively.
5. The majority of Azotobacter strains isolated from site (A) showed multiple resistance to different metals ions combinations.
6. Screening for the herbicide 2,4-D tolerance of Azotobacter spp. indicated that the isolates recovered from site (A) exhibited high incidence of herbicide tolerance compared with strains isolated from site (B), as among 64 isolates of Azotobacter spp. recovered from site (A): 3.1, 6.2, 7.8, 15.6, 60.9 and 6.2% were found to be resistant to 100, 200, 400, 600, 800 and 1000 μg/ml of 2, 4- D, respectively. On the other hand, among 56 isolates from site (B): 25, 35.7, 10.7, 14.3, 0, 0% were found to be resistant to the same concentrations of the herbicide 2,4-D.
7. The intrinsic antibiotic resistance of the isolated strains from site (A) showed high resistance against Chloramphenicol, Nitrofurantoin and Co-trimoxazole compared to those isolated from site (B) as from 64 isolates of Azotobacter sp. isolated from site (A): 90.6, 82.8, 70.3% were resistant to three previous antibiotics, respectively. whereas 50, 53.6, 21.4% of isolates from site (B) were resistant to the same antibiotics, respectively.
8. Isolates from site (A) showed multiple antibiotic resistance in different combinations.
9. The obtained results revealed that there is a positive correlation between heavy metal resistance, herbicide tolerance and antibiotic resistance among the isolates from site (A).
10. Determination of pH tolerance of Azotobacter spp. isolated from site (A) indicated that neutral pH has no inhibitory effect on the growth of any of the isolates, while all isolates failed to grow at pH less than 6.
11. Ten isolates selected from contaminated soil (Site A) that showed multiresistance to all tested products were identified as Azotobacter chroococcum based on phenotypic characterization of Bergey’s Manual.
12. The nitrogenase activity of the ten identified isolates of A. Chroococcum under stress of two heavy metals and the herbicide 2,4-D was determined. The results revealed that the nitrogenase activities were markedly decreased with increasing the concentrations of the two heavy metals and 2,4-D.
13. Potential N2-fixation was shown to be negatively affected by additions of ammonium chloride.
14. A. chroococcum WS20 was the most dominant and resistant isolate that exhibited the maximum values of MIC for all the tested products. Moreover, this strain had the highest rate of nitrogenase activity among all other isolates.
15. The optical density for A. chroococcum WS20 decreased under heavy metals stress by increasing the levels of heavy metals.
16. The growth curves of A. chroococcum WS20 under herbicide stress indicated that there was apparent stimulation of growth at the rate of 100 μg/ml 2,4-D, while at higher concentrations, the growth was decreased.
17. Our results showed a positive response of growth of A. chroococcum WS20 to various concentrations of ammonium chloride.
18. The optimum growth of A. chroococcum WS20 was obtained at pH (8) and the growth decreased with increasing the acidity.
19. The gene expression of A. chroococcum WS20 under stress of two heavy metals and the herbicide 2,4-D was detected and there was an alteration of protein profiles.
20. The lack of plasmid profiles of the identified isolates of A. chroococcum indicated that the resistance to the tested heavy metals,the herbicide 2,4-D and the tested antibiotics is attributed to chromosomally- encoded genes.