الفهرس 
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Abstract
Bioremediation has been demonstrated as an appropriate and effective alternative to cleaning up hydrocarbon-contaminated environments. The synthesis of nanoparticles with the help of microorganisms has provided a cost-effective and eco-friendly strategy. Due to their unique properties, nanoparticles have a broad range of commercial applications in various industries, and in the fields of enhanced oil recovery and environmental bioremediation.
In the present study, the main target was to synthesize nanoparticles by utilizing some bacterial strains isolated from oil-polluted water samples. In addition, the role of nanoparticles in enhancing the biodegradation of crude oil is investigated.
To achieve this target the following items were studied:
Physico-chemical properties of water samples:
The different physico-chemical parameters of the water samples obtained from 1 and 2 sites at the oil refinery company were determined. The results indicate that the sample from site 2 has a higher electrical conductivity than the site 1 water sample and a high total dissolved solids, density, and salinity.
Physico-chemical characterization of a crude oil sample:
The different physico-chemical parameters of the crude oil samples were analyzed according to ASTM D-1298
Isolation and screening of biodegrader bacterial isolates:
Seventeen bacterial isolates (B1-B17) were detected in oil-contaminated water samples by different culture media. In water sample (1), the bacterial isolates B11 and B5 are considered predominant isolates on BHM media. While in the water sample (2) , the bacterial isolates B1, B11, and B8 are the most predominant isolates on BHM media. The bacterial isolate B13 is considered the predominant isolate on LB and MSM media. So, B1, B5, B8, B11, and B13 are the most predominant isolates on different types of media.
Testing the ability of bacterial isolates for the production of nanoparticles:
In the present study, the screening of bacterial isolates for producing nanoparticles and the selection of the highest nanoparticles producer were investigated under growth conditions (incubation time 24 hr., pH 7.3, precursor concentration 50 ml, inoculum size 5 ml).
Determination of the biosynthesis of Fe oxide nanoparticles:
Extracellular biosynthesis of Fe nanoparticles from Fe3+ solution was investigated using the Seventeen bacterial isolates. The DLS results showed that the smallest particle size among the prepared magnetic nanoparticles was achieved by B8 and B5 (75.62 and 72.41nm, respectively).
Also, gravimetric analysis was used to determine the percentage quantity of Fe-Nps in different supernatant culture media, the highest percentage of Fe-Nps production was observed with B3, B5, and B8.
Testing the production of zinc oxide nanoparticles by different bacterial isolates:
The DLS results showed that the smallest particle size among the prepared nanoparticles was achieved by B2 and B10 (61.57and 77.88nm, respectively).
Gravimetric analysis was used to determine the percentage quantity of Zn-Nps in different supernatant culture media, the highest percentage of Zn-Nps production was observed with B2, B4, and B10.
The production of copper oxide nanoparticles by using different bacterial isolates:
In this study, there was no high production of CuO-Nps was with the lowest nanoparticle size. On the other hand, for the supernatant of culture media of bacterial isolate (B4) has the lowest particle size (111-nm) and the highest production of CuO-Nps compared with the other investigated bacterial isolates.
Phylogenic analysis of the selected oil-degrading bacteria:
The bacterial isolates (B5 and B8) were selected for the production of iron nanoparticles.The bacterial isolate (B5) wasidentified by 16S rRNA as Pseudomonas aeruginosastrain DSM 50071with a similarity of 98.99 %. (B8) is identified by 16S rRNA as Pseudomonas aeruginosa ATCC 10145 with a similarity of 99.53 %.
On the other hand, the bacterial isolates (B2 and B10) were selected for the production of zinc nanoparticles. The bacterial isolate (B2) was identified by16S rRNA as Enterobacter hormaechei subsp. Xiangfangensis strain with a similarity of 99.17 %. (B10) is identified by16S rRNA as Stenotrophomonas pavanii strain LMG 25348 with a similarity of 99.15 %.
The optimum conditions for the production of different nanoparticles:
The effect of different conditions (incubation time, pH, precursor concentration (FeCl3, ZnSO4.7H2O), and inoculum size) on the production of Fe-Nps and Zn-Nps was estimated by gravimetric and DLS size analyses. The optimum conditions for the production of zinc oxide nanoparticles by bacterial strains (B2 and B10) and iron oxide nanoparticles by bacterial strains (B5 and B8) were B, G, and J of different experimental conditions.
Meanwhile, (B2) producedthe lowest ZnO nanoparticles size (61 nm) under condition(J). (B5) produced the lowest FeO nanoparticles size (71nm) under condition (J). The optimum condition (J) is (incubation time: 24 hr.) (pH: 7) (ZnSO4.7H2O precursor conc.: 50 ml) (inoculum size:50 ml).
Characterization of the extracellular biosynthesized iron and zinc nanoparticles:
The color of Fe ions changed from yellowish brown to dark brown , while the color of Zn ions changed from yellow to white precipitate.
The obtained bio-Nps were characterized by different analytical toolsas (XRD, SEM, and EDX).
Using XRD analysis a successful synthesis of the ZnO crystal structure with 2Zn3 (bacterial strain (B2) under experimental conditions (J ), incubation time:24 hr., PH: 7, ZnSO4.7H2O precursor conc.:50 ml, inoculum size:50 ml)and Fe nanoparticles crystal structure with 5Fe3 bacterial strain (B5)under experimental conditions (J ), incubation time:24 hr., PH:7, FeCl3precursor conc.:50 ml, inoculum size :50 ml .
Using the SEM analysis images of the synthesized FeO-NPs and ZnO-Nps at different magnifications, the average particle size of the biosynthesized iron nanoparticles by p. aeruginosa is 43.14nm, and the ZnO-Nps showed pyramidal symmetry with agglomeration among the particles. The average particle size of the biosynthesized zinc nanoparticles by E. hormaechi is 38.59nm. Additionally, the EDX analysis confirmed the presence of zinc oxide nanoparticles grown by the biosynthesized method. The elemental analysis of the ZnO- NPs yielded " ~ " 30 % of zinc and " ~ " 70 % of oxygen, proving that the produced ZnO- NP is in purified form. while the FeO-NPs yielded " ~ " 10 % of iron and " ~ " 90 % of oxygen, which proves the production of FeO-NPs.
Biodegradation of a petroleum oil sample using the degrading bacterial strains:
This work extended to measure the capacity of the different bacterial isolates (B1-B17) to biodegrade the crude oil sample and mixture of bacterial isolates (bacterial consortium) incubated in MSM media with crude oil used as a sole carbon source for 14 days. The percentage of the biodegraded oil was calculated, and the alteration in its chemical composition was detected by GC analysis.
Firstly, the visible observation of different bacterial microcosms and the crude oil sample were studied.
At the end of the incubation period (14 days), the oil sample was aggregated in all different microcosms. The highest oil accumulation was determined in microcosms containing (B1, B11, B7, and, B15) separately.
Gravimetric analysis of the degraded crude oil:
The results revealed that the bacterial strains degraded the crude oil in the range of 18% to 95% after 14 days of incubation period.
Gas chromatographic analysis:
The data obtained showed that the percentage degradation ranged from 16.76 % to 97.85 % with the seventeen bacterial isolates. Additionally, the highest percentage degradation was determined in microcosms containing (B4, B7, B8, B9, B11, B13, B14 and a mixture of bacterial isolates about 90.58, 93.35, 97.7, 95.85, 96.59, 94.17, 97.07 %, and 97.85 respectively).It is evident that, the bacterial consortium had the ability to degrade the crude oil sample more than the individual bacterial isolate.
Biodegradation of a crude oil sample in the presence of iron oxide and zinc oxide nanoparticles:
The biodegradation of crude oil was performed using different bacterial strains (B2,B5, B4and B11) with bio FeO-Nps and bio ZnO-Nps. The effect of different Nps on the degradation of crude oil sample in the absence of different bacterial strains was tested. The bioremediation of crude oil sample by the bacterial consortium was more enhanced in the presence of biosynthesized iron oxide nanoparticles than zinc oxide nanoparticles. Thus, these consortium bacterial isolates have the potential to be applied in the bioremediation of petroleum-contaminated sites using specific concentrations of iron oxide nanoparticles.