الفهرس 
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Abstract
The aim of this study was undertaken to assess six heavy metal levels (Zn+2, Fe+2, Co+2, Cd+2, Cu+2, and Pb+2) in industrial wastewater effluent and could be also isolate promising bacteria from industrial wastewater and Azolla having to decrease or remove it is metals present in the industrial effluent.
Sample collection and isolation of heavy metals tolerated bacteria:
Wastewater samples were collected from the site of the main drain of Abu Kerqas Sugar Factory (27°55’28.8”N 30°49’00.4”E), El-Minia governorate in the month of March, 2017. The samples were prepared and analyzed for determination of it is metals that common, more toxic and compared it to the permissible limits according to WHO.
Also, the desired bacteria that have more tolerance to high concentration of heavy metals were selected by using streak plate technique. The isolated bacteria were subjected to heavy metals resistance assay by using the diameter of inhibition zone (cm) technique.
Among the 30 isolated bacteria which appear resistance to six heavy metals, ten isolates were chosen and tested to bacterial growth curve, (IAA) reaction and antibiosis activities. Out of 3 isolates recorded a promising bacterium for uptake of heavy metals from EIWW.
The three selected isolates (Bacillus) were identified and characterized morphologically and biochemically. Also, these isolates were identified using 16S rRNA with the help of Microbiology Labs., Zhejiang University, Hangzhou city, East China.
The obtained sequences data were compared with the sequences in the databases analyzed using the software DNAMAN 6.0 protein sequences was aligned through the BLAST P program. These bacteria were classified as Bacillus sp. SMMAA-1, Bacillus cereus SMMAA-3 and Bacillus altitudinis SMMAA-4 and documented in GenBank under the accession numbers of LC472522, LC472523 and LC472524 respectively.
Bioremediation of heavy metals:
1- Using bacteria:
The selected bacterial isolates (Bacillus spp, B. cereus and B. altitudinis) were tested for their ability to alleviate or eliminate the heavy metal concentrations for industrial wastewater effluent.
2- Using Azolla filiculoides as fresh
3- Using Azolla filiculoides as dry
4- Using Azolla filiculoides as fresh + selected bacterial isolates
5- Using Azolla filiculoides as dry + selected bacterial isolates
The obtained data revealed that:
I- Isolation of tolerated bacterial to heavy metals toxicity:
1- Six heavy metal ions (Zn+2, Fe+2, Co+2, Cd+2, Cu+2 & Pb+2) were the main founded in the studied industrial wastewater samples (IWW). The conc. of the six heavy metals were arranged between 6ppm (Zn+2) to 1.4 ppm (Co+2). The heavy metals (Co, Cd & Pb) were founded more folds than the guidelines of the WHO.
2- Through the isolation of selected bacteria, we have been observed that the best dilution for IWW to obtained single separated bacterial colonies was the conc. (0.25ml) and we have designated 30 isolates which coded by T1, T2, T3, T4 …to T30.
3- A varying response of 30 bacterial isolates to the six heavy metals and its concentrations were observed. The best tolerated isolates to six heavy metals were recorded with 8 isolates (T1, T2, T3, T4, T5, T6, T7 & T8). Also, T10 isolate gave resistant to Cd, Cu and Pb, and T18 isolate to Zn, Fe and Co metal ion.
II -Toxic effect of IWW on growth curve, IAA production of selected bacterial isolates and antibiosis activities of its bacterial isolates against (F. solani, S. rolfsii & E.coli):
a – Toxic effect of IWW on bacterial growth curve:
from the 10 selected bacterial isolates, the data depicted that three isolates (T1, T3 & T4) were founded to be continuously growth from the day No.1 to the day No. 5 and the optimum growth period was registered in the day No.4. These results considering that the selected bacterial isolates (T1, T3 & T4) a promising bacteria for uptake of heavy metal ions from EIWW.
b- Toxic effect of IWW on IAA production:
from the 10 selected bacterial isolates, the data revealed that three isolates (T1, T3 & T4) were founded to be the best IAA reaction.
c- Antibiosis activities against F. solani, S. rolfsii & E.coli:
The obtained data revealed that the all bacterial isolates have positive reaction against the pathogen microorganisms (F. solani, S. rolfsii & E.coli). While, isolates (T2 and T6) have negative reaction against F. solani & S. rolfsii.
III- Morphological, Biochemical characteristics & Molecular identification of selected bacterial isolates (T1, T3&T4):
from classifying bacterial isolates and the advanced technology such as nucleotide sequences of 16S rRNA gene in Microbiology Lab., Agriculture faculty, Zhejiang University, Hangzhou city, East China, the data exhibited that the coded (T1, T3 & T4) as Bacillus spp., Bacillus cereus and Bacillus altitudinis respectively.
IV- Bioremediation of heavy metals from IWW:
a- By using the isolated bacteria (Bacillus spp., B. cereus and B. altitudinis):
Data showed that all bacterial isolates tested (Bacillus spp., B. cereus and B. altitudinis) have ability for biosorption & detoxification of the six heavy metals and its effects were differed among them. B.cereus is significantly more effective for removal efficiency % than Bacillus spp. and B. altitudinis. Co+2, Cd+2, and Cu+2 ions were more absorbed by bacterial isolates than other heavy metals.
b- By using Azolla filiculoides fronds as fresh (Af):
The highest removal efficiency% was founded in the treatment 50%SIWW+50% tap water + Azolla fresh (56.78%) compared to the treatment 100% SIWW + Azolla fresh (52.39%). Our results demonstrated that the importance for the dilution process for bioremediation to fast bio absorption or detoxifying of the heavy metals from the wastewater polluted to obtain the good results.
The highest removal efficiency % was recorded with Zn+2 (72.26%) even though, the removal efficiency % of Cu+2 and Pb+2 were below (37.09% & 48.47%). The effective treatment was founded when SIWW was diluted by tap water 50% and treated with Azolla filiculoides as fresh.
c- By using Azolla filiculoides fronds as dry (Ad):
The best treatment for removing heavy metal conc. in SIWW with the dilution treatment of 50% SIWW + 50% tap water + Azolla dry (0.687ppm) when compared with 100% SIWW + Ad (0.730ppm). These results gave the important role of Azolla in treated of IWW for decreasing of heavy metals conc. especially when the concentration of heavy metals are below.
The highest removal efficiency % was observed by the diluted treatment (SIWW50%+ tap water 50% +Ad) with Zn+2 ion (82.97%) while the lowest removal % was recorded by the 100% SIWW + Ad with Cu+2 ion (49%).
d- By using the combined of Azolla filiculoides fronds as fresh (Af) with the isolated bacteria (Bacillus spp., B. cereus & B. altitudinis):
1- The highly significance removal % was recorded with combined Azolla as fresh and B. altitudinis (98.58%), Bacillus spp. (98.40%) & B. cereus (98.39%) without significant difference among them.
2- The maximum removal % of heavy metals were observed as followed (Zn+2 > Co+2 > Cd+2 > Pb+2 > Cu+2 > Fe+2) and the values of its metals were (99.68% > 99.61 % > 99.30 % > 99.11 % > 97.67% > 95.38 %) respectively.
3- The best combined treatments for removal % of heavy metals was founded with the combined treatment (Af + Bacillus spp.) with Co+2 ion (99.91%). The lowest removal % was observed with the treatments (Af+ Bacillus spp.) and (Af+ B. cereus) with Fe+2 (95.19%).
e- By using the combined of Azolla filiculoides fronds as dry (Ad) with the isolated bacteria (Bacillus spp., B. cereus & B. altitudinis):
1- The combined treatment of Ad + B. altitudinis was highly removal efficiency for heavy metals (98.63%) than other treatments.
2- The removal % of heavy metals arranged from the high to low (from 99.88% for Co+2 to 94.40% for Fe+2) as followed Co+2 > Cd+2 > Zn+2 > Pb+2 > Cu+2 > Fe+2.
3- The all combined treatments of Bacillus spp., B. cereus and B. altitudinis + Ad with Co+2 showed highly removal % (99.92 %, 99.86% and 99.86%) respectively. The lowest removal % by the above mentioned combined treatments mean were recorded with Fe+2 (94.40%).