الفهرس 
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Abstract
Heavy metals are severe pollutants released into the environment and can form compounds that can be toxic, carcino-genic or mutagenic, even in very low concentrations (Idise et al., 2010). Industries such as mining, metals melting, pesticides, tanneries leather industries among others produce waste water loaded with polluted materials (Joshi et al., 2011). The most hazard pollutants are heavy metals such as mercury, lead, cadmium, selenium, copper, chromium and arsenic which cause great environmental problems (Ronda et al., 2007). An additional dimension to the problem of heavy metal pollution is the lack of reliable, efficient and cost effective method of waste treatments that ensure their removal. Most, if not all of the physicochemical methods of the waste treatment available are fraud with many problems (Ahluwalia and Goyal, 2007).
In recent years, the search for the removal of toxic metals has focused on new technologies rather than conventional methods like ion exchange, chemical precipitation, ultra filtration and so on (Veit et al., 2005) which are expensive, need energy and can produce waste products that require careful disposal (Ahalya et al., 2003).
Therefore, the biological approaches have been considered as an alternative remediation for heavy metals removal from wastewaters (Pena-Castro et al., 2004). One of these methods is the bioremoval which can be defined as the ability of biological materials to accumulate heavy metals through metabolism or physico-chemical pathway of uptake (Fourest and Roux, 1992).
Although the bioremoval of heavy metals is a general character of microbial cells, fungi are superior as they have high ability to accumulate, tolerate and detoxify metals (Zafar et al., 2007) through the metal-binding capacities of their cell walls (Gupta et al., 2000).
The present investigation was planned to throw a light on the study of the great importance of using microorganisms especially fungi in bioremediation of heavy metals from industrial waste water. Because of toxicity of heavy metals antibiotics, using microorganisms has been increased due to their efficiency and safety.Water samples were collected from Outlet the main drain station in industrial area in Port Said.
1) - Physico-chemical analysis were carried out immediately at the sampling sites or immediately on arrival to laboratory as it is possible to determine the properties of this water and average was as follows:
pH 9.5, Turbidity 164 ntu, T.S.S 78, TDS 2450 mg / L dissolved oxygen 7.3 mg / L, COD 337 mg / L, BOD75mg / L which are above the guidelines of the Egyptian Environmental law N (48) standards for the year (1982) for the discharge of industrial effluents into the public sewage network
2) - Microbiological analysis of industrial waste water
2.1)-Isolation of microorganisms from industrial waste water:
A total of 55 isolates were isolated from industrial waste water on 4 types of cultures media .these media were nutrient agar media, starch casein agar, Czapek Dox medium and potato dextrose agar media. These isolates are distributed as, 12 isolates of them are reported to grow on nutrient agar medium, 9 isolates are recorded to grow on starch casein agar medium, 16 isolates are grown on Czapek Dox agar medium and 18 isolates are grown on potato dextrose agar medium.
Macroscopical and morphological examination results also showed that among these isolates 15are belonging to fungi (Syncephalastrum racemosum, Alternaria alternata, Betraniella sp., Byssochalamus niveus, Emericella nidulans , Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus tamarii, Cladosporium spherospermum,Colletotricum sp., Curvularialunata, Penicillium chrysogenum, Penicillium digitatum, Penicillium oxalicum ), 12 isolates are belonging to actinobacteria, and 28 isolates are belonging to bacteria.
2.2) -Screening of selected isolates for heavy metals removal:
The 15 fungal isolates were screened for their ability to degrade some heavy metals reported in industrial waste water. The heavy metals tested were Cu+2, Fe+2, Cd+2, Zn+2 and Cr+5 results proved that the isolates No.14 (A.niger) and isolate No.15 (A.flavus)were shown to be the most active organisms in degrading majority of tested heavy metals.
2.3) - Antagonistic effect:
The antagonistic effect between A. niger and A.flavus showed that there is no antagonistic relation between them.
3) - Effect of different environmental factors on growth of A. niger and A.flavus species mixed culture
3.1)-Effect of incubation period on mixed culture growth of A. niger and A.flavus:
Results showed that the high culture growth (405 mg D.wt /50 mL) was reported at incubation period of 21 days medium while the lowest growth (340mg D.wt /50 mL) was recorded at incubation period of7 days. While after incubation period of 14 days the growth was (374 mg D.wt /50 mL) and it was (384 mg D.wt /50 mL) after incubation period of 30 days
3.2)-Effect of incubation temperature on mixed culture growth of A. niger and A.flavus:
Results showed that the high culture growth (417mg D.wt /50 mL) was reported after incubation period of 21 days at incubation temperature of 30 oC while the lowest growth (94mg D.wt /50 mL) was recorded at incubation temperature of 5 oC. The cultural growth was recorded (344mg D.wt /50 mL) at incubation temperature of 40oC and (235mg D.wt /50 mL) at incubation temperature of 20oC.
3.3)-Effect of pH on mixed culture growth of A. niger and A.flavus:
The data indicated that the high culture growth (512 mg D.wt /50 mL) was reported after incubation period of 21 days at incubation pH 5.4 while the lowest growth (211 mg D.wt 50 mL ) was recorded at incubation pH of 10.8.While at pH 3.6 the cultural growth was (372 mg D.wt /50 mL), at pH 4.6 the cultural growth was(425 mg D.wt /50 mL), at pH 6.4 the cultural growth was recorded (456 mg D.wt /50 mL), at pH 8 the cultural growth was(403 mg D.wt /50 mL)and at pH 9.5 the cultural growth was recorded(289 mg D.wt /50 mL).
3.4) - Effect of different concentrations of heavy metals on mixed culture growth of A. niger and A.flavus:
The results indicated that for Cu+2 the high culture growth (405 mg D.wt /50 mL) reported after incubation period of 21 days at concentration 10 ppm while the lowest growth (308mg D.wt /50mL) was recorded at concentration 100 ppm. The cultural growth was recorded (329mg D.wt /50mL) at concentration of 20 ppm and (311mg D.wt /50mL) at concentration of 50 ppm.
But for Zn+2 the high culture growth (442mg D.wt /50mL) was reported after incubation period of 21 days at concentration 10 ppm while the lowest growth (359mg D.wt /50 mL) was recorded at concentration 100 ppm. The cultural growth was recorded (403mg D.wt /50 mL) at concentration of 20 ppm and (326mg D.wt /50 mL) at concentration of 50 ppm.
For Fe+2 the high culture growth (382mg D.wt /50 mL) was reported after incubation period of 21 days at concentration 10 ppm while the lowest growth (324mg D.wt /50 mL) was recorded at concentration 100 ppm. The cultural growth was recorded (342mg D.wt /50 mL) at concentration of 20 ppm and (326mg D.wt/50mL) at concentration of 50 ppm.
That for Cd +2the high culture growth (403mg D.wt /50 mL) was reported after incubation period of 21 days at concentration 10 ppm while the lowest growth (332mg D.wt /50 mL) was recorded at concentration 100 ppm. The cultural growth was recorded (376mg D.wt /50 mL) at concentration of 20 ppm and (336mg D.wt /50 mL) at concentration of 50 ppm.
And for Cr+5 the high culture growth (398mg D.wt /50 mL) was reported after incubation period of 21 days at concentration 10 ppm while the lowest growth (342mg D.wt /50 mL) was recorded at concentration 100 ppm. The cultural growth was recorded (396mg D.wt /50 mL) at concentration of 20 ppm and (348mg D.wt /50 mL) at concentration of 50 ppm.
4)-Effect of different environmental factors on heavy metals removal by mixed culture growth of A. niger and A.flavus:
4.1)-Effect of incubation period on heavy metals removal by mixed culture growth of A. niger and A.flavus:
Results showed that the highest heavy metals removal was reported after incubation period of 21 days while the lowest heavy metals removal was recorded after 7 days.
The data show that Cu+2was recorded the highest bio removal (67%) after incubation period of 21 days while the lowest bio removal (31%) after incubation period of 7 days. The result also indicate that the bio removal of Cu+2 was (55%) at incubation period of 15 days and (61.9%) at incubation period of 30 days.
Fe +2 recorded the highest bio removal (76.5%) after incubation period of 21 days while the lowest bio removal (57.4%) after incubation period of 7 days. The result also indicate that the bio removal of Fe+2 was (73.19%) at incubation period of 15 days and (75%) at incubation period of 30 days.
The highest bio removal (53%) for Cd+2 was recorded after incubation period of 21 days while the lowest bio removal (19.8%) after incubation period of 7 days. The result also indicate that the bio removal of Cd+2 was (42%) at incubation period of 15 days and (45%) at incubation period of 30 days.
The data presented show that Zn+2 was recorded the highest bio removal (88.4%) after incubation period of 21 days while the lowest bio removal (79.9%) after incubation period of 7 days. The result also indicate that the bio removal of Cu+2 was (83.9%) at incubation period of 15 days and (86%) at incubation period of 30 days.
Cr+5was recorded the highest bio removal (58.9%) after incubation period of 21 days while the lowest bio removal (15%) after incubation period of 7 days. The result also indicate that the bio removal of Cr+5 was (35%) at incubation period of 15 days and (50 %) at incubation period of 30 days.
4.2)-Effect of temperature on heavy metals removal by mixed culture growth of A. niger and A.flavus:
Results showed that the high heavy metals removal was reported after incubation period of 21 days at incubation temperature of 30oC while the lowest removal was recorded at incubation temperature of 5oC.
The data show that Cu+2 was recorded the highest bio removal (69%) after incubation period of 21 days at incubation temperature of 30oC while the lowest bio removal (7.8%) at incubation temperature of 5oC. The result also indicate that the bio removal of Cu+2 was (33.3%) at incubation temperature of 40oC and (27.7%) at incubation temperature of 20oC.
The highest bio removal (79%) for Fe+2 was recorded after incubation period of 21 days at incubation temperature of 30oC while the lowest bio removal (18.7%) at incubation temperature of 5oC. The result also indicate that the bio removal of Fe+2 was (60%) at incubation temperature of 40oC and (43%) at incubation temperature of 20oC.
The data presented also show that Cd+2was recorded the highest bio removal (55%) after incubation period of 21 days at incubation temperature of 30oC while the lowest bio removal (7%) at incubation temperature of 5oC. The result also indicate that the bio removal of Cd+2 was (29.6%) at incubation temperature of 40oC and (21%) at incubation temperature of 20oC.
The results also indicates that the highest bio removal for Cr+5 (60%) after incubation period of 21 days at incubation temperature of 30oC while the lowest bio removal (5%) at incubation temperature of 5oC. The result also indicate that the bio removal of Cr+5 was (22%) at incubation temperature of 40oC and (18%) at incubation temperature of 20oC.
The results also shows that the highest bio removal for Zn+2 (89.6%) after incubation period of 21 days at incubation temperature of 30oC while the lowest bio removal (15%) at incubation temperature of 5oC. The result also indicate that the bio removal of Zn+2 was (80%) at incubation temperature of 40oC and (59%) at incubation temperature of 20oC.
4.3) - Effect of pH on heavy metals removal by mixed culture growth of A. niger and A.flavus:
Results showed that the high heavy metals removal was reported after incubation period of 21 days at incubation pH 5.4 while the lowest heavy metals removal was recorded at incubation pH of 10.8.
The data presented show that Cu+2was recorded the highest bio removal (83.7%) after incubation period of 21 days at incubation pH of (5.4) while the lowest bio removal (27%) at incubation pH of (10.8) .The result also indicate that the bio removal of Cu+2 was (58.3%, 77.6%, 80%, 70% and 38%) at incubation pH of (3.6, 4.6, 6.4,8 and 9.5) respectively.
The results show that Zn+2was recorded the highest bio removal (91%) after incubation period of 21 days at incubation pH of (5.4) while the lowest bio removal (56%) at incubation pH of (10.8) .The result also indicate that the bio removal of Zn+2 was (69%, 80%, 88%, 77% and 64%) at incubation pH of (3.6, 4.6, 6.4,8 and 9.5) respectively.
The data show that Fe+2was recorded the highest bio removal (88%) after incubation period of 21 days at incubation pH of (5.4) while the lowest bio removal (55%) at incubation pH of (10.8) .The result also indicate that the bio removal of Fe+2 was (68%, 84%, 87%, 82% and 63%) at incubation pH of (3.6, 4.6, 6.4,8 and 9.5) respectively.
The data show that Cd+2 was recorded the highest bio removal (59%) after incubation period of 21 days at incubation pH of (5.4) while the lowest bio removal (42.7%) at incubation pH of (10.8) .The result also indicate that the bio removal of Cd+2 was (45%, 56%, 58%, 52% and 43.6%) at incubation pH of (3.6, 4.6, 6.4,8 and 9.5) respectively.
The result also indicate that Cr+5was recorded the highest bio removal (62.8%) after incubation period of 21 days at incubation pH of (5.4) while the lowest bio removal (1.3%) at incubation pH of (10.8) .The result also indicate that the bio removal of Cr+5 was (26%, 36.5%, 51.8%, 32%and4.8%) at incubation pH of (3.6, 4.6, 6.4,8 and 9.5) respectively.
Conclusion and recommendation
• Industrial waste water is a source of water surface pollution by heavy metals which cause negative effects on the environment
• Many microorganisms proved to be able to remove heavy metals from industrial waste water
• Using a mixed culture population of fungi in the treatment processes is given better than the application of mono fungal culture
• Environmental culture conditions proved to affect greatly the process of heavy metal removal by mixed culture population
• We recommended to apply a further study on heavy metals bioremediation by mixed culture of fungi to convert this technique from the lab scale to large field scale, this will produce a possibility to environmentally reuse.