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Abstract The present work was designed to investigate the fungal endophytes of some Egyptian plants irrigated with industrial waste water as a new biosorbent of cadmium, copper and lead. The study included the following items: 1- Collecting samples of industrial waste water effluents from a certain factories. 2- Analysis of these industrial waste water samples to determine the type of metal ions. 3- selecting of some plants grow in contaminated area, which irrigated by industrial waste water. 4- Isolation of some fungal endophytes of these plants. 5- Determination of cadmium, copper and lead tolerance of endophytic fungi. 6- Screening the uptake capacities of various selected metal ions by these endophytic fungi from industrial waste water. 7- Determination the biosorption of the selected metal ions after the treatment. 8- Identification of the selected fungal endophytes. 9- IR measurements. - 193 - Summary The obtained results could be summarized as follows: 1- Different industrial waste water samples were collected from different companies of different industries in Egypt such as food (Green Land Co., Halwany Co. and Bisco Misr Co.), painting(Sipes Co.), pharmaceuticals (Gedco Co.) , service (Cairo International Air Port Co., central and middle workshop) and water valves industries (Jacop Delafon Co.) which are drained on sewage waste water as well as packing materials (Misr pack Co.), (Infit, International Co for casting & fitting Co.) and cartoons and inks industries (El-Baddar Co.) that drained on underground water were contain different harmful concentrations of copper, cadmium and lead. These water samples proved to be the best source for cadmium, copper and lead ions. 2- All selected biosorbent plants were collected from garden of fruits in industrial region located at Gisr El Suez area irrigated by the industrial wastewater of (El-Baddar Co.) factory. The highest number of endophytic fungal isolates were recorded from Rhamnus sp. (Monacrosporium elegans, Penicillium duclauxi and Curvularia lunata). And from Saccharum sp. (Rhizopus oryzea, Aspergillus luchuensis and Aspergillus tubingensis) followed by Morus alba (Drechslera hawaiiensis and Verticillium Fungicola), - 194 - Summary Morus rubra (Penicillium lilacinum) and Ricinus communis (Pestalotiopsis clavispora). 3- All endophytic fungal strains under study showed cadmium, copper and lead tolerance with varying level as evident by the minimum inhibition concentration (MIC) values of these metals. As noticed by the highest MIC reported for isolated strains; it was Curvularia lunata and Penicillium lilacinum toward copper, Monacrosporium elegans and Rhizopus oryzea toward cadmium with Penicillium lilacinum and Drechslera hawaiiensis toward lead. Data supported Penicillium lilacinum as potent multi-metal resisted fungus. 4- The biomass of the endophytic Rhizopus oryzea, Monacrosporium elegans, Penicillium duclauxi, Aspergillus luchuensis and Penicillium lilacinum supported the highest uptake of cadmium. The biomass of the endophytic fungi Penicillium lilacinum, Drechslera hawaiiensis and Pestalotiopsis clavispora were the best in copper uptake. On the other hand, Drechslera hawaiiensis, Penicillium lilacinum, Monacrosporium elegans and Penicillium duclluxi supported the highest uptake of lead. Due to Penicillium lilacinum and Drechslera hawaiiensis exhibited the highest removal of copper and lead with good - 195 - Summary removal of cadmium they were selected for further studies as the most promising biosorbent strains. 5- Two isotherm equations have been tested in the present study, namely Langmuir and Freundlich equations. The Langmuir and Freundlich constants have been calculated from the corresponding plots for biosorption of Cd ions , Cu ions and Pb ions ions on the biosorbents (Penicillium lilacinum and Drechslera hawaiiensis). The Langmuir model was able to describe the experimental equilibrium data for biosorption of Cu ions , Cd ions and Pb ions ions on fungal biomass Penicillium lilacinum and Drechslera hawaiiensis under given experimental conditions (biomass weight equal to 30 mg, pH 2 and 3 hrs contact time ). The values of KF in Freundlich isotherm model for Cu ions , Cd ions and Pb ions ions by Penicillium lilacinum and Drechslera hawaiiensis were determined. Data shows the highest value of n (The other Freundlich constant) in the case of Pb ions , which indicating that lead metal ion is favorably adsorbed by the biosorbents than Cd ions or Cu ions . 6- Removal and uptake of cadmium by Drechslera hawaiiensis and Penicillium lilacinum was studied in batch experiments at varying pH values ranging from pH 2 to 8. - 196 - Summary a) Acidic pH below 4 was not fit for Cd ions removal by Drechslera hawaiiensis. There was a correlation between the pH and the time of biosorption process, PH 4.0, 6.0, 7.0 and 8.0 exhibited 100 % removal of Cd ions after process time of 1440, 120, 120 and 10 min, respectively. b) Acidic pH below 4 was not fit for Cd ions removal by Penicillium lilacinum. pH 6.0, 7.0 and 8.0 exhibited 100 % removal of Cd ions after process time of 180, 30 and 30 min. Data clearly indicated that alkali pH (8) is favorable for Cd ions removal by Penicillium lilacinum so in this study we supported alkali pH for the best removal of cadmium by Penicillium lilacinum. 7) Removal and uptake of copper by Drechslera hawaiiensis and Penicillium lilacinum was studied in batch experiments at varying pH values ranging from pH 2 to 8. a) Removal of Cu ions by Drechslera hawaiiensis was increased with increasing pH. pH 2.0, 4.0, 6.0 and 7.0 exhibited 100 % removal of Cu ions after process time of 1440, 1440, 180 and 180 min. Data clearly indicated that neutral pH (7) is favorable for Cu ions removal by Drechslera hawaiiensis so in this study we supported - 197 - Summary neutral pH for the best removal of Copper by Drechslera hawaiiensis at the shortest time. b) Penicillium lilacinum exhibited 100 % removal of Cu ions at pH 6 after 120 min of treatment. There was a correlation between the pH and the time of biosorption process need for 100 % removal. PH 6.0, 7.0 and 8.0 exhibited 100 % removal of Cu ions after process time of 120, 120 and 120 min. Data clearly indicated that pH (6) is favorable for Cu ions removal by Penicillium lilacinum. 8) Removal and uptake of lead by Drechslera hawaiiensis and Penicillium lilacinum was studied in batch experiments at varying pH values ranging from pH 2 to 8. a) Acidic pH below 4 was not fit for Pb ions removal by Drechslera hawaiiensis. pH 2.0, 4.0, 6.0 and 7.0 exhibited 100 % removal of Pb ions after process time of 1440, 1440, 180 and 180 min. Data clearly indicated that neutral pH (7) is favorable for Pb ions removal by Drechslera hawaiiensis so in this study we supported neutral pH for the best removal of lead by Drechslera hawaiiensis at the shortest time . b) Penicillium lilacinum exhibited 100 % removal of Pb ions at pH 6 after 120 min of treatment. There was a - 198 - Summary correlation between the pH and the time of biosorption process. PH 6.0, 7.0 and 8.0 exhibited 100 % removal of Pb ions after process time of 120, 120 and 120 min. Data clearly indicated that pH (6) is favorable for Pb ions removal by Penicillium lilacinum. 9- The effect of different cadmium ion concentrations versus different contact time by Penicillium lilacinum and Drechslera hawaiiensis was investigated. a) Drechslera hawaiiensis is a potent biosorbent for cadmium metal ion from aqueous solution at its lower and higher concentrations under study of cadmium. The equilibrium time was detected to be 10 min with 30mg/L and 180 min with 50, 70 and 90 mg/L to achieve 100% removal but it was 1440 min contact time to totally removal of 110 mg/L of cadmium from aqueous solution. b) The total removal of 10, 30, 50, 70, 90 and 110 mg/L of cadmium by Penicillium lilacinum was achieved after 1440 min contact time. So Drechslera hawaiiensis exhibited faster complete removal of cadmium with the shortest equilibrium time than Penicillium lilacinum. - 199 - Summary Increasing cadmium ion concentrations resulted in an increase in cadmium removal and uptake per unit dry weight of biomass. 10- The effect of different copper ion concentrations versus different contact time by Penicillium lilacinum and Drechslera hawaiiensis was investigated. a) The equilibrium time was equal to 30, 30, 30 and 120 min for 100 % removal of 50, 70, 90 and 110 mg/L of copper from the aqueous by Drechslera hawaiiensis. b) The total removal of 10, 30, 50, 70, 90 and 110 mg/L copper was achieved by Penicillium lilacinum after 120, 180, 180, 120, 120 and 60 min contact time, respectively. 11- The effect of different lead ion concentrations versus different contact time by Penicillium lilacinum and Drechslera hawaiiensis was investigated. a) Drechslera hawaiiensis showed 100 % removal after 120 min contact time. The equilibrium time is detected at 10 min with 30 mg/L of lead but it was equal to 30, 30, 30 and 120 min for 100 % removal of 50, 70, 90 and 110 mg/L of lead from the aqueous solutions. b) Penicillium lilacinum is a potent biosorbent for lead metal ion from aqueous solution at its lower and higher concentrations under study, the total removal of 10, 30, 50, - 200 - Summary 70, 90 and 110 mg/L lead was achieved after 120, 180, 180, 120, 120 and 60 min contact time, respectively. 12-The effect of different biomass concentration of Penicillium lilacinum and Drechslera hawaiiensis versus different contact time was investigated. a) The contact time was equal to 1440 min for 100 % removal of 2.86 mg/L of cadmium by using 50, 70, and 90 mg of fungal biomass of Drechslera hawaiiensis from the aqueous solutions. The total removal of 1.97 mg/L of cadmium was achieved after 30 min contact time by using 10 mg of biomass of Penicillium lilacinum. These data clearly indicated that both strains under the present study are potent biosorbents for cadmium but Drechslera hawaiiensis exhibited more powerful toward cadmium than Penicillium lilacinum. b) Drechslera hawaiiensis showed 100 % removal of 6 mg/L of copper after 1440 min contact time and this is considered the equilibrium time. The contact time was equal to 10 min for 100 % removal of 6 mg/L of copper by using 50, 70, and 90 mg of fungal biomass of Drechslera hawaiiensis from the aqueous solutions. - 201 - Summary The total removal of 6 mg/L of copper was achieved after 1440 min contact time by using 5 mg of biomass of Penicillium lilacinum. c) Drechslera hawaiiensis showed 100 % removal of 5 mg/L of lead after 180 min contact time and this is considered the equilibrium time. The contact time was equal to 120 min for 100 % removal of 5 mg/L of lead by using 30 mg of biomass. The contact time was equal to 10 min for 100 % removal of 5 mg/L of lead by using 50, 70, 90 and 110mg of fungal biomass of Drechslera hawaiiensis from the aqueous solutions. The total removal of 5 mg/L of lead was achieved after 180 min contact time by using 10 mg of biomass of Penicillium lilacinum. 13- Adsorption of heavy metals Cu ions , Cd ions and Pb ions from real industrial wastewater, the results could be summarized as follows: a) The maximum biosorption capacity of Cu ions by dead biomass of Drechslera hawaiiensis was 100 % while under the same conditions the bioaccumulation capacity of Cu ions by active biomass of the strain was 98 %. On the other hand the dead biomass of Drechslera hawaiiensis exhibited 100 % removal of Cu ions at pH - 202 - Summary 7.0 compared to 98 % removal of Cu ions at the same condition by living cells of Drechslera hawaiiensis. The biosorption removal capacities of Cd ions from real industrial wastewater by using live and dead fungal biomass of Drechslera hawaiiensis were 94.7% and 100 %, respectively. dead Drechslera hawaiiensis was observed to remove 98.9, 99.6 and 99.5% of Pb ions from the real industrial waste water within 3 hrs at pH 4, 6 and 7, respectively, compared to 99, 99.26 and 99.26 % removal of Pb ions by alive Drechslera hawaiiensis at pH 4, 6 and 7 under the same conditions. b) The maximum biosorption capacity of Cu ions by dead biomass of Penicillium lilacinum was 94.74% at pH 7.0 while under the same conditions the bioaccumulation capacity of Cu ions by active biomass of the strain was 94.43%. The maximum removal capacities of Cd ions ions from real industrial wastewater by using live and dead fungal biomass of Penicillium lilacinum was 93.42% at pH 6.0 and 7.0. dead Penicillium lilacinum was observed to remove 100% of Pb ions from the real industrial waste water within 3 hrs at pH 4, 6 and 7 compared to 99.07 and 100 % removal of Pb ions by alive Penicillium lilacinum at pH 4, 6 and 7 under the same conditions. - 203 - Summary 14- IR measurements, results could be summarized as follows: a) IR spectrum data of Drechslera hawaiiensis after the removal of cadmium refers to typical of C – N group, amid N – H linkage, ether or ester C – O bond, aromatic structure, methoxyl group, C=C double bond, N – H group, primary amine and / or several O – H groups, quinines and double bond. b) IR spectrum data of Penicillium lilacinum after the removal of cadmium refer to C– N group, aromatic fingerprint, OH or NH stretching vibration bonds, C=C double bonds and primary amine and / or several hydroxyl groups. c) After the removal of copper IR spectrum data of Drechslera hawaiiensis suggested the presence of benzene ring, C – N group, aromatic system, C=C double bond, primary amine and / or several O – H groups and O – H or NH2 groups. d) IR spectrum data of Penicillium lilacinum after the removal of copper characteristic to C−N group, aromatic proton, NO2 group, C=O and double bond, C−H stretching of an aliphatic system, OH group and N−H. e) IR spectrum data of Drechslera hawaiiensis after the removal of lead representing the presence of active groups (O –H, C – O, C– N), benzene ring, aromatic fingerprint, C - 204 - Summary – N group, C – O group, possibly form ether or ester system, C=C double bond. , OH, aromatic structure and methoxyl groups, OH or NH stretching vibration bonds and OH or NH2 groups. f) IR spectrum data of Penicillium lilacinum suggested to C – N group, carbonyl amid group , primary amine and / or several OH groups, presence of carboxylic system containing mostly aliphatic and aromatic C – H stretching bands as well as probably some acidic groups (OH and / or NH), benzene ring, C – H group and OH or NH stretching vibration bonds |