Research Article © ISISnet Publishers Bioscience Research, 3(1): 189-204, 2006 Bioremediation of Zn +2 , Cu +2 and Fe +2 using Bacillus subtilis D 215 and Pseudomonas putida biovar A D 225 . S. Z. Sabae 1 *, M. Hazaa 2 , S. A. Hallim 2 , N. M. Awny 4 and S. M. Daboor 1 1 National Institute of Oceanography and Fisheries, Inland Waters Branch, Egypt. 2 Faculty of Science, Benha University, Egypt 3 Faculty of science, Zagazig University, Egypt *Corresponding author This study provides a step for possible use of bacterial strains in removing heavy metals from effluents. However, bioremediation processes are dependent on several factors including temperature, pH, incubation periods and inoculum size. The results revealed that the tested bacteria Pseudomonas putida biovar A D 225 could remove Zn +2 , Cu +2 and Fe +2 from its solutions, with values of 69.83 %, 67.75 % and 67.65 % at incubation temperature 30 o C, pH 7.0, incubation period 48 hr and with different inoculum size; 200µL L -1 for Cu +2 and 600µl for both Zn +2 and Fe +2. Also, Bacillus subtilis D 215 could remove the same heavy metals with values of 63.73 %, 67.18 % and 70.35 % at incubation temperature 30 o C, pH 7.0, incubation period 48 hr and with different inoculum size; 400µL L -1 for Cu +2 and 600µL L -1 for both Zn +2 Fe +2. On the other hand, the dead cells of Bacillus subtilis D 215 remove Cu +2 , Fe +2 and Zn +2 from its solutions by 25.86 %, 21.30 % and 26.83 %, respectively, while dead cells of P. putida biovar A D 225 remove the same ions with values of 26.70%, 23.70 % and 22.18 %, respectively. The present results showed that the removal percentages of the tested ions by different sorbent materials (cellulose, silica, sponge and sawdust) were much lower than in the case of the isolates. The protein electrophoresis for both of the two isolates treated with copper at sub-lethal concentration (250 mg/l) and non- treated isolates showed that there were differences in the protein bands between the treated and non- treated isolates. However, the detection of plasmid DNA revealed the presence of plasmid DNA at the treated and non-treated bacterial isolates with different size and forms. The use of the tested bacterial isolates, B. subtilis D 215 and P. putida biovar A D 225 , either living cells or dead, as a bioremediators for heavy metals (Cu +2 , Fe +2 and Zn +2) from wastewater was recommended, as well as the use of different sorbent materials. Key words: Bioremediation, heavy metals, Pseudomonas putida, Bacillus subtilis H eavy metals were widespread pollutants of great environmental concern as they are non-degradable and thus persistent in the ecosystem (Sratton, 1987). The problem associated with heavy metals in wastewater entering natural waters had been well documented; these metals exerted a possible human health risk (Sag and Kutsal, 1989). Rapid industrialization had led to increase disposal heavy metals from certain industries into aquatic environment. Unlike many organic wastes, heavy metals cannot be degraded biologically into harmless products. As a result, heavy metals tended to accumulate in the food chain and the environment, (Huang et al., 1991). Untreated effluents containing heavy metals from many industries had an adverse impact on the environment, generating large quantities of such aqueous effluents poses environmental disposal problems, these heavy metals was accumulated in the food chain (Sanchez et al., 1999). The increasing problem of heavy metal contaminating soil, water and other environmental aspects had stimulated as search for new mechanisms to remove these pollutants. Environmental contamination by toxic metals was a serious problem worldwide due to their incremental accumulation in food chain and continued persistence in ecosystem (Hong and Shan-shan, 2005). Bioremediation is an option that offers the possibility to destroy or render harmless various contaminants using natural biological activity. 189