1. Twenty four Cr-resistant (CMBL Crl-Cr24), six copper resistant (CMBL Cul Cu6), six cadmium resistant (CMBL Cdl-Cd6), six mercury resistant (CMBL HgI-Hg6) and eight lead resistant (CMBL Pbl-Pb8) bacteria were isolated from industrial wastewater from Kasur tanneries. In addition, eight Cr-resistant (CMBL Y Cr1-Cr8), six copper resistant (CMBL Y Cul-Cu6), eight cadmium resistant (CMBL Y Cd1-Cd8), six mercury resistant (CMBL Y Hg1-Hg6) and six lead resistant (CMBL Y Pb1-Pb6) yeasts were also isolated. Four species of ciliate protozoan were also found to be dominant in the wastewater samples of tanneries viz., Stylonychia, Hypotrich, Paramecium and Vorticella.
2. Chromium resistant bacterial and yeast isolates could resist 0.9-2.0 and 1.0-2.0 mg/mL of Cr+6, respectively, whereas the protozoa could tolerate 50-70 ug/mL of Cr+6. The isolated bacteria could also tolerate 1.3-2.2 mg/mL of Cu+2, 0.5-0.6 mg/mL of Cd+2, and 0.4-0.5 mg/mL of Hg+2 and 1.7-3.0 mg/mL of Pb+2. The yeast isolates, likewise, tolerated 1.5-2.0 mg/mL of Cu+2, 0.8-1.0 mg/mL of Cd+2, 0.6-0.7 mg/mL of Hg+2 and 3.5-4.0 mg/mL of Pb+2. The protozoa tolerated 20-25 mg/mL of Cu+2, 40-45 mg/mL of Cd+2, 2025 mg/mL of Hg+2 and 65-75 mg/mL of Pb+2.
3. The cross resistance of above bacteria and yeast selected on Cr+6, Cd+2, Cu+2, Pb+2 and Hg+2 were determined against other heavy metals such as C0+2, Cr+6, Hg+2, Cu+2, Cd+2, Pb+2 Ni+2 and Zn+2. Chromium resistant bacterial isolate (CMBL-Crl7) showed maximum resistance against Cr6+ (2.0 mg/mL) and minimum resistance against Hg2+ (0.2 mg/mL). Copper resistant bacterial isolate (CMBL-Cul) showed maximum resistance against Cu+2 (1.7 mg/mL), Cr+6 (1.6 mg/mL) and Pb+2 (1.3 mg/mL) and minimum resistance against Cd+2 and C0+2 (each 0.3 mg/mL). The lead resistant bacteria (CMBLPb8) showed maximum resistance against Pb+2 (2.9 mg/mL) and minimum resistance against Hg+2 (0.2 mg/mL). Chromium resistant yeast isolates, CMBL Y -Crl, Cr4 and Cr8, showed maximum resistance against Cr+2 (2.0 mglmL) and minimum against Hg+2 (O.2mglmL). The lead resistant yeast, CMBL Y Pb 2 and Pb3, could tolerate 4.0 mg/mL of Pb+2 and showed minimum resistance against Hg+2 (0.2mglmL).
4. On the basis of various biochemical tests, 24 Cr++6 resistant, 6 Cu+2 resistant, 6 Cd+2 resistant, 6 Hg+2 resistant and 8 Pb+2 resistant bacterial isolates were identified. Besides that 8 Cr+6 resistant, 6 Cu+6 resistant, 8 Cd+2 resistant, 6 Hg+2 resistant and 6 Pb+2 resista11t yeast isolates were identified.
5. The most suitable temperature for all the heavy metal resistant bacterial isolates was found to be 37°C for optimum growth. Some Cr-resistant and Cu-resistant isolates showed optimum growth at 30°C. The optimum pH for bacterial isolates ranged between 7.0-7.5, whereas, it ranged between 5.5-6.5 for yeast isolates. The suitable temperature for all the metal resistant yeast isolates was 30°C.
6. In order to locate the heavy metal processing gene, the bacterial isolates were cured of plasmid and then tested for heavy metal resistance characteristics. In addition, competent cells of Ecoli C600 were transfom1ed with the plasmids isolated from the resistant strains to see if the metal resistance property was transferred to Ecoli strain. All the bacterial isolates were found to have 23 Kb plasmids. The bacterial isolates were cured of plasmids using ethidium bromide. The success rate of curing of Cr+6, Cu+2, Cd+2 and Hg+2 resistant isolates was 70-100%, while Pb+2 resistant isolates could not be cured of the heavy metal resistance characteristic. To confirm "the location of Cr+6, Cd+6, Cu+2, Pb+2 and Hg+2 processing gene on plasmids, the competent cells of E. coli C600 were transformed with plasmids of bacterial isolates. Colonies appeared after 24 hours on selective plates containing 100 mg/mL of Cr+6, Cd+2, Cu+2, Pb+2 and Hg+2. It was concluded from these experiments that heavy metal resistance genes were located on plasmid, except for that of Pb+2 which was located on the chromosomes as well as the plasmid.
7. The metal resistant microorganisms, isolated from the industrial wastewater, were evaluated for their metal processing capability viz., bio-adsorption and bio-absorption. (i) Chromium resistant bacterial and yeast isolates could remove' 65-82% Cr+6 from the medium after 72 hours of incubation. Copper resistant bacterial and yeast isolates could remove 70-82% and 60-75% of Cu+2, respectively, from the medium after 72 hours of incubation. Cadmium resistant bacterial and yeast isolates could remove 7476% and 76-82% of Cd+2, respectively, from the medium after 72 hours of incubation, while mercury resistant bacterial isolates could remove 70-73% and 80% of Hg+2 respectively, from the medium after 72 hours of incubation. The lead resistant bacterial isolated removed 65-82% and yeast isolates removed 75-85% of Pb+2 from the medium after 72 hours of incubation. The ciliate protozoans Stylonychia and Hypotrich were able to remove 80% of Cr+2 from the medium, while Paramecium and Vorticella could remove 75% of Cr+2 each, after 96 hours of incubation.
The removal of Cd+2 Cu+2, Hg+2 and Pb+2 from the culture medium by Stylonychia was 66%, 65%, 58% and 80%, respectively; while Hypotrich and Paramecium could remove 65%, 60%, 50% and 75% ofCd+2, Cu+2, Hg21 and Pb+2, respectively, after 96 hours of incubation. Vorticella could remove 70%, 60%, 70% and 85% of Cd21, CU21, Hg2+ and Pb2+, respectively, after 96 hours of incubation.
(ii) When microorganisms were used in different combinations, metal processing ability was apparently not much affected. The cadmium (CMBL-Cd3 and CMBL Y -Cd8) and copper resistant bacteria and yeast (CMBL-Cu 1 and CMBL Y -Cu3) could remove 86% of Cd+2 and Cu+2 after 48 hours of incubation. Chromium resistant bacteria and yeast (CMBL-Crl7 and CMBL Y -Crl) removed 78% of Cr+6. Mercury resistant bacteria and yeast (CMBL-Hg l and CMBL Y-Hg2) removed 74% of Hg+2. Lead resistant bacteria and yeast (CMBL-Pb8 and CMBL Y -Pb3) removed 90% of Pb+2 after 48 hours of incubation.
(iii) Chromium resistant yeast and protozoa (CMBLY -CrI and Hypotrich) removed 82% Cr+2, lead resistant yeast and protozoa (CMBL Y -Pb3 and Stylonychia) removed 88% of Pb+2, mercury resistant yeast and protozoa (CMBL Y-Hg2 and Paramecium) removed 77% of Hg+2, cadmium resistant yeast and protozoa (CMBLYCd8 and Vorticella) removed 78% of Cd21 and copper resistant yeast and protozoa (CMBLY -Cu3 and Paramecium) removed 821Yo of Cu2+ after 96 hours of incubation. (iv) In the case of bacteria and protozoa combination, the Cr+6 (CMBL-Cr17 and Hypotrich) and cadmium (CMBL-Cd3 and Vorticella) resistant bacteria and protozoa removed 82% of Cr+6 and Cd+2 respectively, copper (CMBL-Cul and Paramecium) and mercury (CMBL-Hg I and Paramecium) resistant bacteria and protozoa removed 85% of Cu+2 and Hg+2. The lead resistant bacteria and protozoa (CMBL-Pb8 and Stylonychia) removed 83% of Pb+2 after 96 hours of incubation.
(v) The Cr+6 (CMBL-Cr17, CMBLY-Crl and Hypotrich) and copper (CMBL CuI CMBL Y -Cu3 and Paramecium) resistant microorganisms, used as consortia, removed 90% of Cr+2 and Cu+2 from the medium. A consortium of cadmium (CMBL Cd3, CMBLY-Cd8 and Vorticella) and mercury (CMBL-Hg I, CMBLY-Hg2 and Paramecium) resistant bacteria, yeast and protozoa removed 88% of Cd+2 and Hg2. The lead resistant bacteria, yeast and protozoa (CMBL-Pb8, CMBLY -Pb3 and Stylonychia) removed 95% of Pb+2 after 96 hours of incubation.
It was concluded that bacteria, yeast and protozoa showed higher MICs against Cr+6, Cd+2, Cu+2 Hg+2 and Pb+2. They however, showed multiple resistances to other metals such as COH, Ni+2 and Zn+2. The genes for Cr+6, Cd+2, Cu+2 and Hg+2 resistances are located on plasmid. In the case of different combinations, bacteria and yeast individually and in combinations are the best candidates for the removal of various heavy metals from the industrial wastewater.