International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 4(4), 64-73, April (2015) Int. Res. J. Environment Sci. International Science Congress Association 64 Identification and Characterization of Microbes from Industrial area for their Heavy metal Tolerance against Cadmium, Lead and MercurySahoo DebasishBarik Girijanandan and Sahoo Subhasmita3 Nitza Biologicals (P) Ltd., Secunderabad, 500056, Telangana, INDIA Siksha O Anusandhan University, Khandagiri, Bhubaneswar, 751003, Odisha, INDIA Trident Academy of Creative Technology, Patia, Bhubaneswar, 751024, Odisha, INDIAAvailable online at: www.isca.in, www.isca.me Received 5th February 2015, revised 9th March 2015, accepted 19th April 2015 AbstractThe search for bacteria and fungus capable against metal toxicity starts with their isolation from the waste water released from the industries that uses either the heavy metals in their process or releases heavy metals as their waste product. The waste effluent released from paper, textile, paint and iron processing industries were collected and different microbial colonies were isolated from those waste water by standard plating methods, identified by their colony morphology, staining methods and different biochemical procedure. Those isolates were then screened for their antibiotics sensitivity and heavy metal toxicity test. From the antibiotics sensitivity test, Erythromycin and Streptomycin proved to be better antibiotics against isolated bacteria and Tetracycline and Ampicillin proved to be better against fungal isolates. Those antibiotics can be used as good selection markers in the molecular biology techniques. For heavy metal toxicity test, three heavy metals such as Cadmium, Mercury and Lead were analyzed at different concentrations such as 1mM, 5mM, 10mM and 20mM for up to 72 hours for bacterial isolates and 144 hours for fungal isolates. The potential isolates were selected over their growth rate at higher concentration of heavy metals. Bacterial isolates such as Bacillus megaterium, Bacillus licheniformis, Pseudomonas fluoroscence, Pseudomonas syringae, Bacillus subtilis, Corynebacterium xerosis, Bacillus macerans and fungal isolates such as Fusarium, Aspergillus niger, Aspergillus flavus, Cladosporium proved to be the better isolates that can be exploited at their molecular level for the bioremediation of heavy metal contamination. Keywords: Biochemical methods, antibiotics sensitivity test, heavy metal toxicity test. Introduction The influx of heavy metals into waste is mainly due to the intended use of heavy metals in industrial products. At the end, these either will end up in waste to the extent they are not attractive for recycling. Heavy metals may also channel to waste during production and utilization phases. The loss in the manufacturing process is often disposed of as manufacturing waste, while products may be exposed to wear and tear and inclusive corrosion during the use phase. Ongoing research and development in the different processes such as speciation of metals, their toxicity, bioaccumulation, biomagnification, bioindication, migration, removal, biomonitoring must be conducted that enable optimal usage, reusability and bioremediation of these heavy metals. The new methodology of using microbial cultures other than phytoremedial procedures for bioremediation proved to be a good alternative to chemical and other conventional methods of reducing the heavy metal contamination in the soil and water in-effect to rapid industrialization process. The microbial bioremediation is simple, cost effective, safe and comparatively a faster process2, 3, . Methodology Water sample collection: The waste water sample from the four sampling points such as sample i. From near paper industries, sample ii. From near textile industries, sample iii. From near paint industries and sample iv. From iron processing industries were collected in sampling bottles. Isolation and Identification of micro-organisms: The water sample was collected from different sites near to that of industries were first serial diluted, 100l of the diluents from the samples were taken and then they were spread plated on Nutrient Agar Media, and Sabouraud Dextrose agar (containing 0.5% Chloramphenicol antibiotics). The Nutrient Agar Medium was incubated in incubator at 37C for 24 hours. The Sabouraud Dextrose agar plates were incubated at 30C for 96 hours. The organisms isolated from Nutrient Agar Media were first screened by their colony morphology and then were gram stained7 to identify their structure. Then they were identified by different biochemical test as suggested in Bergey's Manual of Determinative Bacteriology, 9th Edition. The unknown bacteria were identified by different staining methods and biochemical tests9-12. The fungus were identified as in James, G. C. and Natalie, S.13. Antibiotics sensitivity test for bacteria: The antimicrobial activity of different antibiotics was determined in accordance with agar-well diffusion method as described by Rious et al.14. International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(4), 64-73, April (2015) Int. Res. J. Environment Sci. International Science Congress Association 65 The bacterial and fungal isolated were first grown in Nutrient broth and Sabouraud dextrose broth (containing 0.5% Chloramphenicol antibiotics) respectively and standardized to 0.5 McFarland standards (106 cfu/mL). 200µL of standardized cell suspension of bacterial and fungal isolates were spread over Mueller-Hinton agar and Potato Dextrose agar (containing 0.1% Streptomycin antibiotics) respectively. Wells were then bored into the agar using a sterile 6mm diameter cork borer. Then 100µL of standardized solutions of Ampicillin (5000g/ml), Chloromphenicol (5000g/ml), Tetracyclin (5000g/ml), Kanamycin (5000g/ml), Erythromycin (5000g/ml), Streptomycin (5000g/ml) and Nalidixic acid (5000µg/ml) were pipette into the wells. The bacterial plates were incubated at 37ºC for 36 hours and fungal plates were incubated at 30C for 72 hours. Inhibition zones in diameters were measured in mm using a calibrated calliper. Metal toxicity test for isolated microorganisms: The metal toxicity test for different bacterial and fungal isolates were done against three different heavy metals such as Cadmium (Cd), Mercury (Hg) and Lead (Pb) at different concentration such as 1mM, 5mM, 10mM, 20mM. The metal toxicity was assayed for 24 hours, 48 hours and 72 hours for bacterial isolates and 48 hours, 96 hours and 144 hours for fungal isolates respectively for all the concentrations of the different heavy metals. The absorbance was calculated for each parameters at 620nm for bacterial isolates and 405nm for the fungal isolates by a double beam UV-VIS spectrophotometer with double distill water as blank (absorbance = 0) . Results and DiscussionThe bacteria were isolated and identified according to the colony morphology, different staining procedures and biochemical tests. From the four sampling points, 14 bacterial colonies were selected for the biochemical tests and their identification were done by Bergey’s manual of determinative bacteriology as in table-1. Different fungal species were isolated basing upon their observation in Lactophenol cotton blue staining and their colony morphology on the plates such as hyphae structure, colour, etc. Five fungal colonies were isolated as Aspergillus niger, Aspergillus flavus, Penicillum species., Cladosporium andFusarium. The different microorganism were analysed for the antibiotics sensitivity test where zone of inhibition (in mm) was calculated against antibiotics such as Ampicillin, Tetracycline, Chloramphenicol, Kanamycin, Erythromycin, Streptomycin and Nalidixic acid. table-2, figure-1 and figure-2. The different microbial isolates were subjected to heavy metal toxicity test against three heavy metals such as Cadmium (Cd) as in figure-3 and figure-4, Mercury (Hg) as in figure-5 and figure-6 and Lead (Pb) as in figure-7 and figure-8 at different concentration such as 1mM in table-3, 5mM in table-4, 10mM in table-5 and 20 mM in table – 6 for three consecutive days for bacterial species and six consecutive days for fungal species. Discussion: Different bacterial and fungal colonies isolated from different industrial sites were analyzed for their ability against antibiotics and tolerance against heavy metals such as Cadmium (Cd), Mercury (Hg) and Lead (Pb). While testing the antibiotics sensitivity test of isolates, B.megaterium showed highest zone of inhibition of 44mm with Erythromycin while Fusariumsp. showed highest zone of inhibition against Kanamycin. Highest resistance was seen in case of S.saprophyticus against Nalidixic acid while Penicillum sp. was sensitive against Chloramphenicol. Erythromycin was the potential antibiotics as it has good sensitive reaction against P.fluorescence (41mm), P.syringae (31mm), B.subtilis (42mm) while Tetracycline was better antimicrobial activity against A.niger and A.flavus. For Metal toxicity test against Cadmium, bacterial colonies such as B.licheniformis, M.varians, S.saprophyticus, P.fluorescence, C.xerosis, B.insolitus, B.megaterium proved better isolates. Fungal colonies such as Fusarium, A.niger and A.flavus were well tolerant to Cadmium toxicity at higher concentration. P.fluorescence, B.megaterium, B.licheniformis, P.syringae, B.subtilis, C.xerosis, B.macerans were effective at higher concentration (20mM) of Mercury toxicity. Likewise Aspergillus niger, Fusarium, Aspergillus Flavus were well-tolerant to Mercury toxicity. Bacteria that were tolerant to Lead toxicity at higher level (20mM) were B.megaterium, B.licheniformis, P.fluorescence, S.saprophyticus, P.syringae, B.macerans, B.subtilis. Fusarium, A.niger, Cladosporium showed good tolerant result for the Lead toxicity. Conclusion The different isolates that have a good tolerance level of heavy metal toxicity against test heavy metals such as Cadmium, Lead and Mercury can be good potentials for the bioremediation and the genes and genetics of those bacteria can be well exploited in future at molecular level to prove as a bio-machine against environmental pollution. Reference1.Szyczewski P., Siepak J., Niedzielski P. and Sobczyski T., Research on Heavy Metals in Poland, (2009)2.Vijendra singh and Singh Chandel C.P., Analytical Study of Heavy Metals of Industrial Effluents, at Jaipur, Rajasthan, (2006)3.Ahmad Varish, Dhama Gaurav, Singh Ajeet, Prakash Tej and Taqui Abbas, Isolation of potential pathogenic fungi from industrial effluent, (2011) International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(4), 64-73, April (2015) Int. Res. J. Environment Sci. International Science Congress Association 66 Table-1 Biochemical test for identification of bacterial isolatesBiochemical test C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 Gram staining +ve rod +ve rod -ve rod -ve rod +ve rod -ve rod +ve rod +ve rod +ve rod +ve rod +ve coccus -ve rod +ve rod +ve rod Endospore staining -ve -ve *NA *NA +ve *NA +ve +ve -ve +ve *NA *NA *NA -ve Acid Fast staining -ve -ve *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA Catalase test +ve +ve *NA *NA *NA *NA +ve +ve *NA*NA +ve *NA +ve*NA Mannitol fermentation test *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA -ve *NA -ve*NA Glucose fermentation *NA *NA +ve +ve +ve -ve *NA +ve *NA*NA+ve -ve *NA*NA VP test *NA *NA -ve +ve -ve *NA -ve -ve +ve +ve*NA *NA *NA -ve Indole test *NA *NA -ve *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA Amylase production test - ve +ve *NA *NA +ve *NA -ve -ve +ve +ve*NA *NA *NA +ve Motility test *NA *NA -ve *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA Citrate test *NA *NA *NA*NA *NA *NA *NA *NA +ve+ve*NA *NA *NA +ve Nitrate reduction test *NA *NA *NA *NA *NA +ve +ve *NA *NA *NA *NA -ve *NA*NA Lecithinase test *NA *NA *NA *NA *NA +ve *NA *NA *NA *NA *NA -ve *NA*NA Oxidase Test *NA *NA +ve +ve *NA +ve *NA *NA *NA *NA*NA -ve *NA*NA 6.5 % NaCl Test *NA *NA -ve +ve +ve *NA *NA *NA +ve+ve *NA *NA *NA *NA Urease Test *NA *NA +ve *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA Luminiscent agar Test *NA *NA *NA -ve *NA *NA *NA *NA *NA *NA*NA *NA *NA *NA Pour plate *NA *NA *NA *NA -ve *NA -ve *NA *NA *NA *NA *NA *NA *NA Pseudo P agar *NA *NA *NA *NA *NA +ve *NA *NA *NA *NA *NA +ve *NA*NA Growth at 55 0 C *NA *NA *NA *NA *NA *NA *NA *NA +ve-ve *NA*NA *NA *NA Novobiocin sensitivity *NA *NA *NA *NA *NA *NA *NA *NA *NA*NA*NA *NA -ve*NA Pigmented colony 37C *NA *NA *NA *NA *NA *NA *NA *NA *NA *NA+ve *NA -ve*NA Identified micro - organism Corynebacterium xerosis Corynebacterium kutsceri Aeromonas caviae Vibrio alginolyticus Bacillus macerans Pseudomonas fluorescence Bacillus pasteurii Bacillus insolitus Bacillus licheniformis Bacillus subtilis Micrococcus varians Pseudomonas syringae S. saprophyticus Bacillus megaterium +ve – organisms show positive result to test; -ve - organisms do not show results. *NA- biochemical tests/methodology not acquired or required. International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(4), 64-73, April (2015) Int. Res. J. Environment Sci. International Science Congress Association 67 Table-2 Antibiotics sensitivity test of isolated microorganismsZone of Inhibition (in mm) Micro-organism Ampicillin Tetracyclin Chloramphenicol Kanamycin Erythromycin Streptomycin Nalidixic acid Bacterial Isolates C.xerosis 35 mm 27 mm 34 mm 36 mm 18 mm 42 mm 38 mm C.kutsceri 40 mm 31 mm 27 mm 17 mm 29 mm 35 mm 43 mm A.caviae 08 mm 14 mm 42 mm 23 mm 30 mm 29 mm 32 mm V.alginolyticus 22 mm 35 mm 24 mm 40 mm 16 mm 24 mm 23 mm B. macerans 27 mm 28 mm 34 mm 41 mm 35 mm 22 mm 18 mm P.fluorescence 12 mm 33 mm 13 mm 22 mm 41 mm 17 mm 04 mm B.pasteurii 33 mm 23 mm 38 mm 33 mm 22 mm 43 mm 34 mm B.insolitus 37 mm 29 mm 40 mm 25 mm 26 mm 15 mm 27 mm B.licheniformis 29 mm 19 mm 14 mm 32 mm 33 mm 37 mm 29 mm B.subtilis 25 mm 28 mm 34 mm 37 mm 42 mm 27 mm 37 mm M.varians 19 mm 32 mm 39 mm 21 mm 25 mm 20 mm 19 mm P.syringae 13 mm 19 mm 30 mm 20 mm 31 mm 22 mm 06 mm S.saprophyticus 22 mm 39 mm 31 mm 38 mm 21 mm 43 mm 03 mm B.megaterium 31 mm 30 mm 39 mm 26 mm 44 mm 36 mm 21 mm Fungal Isolates A.niger 33 mm 42 mm 08 mm 24 mm 13 mm 12 mm 19 mm A.flavus 26 mm 35 mm 07 mm 13 mm 20 mm 16 mm 14 mm Penicillum 36 mm 39 mm 05 mm 22 mm 23 mm 22 mm 17 mm Cladosporium 22 mm 21 mm 12 mm 15 mm 21 mm 19 mm 10 mm Fusarium 29 mm 27 mm 15 mm 32 mm 18 mm 27 mm 26 mm Figure-1Antibiotics sensitivity test for different isolated bacterial species International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(4), 64-73, April (2015) Int. Res. J. Environment Sci. International Science Congress Association 68 Figure-2 Antibiotics sensitivity test for different isolated fungal speciesTable-3 Metal toxicity test against three heavy metals at 1mM concentrationAbsorbance at 620nm Heavy metals Cadmium (Cd) Mercury (Hg) Lead (Pb) Bacterial Isolates Hours 24 hrs 48hrs 72hrs 24 hrs 48hrs 72hrs 24 hrs 48hrs 72hrs C.xerosis 0.31 0.37 0.42 0.23 0.31 0.39 0.36 0.44 0.52 C.kutsceri 0.24 0.27 0.30 0.22 0.28 0.33 0.33 0.40 0.48 A.caviae 0.25 0.29 0.33 0.21 0.25 0.29 0.31 0.34 0.39 V.alginolyticus 0.22 0.25 0.27 0.22 0.27 0.31 0.33 0.37 0.42 B. macerans 0.27 0.31 0.35 0.24 0.32 0.41 0.38 0.46 0.55 P.fluorescence 0.32 0.39 0.45 0.28 0.37 0.48 0.39 0.51 0.63 B.pasteurii 0.26 0.30 0.34 0.21 0.27 0.33 0.34 0.42 0.49 B.insolitus 0.30 0.36 0.42 0.23 0.29 0.34 0.35 0.44 0.53 B.licheniformis 0.34 0.42 0.50 0.25 0.34 0.42 0.41 0.53 0.65 B.subtilis 0.27 0.32 0.37 0.23 0.32 0.40 0.37 0.47 0.56 M.varians 0.33 0.43 0.49 0.23 0.30 0.39 0.30 0.32 0.34 P.syringae 0.28 0.34 0.39 0.26 0.35 0.32 0.38 0.49 0.58 S.saprophyticus 0.33 0.40 0.48 0.24 0.30 0.37 0.39 0.50 0.59 B.megaterium 0.29 0.35 0.41 0.27 0.35 0.45 0.42 0.55 0.64 Fungal Isolates (Absorbance at 405nm Hours 48 hrs 96hrs 144hrs 48 hrs 96hrs 144hrs 48 hrs 96hrs 144hrs A.niger 0.25 0.29 0.34 0.27 0.32 0.37 0.28 0.32 0.38 A.flavus 0.24 0.28 0.31 0.22 0.26 0.31 0.21 0.25 0.28 Penicillum 0.21 0.23 0.26 0.21 0.23 0.26 0.23 0.25 0.32 Cladosporium 0.20 0.21 0.22 0.20 0.22 0.24 0.27 0.31 0.36 Fusarium 0.26 0.31 0.35 0.25 0.29 0.35 0.30 0.36 0.41 International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(4), 64-73, April (2015) Int. 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International Science Congress Association 69 Table-4 Metal toxicity test against three heavy metals at 5mM concentration Absorbance at 620nm Heavy metals Cadmium (Cd) Mercury (Hg) Lead (Pb) Bacterial Isolates Hours 24 hrs 48hrs 72hrs 24 hrs 48hrs 72hrs 24 hrs 48hrs 72hrs C.xerosis 0.28 0.33 0.37 0.21 0.28 0.34 0.32 0.41 0.48 C.kutsceri 0.22 0.23 0.22 0.20 0.25 0.31 0.32 0.36 0.40 A.caviae 0.22 0.21 0.22 0.17 0.18 0.18 0.29 0.30 0.31 V.alginolyticus 0.21 0.21 0.20 0.18 0.19 0.21 0.31 0.32 0.34 B. macerans 0.25 0.31 0.35 0.22 0.29 0.36 0.36 0.44 0.54 P.fluorescence 0.2 9 0.34 0.39 0.26 0.34 0.43 0.37 0.49 0.60 B.pasteurii 0.23 0.26 0.29 0.19 0.24 0.27 0.32 0.37 0.41 B.insolitus 0.28 0.31 0.35 0.21 0.26 0.31 0.33 0.40 0.48 B.licheniformis 0.31 0.38 0.46 0.23 0.31 0.39 0.39 0.52 0.65 B.subtilis 0.25 0.29 0.31 0.21 0.28 0.35 0.35 0.44 0.53 M.varians 0.30 0.36 0.42 0.21 0.27 0.30 0.28 0.28 0.29 P.syringae 0.27 0.30 0.33 0.24 0.31 0.38 0.36 0.47 0.58 S.saprophyticus 0.29 0.35 0.41 0.22 0.28 0.33 0.37 0.48 0.60 B.megaterium 0.27 0.31 0.35 0.25 0.33 0.41 0.40 0.53 0.65 Fungal Isolates ( Absorbance at 405nm ) Hours 48 hrs 96hrs 144hrs 48 hrs 96hrs 144hrs 48 hrs 96hrs 144hrs A.niger 0.24 0.29 0.32 0.25 0.30 0.36 0.25 0.29 0.33 A.flavus 0.23 0.25 0.28 0.20 0.24 0.29 0.19 0.24 0.26 Penicillum 0.18 0.19 0.20 0.18 0.20 0.26 0.21 0.23 0.25 Cladosporium 0.19 0.18 0.20 0.17 0.18 0.20 0.24 0.27 0.31 Fusarium 0.25 0.32 0.34 0.23 0.27 0.32 0.27 0.32 0.38 Table-5 Metal toxicity test against three heavy metals at 10mM concentration Absorbance at 620nm Heavy metals Cadmium (Cd) Mercury (Hg) Lead (Pb) Bacterial isolates Hours 24 hrs 48hrs 72hrs 24 hrs 48hrs 72hrs 24 hrs 48hrs 72hrs C.xerosis 0.25 0.29 0.32 0.18 0.21 0.23 0.29 0.34 0.38 C.kutsceri 0.20 0.21 0.20 0.17 0.18 0.17 0.29 0.31 0.33 A.caviae 0.20 0.21 0.21 0.16 0.15 0 .15 0.26 0.25 0.25 V.alginolyticus 0.21 0.20 0.20 0.16 0.16 0.15 0.28 0.29 0.30 B. macerans 0.22 0.22 0.23 0.19 0.22 0.24 0.33 0.45 0.53 P.fluorescence 0.27 0.31 0.32 0.23 0.28 0.33 0.34 0.43 0.54 B.pasteurii 0.21 0.22 0.22 0.16 0.17 0.17 0.29 0.32 0.3 4 B.insolitus 0.24 0.27 0.30 0.18 0.20 0.22 0.30 0.35 0.39 B.licheniformis 0.28 0.33 0.37 0.20 0.24 0.26 0.36 0.47 0.57 B.subtilis 0.23 0.24 0.25 0.19 0.23 0.26 0.32 0.39 0.45 M.varians 0.27 0.32 0.35 0.17 0.19 0.20 0.25 0.26 0.26 P.syringae 0.24 0.25 0.27 0.21 0.25 0.27 0.33 0.40 0.48 S.saprophyticus 0.26 0.29 0.34 0.19 0.21 0.23 0.34 0.43 0.52 B.megaterium 0.24 0.26 0.29 0.22 0.27 0.32 0.37 0.48 0.59 Fungal Isolates ( Absorbance at 405nm ) Hours 48 hrs 96hrs 144hrs 48 hrs 96hrs 144hrs 48 hrs 96hrs 144hrs A.niger 0.22 0.28 0.30 0.24 0.28 0.31 0.23 0.26 0.30 A.flavus 0.20 0.24 0.27 0.18 0.22 0.25 0.17 0.17 0.19 Penicillum 0.16 0.17 0.18 0.16 0.18 0.19 0.18 0.20 0.23 Cladosporium 0.17 0.19 0.20 0.15 0.16 0.16 0.20 0.24 0.27 Fusarium 0.23 0.28 0.31 0.21 0.26 0.29 0.24 0.28 0.33 International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(4), 64-73, April (2015) Int. 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International Science Congress Association 70 Table-6 Metal toxicity test against three heavy metals at 20mM concentrationAbsorbance at 620nm Heavy metals Cadmium (Cd) Mercury (Hg) Lead (Pb) Bacterial Isolates Hours 24 hrs 48hrs 72hrs 24 hrs 48hrs 72hrs 24 hrs 48hrs 72hrs C.xerosis 0.23 0.24 0.26 0.18 0.20 0.22 0.27 0.26 0.27 C.kutsceri 0.20 0.21 0.20 0.15 0.16 0.16 0.27 0.26 0.26 A.caviae 0.20 0.21 0.21 0.16 0.15 0.16 0.26 0.26 0.25 V.alginolyticus 0.20 0.20 0.21 0.16 0.15 0.17 0.26 0.25 0.25 B. macerans 0.21 0.21 0.22 0.17 0.19 0.20 0.33 0.37 0.41 P.fluorescence 0.23 0.25 0.27 0.22 0.25 0.27 0.34 0.39 0.44 B.pasteurii 0.20 0.21 0.21 0.16 0.15 0.15 0.28 0.27 0.26 B.insolitus 0.23 0.25 0.26 0.16 0.16 0.15 0.28 0.28 0.27 B.licheniformis 0.27 0.30 0.34 0.20 0.23 0.25 0.36 0.41 0.46 B.subtilis 0.22 0.22 0.23 0.18 0.21 0.23 0.32 0.36 0.40 M.varians 0.25 0.28 0.30 0.15 0.16 0.15 0.25 0.25 0.26 P.syringae 0.23 0.24 0.25 0.19 0.21 0.23 0.33 0.38 0.43 S.saprophyticus 0.24 0.27 0.29 0.15 0.16 0.16 0.34 0.39 0.42 B.megaterium 0.22 0.24 0.24 0.21 0.24 0.26 0.37 0.48 0.59 Fungal Isolates (Absorbance at 405nm Hours 48 hrs 96hrs 144hrs 48 hrs 96hrs 144hrs 48 hrs 96hrs 144hrs A.niger 0.19 0.22 0.24 0.21 0.24 0.28 0.18 0.20 0.22 A.flavus 0.17 0.19 0.21 0.18 0.22 0.25 0.16 0.16 0.17 Penicillum 0.16 0.17 0.16 0.16 0.16 0.17 0.16 0.17 0.19 Cladosporium 0.16 0.17 0.17 0.16 0.15 0.16 0.17 0.18 0.21 Fusarium 0.21 0.24 0.25 0.19 0.23 0.26 0.20 0.23 0.25 Figure-3 Comparative analysis for growth of different isolated bacterial strain against different concentration of Cadmium (Cd) at different time intervals International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(4), 64-73, April (2015) Int. Res. J. Environment Sci. International Science Congress Association 71 0.050.10.150.20.250.30.350.4A.nigerA.flavusPenicillumCladosporiumFusariumABSORBANCE AT 405nm 1m M- 48 hrs 1m M- 96 hrs 1m M- 144 hrs 5m M- 48 hrs 5m M- 96 hrs 5m M- 144 hrs 10m M- 48 hrs 10m M- 96 hrs 10m M- 144 hrs 20m M- 48 hrs 20m M- 96 hrs 20m M- 144 hrs Figure-4 Comparative analysis for growth of different isolated fungal isolated strain against different concentration of Cadmium (Cd) at different time intervals 0.10.20.30.40.50.6 ABSORBANCE AT 620nm 1mM-24 hrs 1mM-48hrs 1mM-72hrs 5mM-24 hrs 5mM-48hrs 5mM-72hrs 10mM-24 hrs 10mM-48hrs 10mM-72hrs 20mM-24 hrs 20mM-48hrs 20mM-72hrsFigure-5 Comparative analysis for growth of different isolated bacterial strain against different concentration of Mercury (Hg) at different time intervals International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(4), 64-73, April (2015) Int. Res. J. Environment Sci. International Science Congress Association 72 0.050.10.150.20.250.30.350.4A.nigerA.flavusPenicillumCladosporiumFusariumABSORBANCE AT 405 nm 1m M-48 hrs 1m M-96 hrs 1m M-144 hrs 5m M-48 hrs 5m M-96 hrs 5m M-144 hrs 10m M-48 hrs 10m M-96 hrs 10m M-144 hrs 20m M-48 hrs 20m M-96 hrs 20m M-144 hrsFigure-6 Comparative analysis for growth of different isolated fungal strain against different concentration of Mercury (Hg) at different time intervals 0.10.20.30.40.50.60.7 ABSORBANCE AT 620nm 1mM-24 hrs 1mM-48hrs 1mM-72hrs 5mM-24 hrs 5mM-48hrs 5mM-72hrs 10mM-24 hrs 10mM-48hrs 10mM-72hrs 20mM-24 hrs 20mM-48hrs 20mM-72hrsFigure-7 Comparative analysis for growth of different isolated bacterial strain against different concentration of Lead (Pb) at different time intervals International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(4), 64-73, April (2015) Int. Res. J. Environment Sci. International Science Congress Association 73 Figure-8 Comparative analysis for growth of different isolated fungal strain against different concentration of Lead (Pb) at different time intervals 4.Nanda Manisha, Sharma Dinesh and Arun Kumar, Removal of Heavy Metals from Industrial, Effluent Using Bacteria, (2011) 5.Downes F.P. and Ito K.(Ed.), Compendium of Methods for the Microbiological Examination of Foods, 4th Ed., American Public Health Association, Washington, D.C, 2001) 6.Guinea J, Peláez T, Alcalá L, Bouza E, "Evaluation of Czapeck agar and Sabouraud dextrose agar for the culture of airborne Aspergillus conidia, Diagnostic microbiology and infectious disease, 53(4), 333–4 (2005) 7.Gram, HC, Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten, Fortschritte der Medizin (in German), :185–189, English translation in:Brock, T.D. (1999), Milestones in Microbiology 1546–1940 (2 ed.). 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