The Authors are a Research Scholar¹ and a Lecturer² at the Department of Environmental Sciences, University of Kerala, in Kariavattom, 695 581, India.
Heavy metals are some of the major environmental pollutants since, day by day increasing amounts of them are being released into the water supplies as a result of various industrial processing. Microbial interactions with heavy metals have several implications in the environment. When heavy metals are introduced in the environment, biological activity of microorganisms play an important natural role in the metal sequestration and immobilization. The Travancore Titanium Products Ltd. effluent was reported to be characterized by certain heavy metals¹. Hence an attempt was made to isolate bacterial cultures from the effluent and sediments of effluent discharge point and test verify their heavy metal bioremediation invitro.
Sample collection: Water samples were collected in sterile bottles; sediments were also collected from the effluent discharge point.
Isolation of bacterial cultures: Bacterial cultures were isolated using pour plate method in nutrient agar medium. The cultures were purified by repeated sub culturing.
Heavy metal biosorption study: Stock solutions of heavy metals were prepared in nutrient broth using cadmium chloride and chromium trioxide respectively. The stock was used to prepare different dilutions. The bacterial strains viz., Vibrio sp strainHM1, Thiobacillus sp HM2 and Vibrio sp strain HM3 and Pseudomonas fluorescens which was used as positive control were inoculated under aseptic conditions and incubated for 4 days. The cultures were centrifuged and the supernatant analyzed for heavy metals by Atomic Absorption Spectrophotometry. Heavy metal absorption studies were done at two different pH viz., 7 and 5.
For half plate technique, nutrient agar medium with the respective heavy metals and agar medium without heavy metals were prepared and sterilized. Initially, about 7 ml of nutrient medium with heavy metals were poured in petri plates under aseptic conditions. The plates were positioned such that nutrient agar medium with heavy metals filled half the plates and left for solidification. After the media has solidified, about 7ml of the nutrient medium without heavy metals was poured on the half plate to fill the other half and left for solidification. The half-plates had increasing concentration of the respective heavy metals from the center of Petri plates on one side and no heavy metal on the other side. A loopfull of the different bacterial strains and Pseudomonas fluorescens were streaked throughout the half-plates starting from the side where there is no heavy metal. The plates were incubated at 37°C overnight and observed for the growth of bacteria.
Three bacterial strains viz., Vibrio sp strain HM1, Thiobacillus sp strain HM2 and Vibrio sp strain HM3 were isolated from the highly acidic Travancore Titanium Products Ltd., effluent and sediments of effluent discharge point. The strains were naturally acidophilic, could tolerate a pH of 2 and showed good growth at a pH of 3.2. Three bacterial strains andPseudomonas fluorescens which was used as the positive control were grown in nutrient broth containing different concentrations of cadmium and chromium. The bacterial growth in terms of cell protein content (ppm) and percentage heavy metal removed were observed after a week. The results are presented in Tables (1-8).
In the presence of cadmium strain Thiobacillus sp showed maximum growth than the other two strains at all concentrations. All the three strains showed maximum growth at pH 5 than pH7 . Due to higher growth, more number of ligands with negative charges would be exposed which in turn attract more positive charged metal ions from the aqueous solution². The growth of P.fluorescens was low at lower pH which resulted in the low metal removal from the medium. Lower absorption of lead and copper by Pseudomonas, Saccharomyces and E.coli was reported when the pH was reduced below neutral³.
In the case of chromium P.fluorescens showed very high percentage removal (Table 3). Vibrio sp strain S1, showed maximum absorption in both pH. Thangavel et al (1997) reported a decreased sorption of chromium in soil with increasing pH³.
Growth of microbes in presence of heavy metals in solid medium is shown in Plates 1 – 4. When added with cadmium the three strains showed good growth in the metal and non metal part. But in the case of positive control P.fluorescens no growth was found in the metal added part. When added with chromium the three strains and P.fluorescens showed good growth both in the metal and non metal part.
The present study showed that the positive control P.fluorescens showed good growth as well as biosorption of heavy metals at neutral pH where as acidophilic bacterial strains isolated from Titanium effluent showed good growth and biosorption of the heavy metals at acidic pH. These bacterial isolation after characterization can be positively used for heavy metal bioremediation.
Concentration | Vibrio sp strain HM1 | Thiobacillus sp HM2 | Vibrio sp strain HM3 | Pseudomonas fluorescens |
50 | 45.58 | 23.18 | 35.46 | 62.20 |
100 | 42.64 | 23.26 | 35.98 | 60.16 |
Concentration | Vibrio sp strain HM1 | Thiobacillus sp HM2 | Vibrio sp strain HM3 | Pseudomonas fluorescens |
50 | 72.96 | 78.36 | 71.11 | 40.18 |
100 | 69.39 | 70.09 | 65.32 | 19.1 |
Concentration | Vibrio sp strain HM1 | Thiobacillus sp HM2 | Vibrio sp strain HM3 | Pseudomonas fluorescens |
50 | 24.12 | 27.92 | 22.62. | 68.9 |
100 | 20.96 | 22.18 | 20.66 | 65.62 |
Concentration | Vibrio sp strain HM1 | Thiobacillus sp HM2 | Vibrio sp strain HM3 | Pseudomonas fluorescens |
50 | 79.36 | 76.25 | 74.42 | 22.52 |
100 | 68.22 | 65.33 | 61.3 | 17.17 |
Concentration | Vibrio sp strain HM1 | Thiobacillus sp HM2 | Vibrio sp strain HM3 | Pseudomonas fluorescens |
50 | 32.6 | 15.55 | 24.55 | 41.45 |
100 | 27.95 | 14.55 | 23.4 | 39.6 |
Concentration | Vibrio sp strain HM1 | Thiobacillus sp HM2 | Vibrio sp strain HM3 | Pseudomonas fluorescens |
50 | 62.25 | 65.02 | 60 | 13.8 |
100 | 56.66 | 59.33 | 54.47 | 11.2 |
Concentration | Vibrio sp strain HM1 | Thiobacillus sp HM2 | Vibrio sp strain HM3 | Pseudomonas fluorescens |
50 | 10.02 | 11.1 | 16.45 | 45.9 |
100 | 8.83 | 9.32 | 15.25 | 44.15 |
Concentration | Vibrio sp strain HM1 | Thiobacillus sp HM2 | Vibrio sp strain HM3 | Pseudomonas fluorescens |
50 | 70 | 68.21 | 67.14 | 15.4 |
100 | 65.58 | 62.21 | 62.21 | 14.5 |
Plate 1 · Growth of bacterial isolates in solid media added with 50 ppm of Cadmium
Plate 2 · Growth of bacterial isolates in solid media added with 100 ppm of Cadmium
Plate 3 · Growth of bacterial isolates in solid media added with 50 ppm of Chromium
Plate 4 · Growth of bacterial isolates in solid media added with 100 ppm of Chromium
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