Biosorption of Cadmium and Chromium by Acidophilic Bacterial Strains Isolated from Waste Waters

By M. Lekshmi¹ & Dr. Salom Gnana Thanga²
January 2011

The Authors are a Research Scholar¹ and a Lecturer² at the Department of Environmental Sciences, University of Kerala, in Kariavattom, 695 581, India.

Abstract
Heavy metal pollution has become one of the most serious environmental problems today. The heavy meals are highly toxic in nature and are normally non-biodegradable. In recent years, increasing awareness of the environmental impact of heavy metals has prompted a demand for the purification of industrial waste waters prior to discharge into natural waters. Conventional methods for the removal of these toxic metals are chemical precipitation, chemical oxidation, chemical reduction, ion exchange, filtration, electrochemical treatment, evaporation etc. All these procedures have significant disadvantages. Alternative methods include the biosorption of heavy metal ions on microbial biomass. The microbes can play a significant role in reducing the toxic heavy metal ions from contaminated waste waters to an environmentally acceptable limit in an ecofriendly manner. The present study is aimed to isolate cadmium and chromium resistant acidophilic bacterial strains from polluted industrial effluents and to investigate the capacities of metal sorption invitro. Three bacterial strains viz Vibrio sp strain HM1, Thiobacillus sp HM2 and Vibrio sp strain HM3 were isolated from the highly acidic effluents of Travancore Titanium Products Ltd, Thiruvananthapuram. All these strains were naturally acidophilic and could tolerate a pH of 2. Heavy metal cadmium and chromium were used for biosorption studies. The biosorption studies were carried out using bacterial strains with different concentrations of cadmium and chromium at two different pH (5 and 7). Pseudomonas fluorescens was used as positive control. All the tested strains showed maximum growth and biosorption at pH 5. Pseudomonas fluorescens showed maximum growth and biosorption at pH 7. All the isolated strains can be positively used for the biosorption of heavy metal.

Introduction

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.

Materials and Methods

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.

Half-plate technique for growth of bacterial isolates in solid medium

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.

Results and Conclusion

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).

Bacterial removal of Cadmium

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.

Bacterial removal of Chromium

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

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.

Conclusion

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.

Table 1 · Percentage removal of cadmium by bacterial isolates at 7 pH
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

Table 2 · Percentage removal of cadmium by bacterial isolates at 5 pH
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

Table 3 · Percentage removal of chromium by bacterial isolates at 7 pH
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

Table 4 · Percentage removal of chromium by bacterial isolates at 5 pH
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

Table 5 · Growth of bacterial isolates in nutrient broth added with different concentrations of Cadmium (7 pH)
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

Table 6 · Growth of bacterial isolates in nutrient broth added with different concentrations of Cadmium (5 pH)
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

Table 7 · Growth of bacterial isolates in nutrient broth added with different concentrations of Chromium (7 pH)
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

Table 8 · Growth of bacterial isolates in nutrient broth added with different concentrations of Chromium (5 pH)
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

References

Baijulal B., Cytotoxic effects of some industrial effluents on Allium cepa. M.Phil dissertation submitted to University of Kerala, Thiruvananthapuram (2000)
Ramraj R., Afshan A., Gowda T.P.H. and Karanth N.G.K. Sorption of Copper (II) and Lead(II) by microbial cultures during growth. Indian journal of experimental Health,42, 95-99 (2000)
Thangavel P., Ramasamy K., Ramaswami P.P., Ramesh P.T. and Naidu R., Sorption of chromium in soils. In “Proceedings of the 6th National Symposium on Environment”, TamilNadu Agricultural University, Coimbatore, 307-311 (1997)

***