Biosorption of Heavy Metals by using Microorganisms
Current problems of heavy metal pollution in urban and rural ground water resources are reducing the potential of drinking water and also there are several ill effects on human/animal health due to toxicity of heavy metals in soil and water. The biological process of accumulation of heavy metals on the surface of bacteria is called as biosorption. Bacteria are effective agent in the removal of heavy metal from soil and water. The main objective of our study was removal of heavy metals from the soil and water by using different of bacterial strains. We further investigated and compared the effect of pH, temperature and incubation time on the removal of heavy metals. There are certain bacterial species which removes heavy metals such as Bacillus sp. and Pseudomonas sp and Staphylococcus sp. However, the accumulation and removal of heavy metals by microbial biomass holds great potential for heavy metal removal in both soluble and particular form. The heavy metals used in this study were Zn and Pb.
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Keywords: Heavy metals, potential, toxicity and biosorption
Today there is need of such a technology which provide us clean and nontoxic environment, due to the disposal of metals, part of water resources have been seriously polluted and caused worldwide concern. These metals are mainly derived from mining, metallurgical and chemical manufacturing industries. The aqueous effluent discharging from these industries contains heavy metals such as lead, cadmium, chromium in dissolve and particulate form. Conventional methods for removing the heavy metals include chemical precipitation, chemical oxidation and reduction ,ion exchange, but the cost of these process are high when heavy metal concentration are low. The heavy metals are a group of elements on the ‘‘heavier’’ end of the periodic table of elements that have particular weight characteristics . The removal of cations of heavy metals from industrial waste water can be accomplished by biotechnological methods which make use of microorganisms as cation collectors .The uptake, absorption and accumulation of heavy metals by microbial biomass is receiving increasing attention [9,10]. The microbes are efficient in removing the heavy metals because microbial cell or cell fraction may contain negative charged groups . The amount of accumulated cations can be very large and depends on various factors such as microbial species and its physiological state to external physio-chemical conditions such as pH, temperature , initial metal concentration and inoculum concentration etc. Other important factors affecting accumulation capacity are the type of cation, its valence, the presence of other cations or anions, complexing agents, inhibitors of microbial growth, resistance to stress factors. Copper contributes markedly to the environmental pollutions, especially of water and soil. It is usually not found in high concentrations but the toxicity of copper is relatively high to that of other heavy metals . Some heavy metals are highly toxic for human such as lead, cadmium, chromium etc. Symptoms of heavy metal toxicity include mental confusion, pain in muscles and joints, headaches, short-term memory loss, gastrointestinal upsets, food intolerances/allergies, vision problems, chronic fatigue, and others. Taking lead poisoning as examples, the nervous system, gastrointestinal system, cardiovascular system, blood production, kidneys, and even reproductive system would be affected. And the accumulation of nickel in the human body will lead to various diseases and disorders such as lung fibrosis, cardiovascular and kidney disease and increased risk of malignant tumors.
There are several advantages of removal of heavy metals using biomass. First, the removal processes are extremely rapid. Second, the costs of removal of heavy metals by biomass are very low as compared to other conventional process such as chemical precipitation, chemical oxidation- reduction, ion-exchange etc. Third, the cell membrane and cell wall of microbes has biopolymers such as polysaccharides, chitin, teichoic acid, phospholipids and proteins which provided significantly enhanced adsorption or complexation of heavy metals on the microbial surface. Fourth, the removal of metals by biomass is a reversible process means we can separate metals for biomass after adsorption by using different techniques .
In this study, we investigated the potential bacterial biomass for removal of heavy metal, Pb, Cd, Zn, Mn from aqueous solution. We performed experiment with bacterial biomass under different pH (pH5, pH7 and pH9), metal concentrations (25ppm, 50ppm and 75ppm), temperature (25°C, 35°C and 45°C) and incubation time (20 min, 40 min and 60 min ).
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Materials and Methods
- Collection of sample and isolation of bacterial culture
The soil sample was collected from VIT University campus and used for the purpose removal of Heavy metals from aqueous medium. The collected soil sample was serially diluted 10-1 to 10-7 times in distilled water. From Serially diluted sample 0.1ml was used for spreading the diluted sample on the nutrient agar medium plate. These bacterial culture plates were incubated at 37°C for 24 hr. The bacterial strain was isolated and sub cultured and it was used for biosorption of heavy metals.
The Isolated bacterial strain was sub-cultured regularly and stored at 4°C. 400ml nutrient broth medium (1.3g nutrient broth in 100ml distilled water) was prepared in a conical flask and sterilized in an autoclave. 20ml of nutrient broth was taken out and stored it as a blank. Medium was then inoculated with a selected bacterial strain of either Strain. Inoculated flask was kept at room temperature on a shaker (100rpm) for 24hr. After 24hr of incubation, O.D. of the culture was measured at 590nm.Then the medium containing bacterial biomass was used for biosorption of the heavy metals in aqueous solution. The heavy metals was used in this study were Zn and Pb. In this paper we studied the factors like temperature, pH and incubation time that influenced absorption of the heavy metals on the surface of the bacteria.
- Pre treatment of bacterial biomass
The isolated bacterial strain was cultured and centrifuged. The obtained bacterial biomass was washed with distilled water for 3-4 times to remove extracellular nutrients before being used .In the case the chemicals were used for treating the bacterial biomass, the washed biomass was mixed with 10 ml aqueous solution of NaOH ( 0.1 mol/L), HCL ( 0.1 mol/L), ethanol (30%) or distilled water. The mixture was kept to react at room temperature ( about 28°C) for 40min ( NaOH, HCl and ethanol ) and 1 day ( distilled water) respectively. This chemically treated bacterial biomass was washed several times with distilled water and then it was centrifugally separated at 4000rpm for 20 min prior to it was used in metal removal experiments. 
- Preparation of standard and test solutions
- Aqueous solution containing heavy metals (Zn or Pb) of different concentrations (25ppm or 50ppm) were prepared.
First prepared 1000ppm metal solution followed by its dilution using distilled water to made test solutions of 25 ppm and 50 ppm. Similarly, prepared 5 ppm, 10 ppm and 15ppm standard solutions with distilled water.
- Twenty four hours old 20ml of bacterial biomass was centrifuged and cell pellet was treated chemically. Cell pellet was washed with distilled water and used for further experiment.
- Added bacterial biomass to 20ml of 25ppm or 50ppm of metal solution.
- Keep the solution containing bacterial biomass at different conditions of pH (5, 7, 9) at different temperatures (25°C, 35°C, 45°C) for different incubation period (20 min, 40 min and 60min). Solutions were properly mixed using shaker.
- Biomass-metal mixture was filterd by using bacteriological filter paper and filtrate was collected. The metals which had not absorbed by the bacteria remained in the filtrate solution.
- This filtrate containing unabsorbed metal was sent for atomic absorption spectroscopy.
Concentration of absorbed metal was calculated using following formula-
(Concn of metal adsorbed on bacterial surface )
= (Concn of metal added in distilled water ) – (Concn of metal in filtrate)
- For 25ppm metal concentration
Effect of different physical parameters on biosorption activity was studied on 25ppm of aqueous metal solution and following results were obtained.
For incubation time
Fig.1. Effect of incubation time on biosorption of heavy metals (Zn, Pb)
It was clear that as incubation time increases biosorption of heavy metal by bacteria also increases. For Zn and Pb metals, maximum biosorption observed at incubation time 60minute (24.07ppm and 20.81ppm respectively).
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Fig 2. Effect of temperature on biosorption of heavy metals(Zn,Pb)
For bacterial strain, maximum biosorption of Zn and Pb metal observed at temperature 35°C. At 35°C, bacteria absorbed 23.26ppm of Zn metal and 19.93ppm of Pb metal.
Fig.3. Effect of pH on biosorption of heavy metals (Zn, Pb)
Bacterial biosorption of heavy metal changed with change in pH of medium. For Zn and Pb metals, maximum biosorption observed at pH 7. 21.48ppm of Zn and 24.27ppm of Pb were absorbed at pH 7 of medium.
- For 50ppm metal concentration
Effect of different physical parameters on biosorption activity was studied on 50ppm of aqueous metal solution and following results were obtained.
For incubation time
Fig.4. Effect of incubation time on biosorption of heavy metals (Zn, Pb)
It was clear that as incubation time increases biosorption of heavy metal by bacteria also increases. For Zn and Pb metals, maximum biosorption observed at incubation time 60minute (48.63ppm and 49.6ppm respectively).
For bacterial strain, maximum biosorption of Zn and Pb metal observed at temperature 35°C . At 35°C, bacteria absorbed 46.57ppm of Zn metal and 49.31ppm of Pb metal.
Fig 5. Effect of temperature on biosorption of heavy metals (Zn, Pb)
Fig 6. Effect of pH on biosorption of heavy metals (Zn, Pb)
Bacterial biosorption of heavy metal changed with change in pH of medium. For Zn and Pb metals, maximum biosorption observed at pH 7. 47.39ppm of Zn and 49.01ppm of Pb were absorbed at pH 7 of medium.
Biosorption of heavy metals (Zn, Pb) was successfully carried out using available bacterial strain. Bacterial strain was efficient in biosorption of Zn and Pb metals. It was observed that physical parameters like pH, temperature and incubation period influenced biosorption rate of both heavy metals. At optimum pH bacterial strain carried out maximum biosorption of both metals. According to Carlos et al.,2007, at low pH value, biosorption of heavy metals is affected may be due binding site competition between cations and acid hydrolysed products. Influence on biosorption process occurred with changed in temperature. According to Carlos et al., 2007, increase in temperature upto 35°C resulted in increase in biosorption of both metals. Maximum biosorption of both metals observed at incubation period of 60minutes. Maximum biosorption occurred at neutral pH (according to Hemambika et al., 2009) and 35° C temperature (according to Carlos et al., 2007). Bacterial strain removed both metals efficiently upto 50ppm concentration.
We sincerely thank the management of VIT University, Vellore for their support and encouragement.
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