Biosorption of Lead Using the Bacterial Strain, Bacillus subtilis (MTCC 2423)

Lead removal efficiency of the bacterial strain, Bacillus subtilis (MTCC 2423) was tested with 200, 400, 600, 800 and 1000 ppm of lead in minimal broth for a period of ten days. Samples were tested for the level of lead every two days in each concentration and maximum removal was observed after six days of treatment. With the increase in lead concentration, both biomass and lead removal efficiency showed an increase. When tested with immobilized, dead and live cells, maximum removal was observed for immobilized cells. Among the sugars tested, monosaccharide sugars enhanced the biomass of B. subtilis during lead treatment and the results are discussed.


Introduction
Heavy metals employed in industries are hazardous to human beings and other organisms. A sudden boost in the industrial activities has contributed quantitatively as well as qualitatively to the alarming increase in the discharge of metal pollutants into environmental sink, especially the aqueous environment [1,2]. Heavy metals reaching aquatic systems undergo food chain concentration and cause disorders in higher trophic levels. This fact renders the removal of heavy metals from aqueous solutions indispensable [3].
Lead is a well-known metal for its extensive industrial applications which is in the environment at the end of the industrial processes such as metal finishing, metallurgical work, electroplating, chemical manufacturing, mining, paint production and battery manufacturing. Overall, only a small amount of lead is present in the environment naturally. But, the level is elevated only because of human activities [4,5]. Most commonly children are exposed to lead through lead-based paints, whereas, adults are exposed mainly through their nature of work. With relevance to the impact of heavy metals on human health, each heavy metal causes diverse effects as well as symptoms [6].
Literature review suggested that lead has no significant role to play inside the human body. At the same time, intake of lead through external sources create a variety of surplus effects like elevation of blood pressure, damage of kidney and brain, infertility in men, learning disability and behavioral distraction in children and others within human body [7][8][9].
Several traditional practices such as ion exchange, filtration, evaporation, solvent extraction, electrochemical treatment, reverse osmosis, chemical precipitation and chemical oxidation or reduction are used to eliminate the toxic heavy metals dissolved in the industrial effluents. All these above mentioned processes are either very much expensive or not that much effective in removal process, in particular when the metal concentration is in the range of 1-100 mg/l in the waste water. An additional drawback of the traditional methods is the fabrication of more amount of toxic

Determination of Metal Inhibition
The maximum concentration of lead allowing bacterial growth was determined in nutrient agar (peptic digest of animal tissue-0.5g, beef extract-0.15g, yeast extract-0.15g, sodium chloride-0.5g and agar-1.5g in 100 ml of distilled water) having 50, 100, 500, 1000, 2000, 3000 and 4000 ppm of lead. Growth was observed after incubating the plates for 24 hrs at 37°C.

Sample Preparation
The organism from the overnight culture

Estimation of Residual Lead Concentration
From each concentration ten ml sample was taken and centrifuged at 2500 rpm for fifteen minutes.
The supernatant was taken in an eppendorf tube and subjected to Atomic Absorption Spectrophotometric (AAS) analysis (Model: MSA030351; Thermo Fisher Scientific Ltd., India) and the readings were recorded.

Biomass Estimation
After centrifugation pellet was collected and dried in a hot air oven at 80°C for 3 hrs. The final dry weight was taken to calculate the biomass.

Calculation of Lead Uptake
Lead uptake by the bacterium was calculated using the following mass balance equation [9]: Where, q = lead uptake (mg metal/g cell dry weight);
The classical Freundlich equation is given below: Where, q = heavy metal adsorbed on the Equation [4] can be linearized as follows: The adsorption constants (Q max and b) were obtained by plotting 1/q as a function of 1/C e .

Results
The   Highest biomass is obtained for all the lead concentrations on the sixth day with respect to the treatment period ( Figure 2). residual concentration of lead due to cell types were not significant at 5% level but significant due to treatment period. The variations in the biomass (g/ml) of B. subtilis due to lead concentration and treatment period were statistically significant at 5% level.
The variation in percent removal of lead due to cell types was not statistically significant at 5% level but significant due to treatment period. The variation in percent removal of lead due to lead concentration was not statistically significant at 5% level but significant due to treatment period ( Table 3).
The Freundlich adsorption isotherms for lead biosorption by B. subtilis after every two days of treatment period are given in Figure 5. The Langmuir adsorption isotherms for lead biosorption by B. subtilis after every two days of treatment period are given in Figure 6. In Freundlich isotherm models, R 2 was the maximum after two days of treatment and it showed a decline with the increase in treatment period.
Kf was the highest after eight days of treatment while 1/n was the maximum after four days of treatment. In the case of Langmuir models, R 2 and Qmax were the highest after two days of treatment while b was the maximum after eight days of treatment (Table 4).

Discussion
Even low levels of lead can cause permanent damage in organisms. The immediate measure to prevent lead poisoning is to avoid exposure to lead.
Removal of the source of lead is critical to reducing lead levels. Biosorption is an alternative to traditional physico-chemical means for removing toxic metals from ground waters and waste waters. Removal of lead from solution was studied using growing cells and washed cells of Bacillus cereus [29]. The ability of the strain, B. subtilis to remove lead from solution was investigated in the present study. B. subtilis was reported to be the best for biosorbing lead among copper, zinc, lead and cadmium [30] When different concentrations of lead were tested, the strain B. subtilis was able to resist upto 1000 ppm. When the walls of B. subtilis were tested for the uptake of eighteen different metals, lead was shown to be taken up in small amounts into the wall [34].