Bioremediation of Zinc using Pseudomonas Species - Mechanistic Studies and Biosensor Applications
Ebinesar, J S S Allwin
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The rivers, lakes and seas are the major water sources for the animal and plant kingdom in this earth. In recent times, the usage and wastage of water have been increasing due to the uncontrolled population growth. In addition to that, the rapid industrialization over the years has led to the gradual depletion of the natural resources like water, soil and air. Some of these industries discharge contaminants like organic products and inorganic (or) toxic heavy metals without treatment into the environment, leading to its degradation. Zinc is the 24th most abundant element present in the earth crust, amounting 75 ppm (0.0075%). The concentration of zinc present in the soil and seawater is about 64 ppm and 30 ppb respectively (Emsley, 2001). Generally, the zinc is found with the base metals such as copper and lead and it has less affinity with oxides and strong affinity with sulphides. Sphalerite, a zinc sulphide ore, is majorly containing 60-62% of zinc. The other sources of zinc from the minerals are smithsonite, hemimorphite, quartzite, and hydro zincate. The major sources of zinc contamination arise from several industrial activities such as mining, coal, waste combustion and steel and iron processing. Drinking water also contains certain amounts of Zn, which may be higher when it is stored in metal tanks. The acute toxicity arises from the ingestion of excessive amounts of zinc salts, either accidentally or as dietary supplement. Vomiting, nausea and stomach cramps usually occur after the consumption of more than 500 mg of zinc sulfate. In addition to that, the higher amounts of zinc affect gastrointestinal tract, liver, bone and prostate glands. Finally, Zn can interrupt the activity in soils, as it negatively influences the activity of microorganisms and earthworms, thus retarding the breakdown of organic matter. To combat this problem, techniques such as chemical precipitation, ion exchange, reverse osmosis, etc. are adopted, but these processes result in a huge amount of secondary sludge formation, inefficient removal of metals and are not cost effective. In recent times, an innovative, eco-friendly, cost-effective method has been introduced to treat the toxic heavy metals namely bioremediation. ―Bioremediation‖ is a process of removal of organic or inorganic contaminants by using bacteria, fungi, algae and its metabolites In this research work, the potential of four bacterial strains of the Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens and the extracellular proteins secreted by these four species for the bio-sorption of zinc has been investigated through batch experiments. The mechanisms of interaction between the zinc ion and the bacterial biomass as well as with the extracellular proteins have been elucidated. Additionally, a carbon paste electrode has been modified by using Pseudomonas sp. and its metabolites to develop biosensors for zinc and the lower limit of detection of zinc in aqueous solution has been determined. The major objectives of this research work are specified below: • To study the potential of Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens for the bio sorption of zinc, in batch systems. • To determine the speciation of zinc with respect to pH in the growth medium and the maximum inhibitory effect of zinc on the growth of the four chosen Pseudomonas sp. • To isolate and characterize the extracellular proteins from the four Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens. • To study the biosorption of zinc by extracellular proteins secreted by the Pseudomonas sp. • To elucidate the mechanisms involved in the biosorption of zinc at the microbe- metal interface and protein-metal ion interface for all the four systems by different characterization studies such as zeta potential, FTIR analysis and EDAX analysis. • To develop a biomass modified CPE using bacterial cells and extracellular protein to detect the concentration of zinc in aqueous solutions adopting voltammetric techniques. The significant results obtained from this research work are summarized as follows: The initial studies were concentrated on the bio sorption of zinc by using four Pseudomonas species such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens. The various factors affecting the bio sorption of zinc by these species were investigated by varying the contact time (10-80 min), pH (2-5±0.2), biomass concentration of the four species in the range of 108- 1011 cells / mL, and the initial zinc concentration from 5 mg/L to 80 mg/L respectively, keeping other parameters such as temperature and agitation speed constant in all the experiments. From the results obtained, the maximum percentage of biosorption achieved by the P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens was found to be 60%, 93%, 70% and 65% respectively for 25 mg/L at pH 5±0.2. The equilibrium time taken by the four species to achieve maximum biosorption was about 10 min and the biosorption kinetics adhered to pseudo-second order reaction and the rate constants were determined for different concentrations of zinc. The biosorption isotherm followed both the Langmuir and Freundlich isotherm models. The Gibbs free energy (ΔG) values determined from the Langmuir isotherm model for all the four systems were found to be -26, -32, -30 and -28 kJ /mole respectively. The Gibbs free energy values indicate that the biosorption of zinc ions onto the bacterial surface is a chemi-sorption process involving co-ordination, complexation or chelation. The characterization studies, namely zeta potential, FTIR analysis and SEM-EDX were also carried out on the bacterial cells before and after interaction with zinc. These studies also provide evidence in support of the complexation of zinc with the functional groups on the bacterial cell surface apart from electrostatic interaction. In the second part of the investigation, the inhibitory effect of zinc on the growth of four Pseudomonas sp. was investigated by varying the concentration of zinc from 50 mg/L to 1000 mg/L and the stability of zinc was analysed with respect to pH (2-12) with different concentrations from 50 - 1700 mg/L. It was found that in the absence of zinc the time taken to reach the exponential phase and the specific growth were almost the same for all the four systems. However, in the presence of zinc ions, the growth of the four Pseudomonas sp. was suppressed beyond 50mg/L of zinc. A control study on the stability of zinc in Luria broth medium showed that zinc was highly stable up to 200 mg/L from pH 2-8. However, the stability of zinc in the growth medium decreased beyond that concentration Additionally, studies on the biosorption of zinc were performed using extracellular proteins isolated from the four Pseudomonas sp. The amount of protein was estimated by the Bradford protein assay method at 594 nm. The biosorption experiments were carried out by varying the protein concentration from 50 to 1000µg/mL and the zinc concentration from 50-1000 mg/L and keeping other parameters fixed, namely such as pH at 5±0.2, reaction time of 20 min, temperature at 30±0.2 and the speed of rotation of 200 rpm. It was found that the maximum percentage of zinc biosorbed by the proteins isolated from P.putida was found to be 91% at 500µg/mL of protein concentration and from the other three species, it was found to be about 60% of biosorption at the same protein concentration. The biosorption isotherms of zinc for extracellular protein adhered to the Giles H1 type for all the four systems. The maximum amount of zinc biosorbed by the protein isolated from P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens was found to be 35.6, 19,18.3 and 10 mg/µg respectively and the Gibbs free energy values were found to be -32, -22,-22 and -23 kJ/mole. The mechanisms involved in protein-zinc interaction were elucidated using FTIR analysis and EDX analysis. The FTIR analysis revealed, that the zinc ions were complexed with carboxylic and amine functional groups. Further, the potential of P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens and their extracellular proteins of P.putida as biosensors for detecting zinc ions in aqueous solutions, using electrochemical methods such as, Cyclic Voltammetry and Differential pulse anodic stripping voltammetry, was assessed. The developed carbon paste electrode coated by the biomass showed an approximately 3-fold increase in the sensing of Zn2+ ion in comparison with the bare electrode. The lower limit of detection of the biosensor for zinc ions by Cyclic voltammetry was found to be 10-6 M, and in case of DPASV the lower limit of detection was about 10-7M. The lower limit of detection of the protein modified biosensor for zinc ions by cyclic voltammetry was found to be 10-7M and in the case of DPASV method the lower limit of detection was found to be 10-9 M.
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