Investigation of the Use of Siderophores from Pseudomonas genus to c helate Heavy Metal ions

1. Investigation of the Use of Siderophores from Pseudomonas genus to chelateHeavy Metal ions Members: VamsiMeka Josh Hasan Shaun Png Johnny Yeung 6/11/2009

2. Problem • Arsenic poising in the water of the village of Bangladesh and formation of lung cancer in patients causes health problems (Renshawetal. 2002). • Heavy metal ions are not biodegradable nor thermodegradable and are toxic. (Tansupo et al. , 2009) • Metal pollution caused by factors such as motor vehicle emissions, sewage sludge applications, and manufacturing has led to formation of hazardous environments

3. Background - siderophores • The property of siderophores in chelating ferric ions have long been known and utilized in scientific industries (Duckworth & Sposito, 2007) • Contains antimicrobial properties (Barry & Challis, 2009). http://www.biw.kuleuven.be/dtp/cmpg/pgprb_images/PseudoGreen.jpg

4. Hypothesis • Siderophores will demonstrate chelation for nickel, copper(II), iron(III) and lead(II) ions. • Temperature and pH for the greatest degree of chelation is 26 degrees Celcius and 7 respectively.

5. Objectives • The objective of the experiment is • To investigate the degree of chelation of heavy metal ions of copper (II), iron (III), nickel and lead(II) by siderophores from Pseudomonas genus • To find the temperature and pH which allows the greatest degree of chelation.

6. Variables

7. Apparatus and Materials

8. Procedures Part I: Investigating which heavy metal ions can be chelated: Phase 1: Culturing the bacteria

9. Procedures Phase 2: introducing siderophores to solution. Step 4: Prepare 3 solutions as follows: Set up 1: Experimental Set up; Set up 2 and 3: controls Set up 2: Determine if concentration of heavy metal ions changes in absence of Pseudomonas bacteria Set up 3: Determine if heavy metal ions affect growth of Pseudomonas bacteria In the experiment, only record the results for set ups 1 and 2 to compare the difference in heavy metal ion concentration.

10. Procedures For set ups 1 and 2, • Measure initial concentrations of 50ppm for the heavy metal ions using a spectrophotometer. • Incubate at 26degC for 2 days to allow bacteria to grow • Measure optical density of 600nm using a spectrophotometer • Plot a standard graph of different concentrations of heavy metal ions. • Measure the final concentration of heavy metal ions • Calculate the different in concentrations for each heavy metal ion.

11. Procedures Part II: Determining optimum temperature for chelation • Repeat Phase 1 • Carry out phase 2 only on set up 1, but instead of fixing the incubation temperature at 26 degC, incubate at 22, 24, 26, 28, 30 degC during step 6. • After finishing step 10, plot a graph of difference in concentration/ppmvs temperature to determine the optimum temperature for chelation.

12. Procedures Part III: Determining optimum pH for chelation • Repeat the same procedures for Part I for 5 sets for each type of bacteria, but during step 3, alter the pH to 5.2, 6.2, 7.2, 8.2, 9.2 for each set. • At the end of step 10, plot a graph of difference in concentration/ppmvs pH to determine the optimum pH for chelation.

13. Data collection • Change in concentration/ppm • The higher the change, the higher the rate of chelation • Statistical tests • Done before and after introduction of siderophores from Psudomonasbacteria • p<0.05 suggests that the difference is insignificant and vice versa. • Sample size=5 • Paired t-test to be done

14. Work distribution and safety issues • Both AOS and HCI will take exactly the same steps (same heavy metal ion, same treatment etc.) • However, AOS would work with Pseudomonas syringae while HCI to work with Pseudomonas fluorescens. Both are at BSL level 1 so they are safe to work with. • Lab coats and gloves are worn at all times during experiments.

15. Timeline

16. Progress • Collected Articles to use in Background Research • Background Research draft completed and peer reviewed by three people • Singaporean team has arrived in AOS and worked out details for project. • Both sides to work on their respective tasks while in AOS and HCI respectively and combine results and explanations when AOS arrives in HCI in August 2010. • A parallel project to ease combination in the future.

17. References • Barry, S.M., & Challis G.L. (2009). Recent advances in siderophore biosynthesis. Current Opinions in Chemical Biology, 13(2), 205-215 • Cook, R. J. 1993. Making greater use of introduced microorganisms for biological control of plant pathogens. Annu. Rev. Phytopathol. 31:53–80. • Duckworth, O.W., & Sposito G. (2007). Siderophore-promoted dissolution of synthetic and biogenic layer-type Mn oxides. Chemical Geology, 242(3-4), 497-508 • Miethke, M., & Marahiel M.A. (2007). Siderophore-based iron acquisition and pathogen control. Microbiology and molecular biology reviews, 71(3), 413-451 • Nielands, J. B. (1995). Siderophores: structure and function of microbial iron transport compounds. The Journal of Biological Chemistry, 270(45), 26723-26726. • Renshaw, J.C., Robson, G.D., Trinci, A.P., Wiebe, M.G., & Livens, F.R. (2002). Fungal siderophores: structures, functions and applications. Mycological Research, 106(10), 1123-1142 • Visca, P., Imperi, F., & Lamont, I.L. (2006). Pyoverdinesiderophores: from biogenesis to biosignificance. TRENDS in Microbiology, 15(11), 22-30

18. Thank you.