1. Members: Vamsi Meka Josh Hasan Shaun Png Johnny Yeung Investigation of the Use of Siderophores from Pseudomonas genus to chelate Heavy Metal ions 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 (Renshaw et al. 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 for both species of Pseudomonas Temperature and pH for the greatest degree of chelation is 26 degrees Celcius and 7.2 respectively for both species of Pseudomonas

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 IndependentDependentControlled Type of heavy metal ionFinal concentration of heavy metal ions Initial ion concentration Temperature of incubation Initial bacteria innoculum pH

7. Apparatus and Materials ApparatusMaterials SpectrophotometerPseudomonas fluorescens ATCC 948 MicropipettesPseudomonas syringae Inoculating loopCopper (II) ions Orbital shakerFerric ions CentrifugeNickel ions Petri dishesLead(II) ions LB medium MSG medium

8. Procedures Phase 1: Culturing the bacteria Part I: Investigating the heavy metal ions that can be chelated: 1. Preculture bacteria in 10ml LB broth in 26 degC 2. Standardise the optical density of at 600nm with spectrophotometer to 1 3. Centrifuge the culture at 8000 rpm for 5 minutes and resuspend the cell pellet in 5ml MSG medium at pH 7.2

9. Procedures Set upMSG medium/broth (Is bacteria present?)Presence of heavy metal ion solution 1MSG medium – bacteria presentPresent 2MSG broth –bacteria absentPresent 3MSG medium –bacteria presentAbsent Phase 2: introducing siderophores to solution. 4. Prepare 3 solutions as follows: Set up 1: Experimental Set up 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 5. Incubate at 26degC for 2 days to allow bacteria to grow 6. Centrifuge the resulting solution at 8000rpm for 5 mins. 7. Measure optical density at 600nm of the filtrate using a spectrophotometer 8. Plot a standard graph of different concentrations of heavy metal ions. 9. Measure the final concentration of heavy metal ions 10. Calculate the different in concentrations for each heavy metal ion.

11. Procedures Part II: Determining optimum temperature for chelation - test only for metal ions that are chelated during Part I Measure difference in heavy metal ion concentration Measure final concentration of heavy metal ions with spectrophotometer and standard curve. Centrifuge to remove bacteria and obtain supernatant Prepare 5 sets of setup 1, and incubate the different setups at temperatures of 22, 24, 26, 28 and 30 degrees Celcius for 2 days Repeat Phase 1

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 preparation of MSG medium, alter the pH to 5.2, 6.2, 7.2, 8.2, 9.2 for each set. After obtaining absorbance values, plot a standard graph to find the final concentration of the set up and thus calculate the change in concentration Plot a graph of the change in concentration/ppm vs pH for various values of pH.

13. Data collection Change in concentration/ppm The higher the change, the higher the rate of chelation Graph plotting Plot graphs for 3 different experiments Change in concentration against type of metal Change in concetration against temperature Change in concentration against pH Statistical tests Conducted with concentration obtained before and after introduction of siderophores from Pseudomonas bacteria p<0.05 suggests that the difference is significant 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). Pyoverdine siderophores: from biogenesis to biosignificance. TRENDS in Microbiology, 15(11), 22-30

18. Thank you.