Overview

1. Analytical approaches to study the complexation of Uranium by bacterial cells: EXAFS, XPS, vibrational spectroscopy and potentiometric titrations Jesus J. Ojeda, Maria E. Romero-Gonzalez, Mohamed Merroun, Marta Nedelkova, Christoph Hennig, Andre Rossberg, Sonja Selenska-Pobell

2. Overview Introduction Potentiometric titrations Vibrational spectroscopy X-ray Photoelectron Spectroscopy X-ray absorption spectroscopy Conclusions and Further work

3. Introduction • Radioactive waste has been stored underground for decades. However, there is concern that this waste can escape and migrate into groundwater and sediments.

4. Introduction • Radioactive waste has been stored underground for decades. However, there is concern that this waste can escape and migrate into groundwater and sediments. • Indigenous bacterial population can interact with radionuclides via different mechanisms, causing immobilization of the metals.

5. Introduction • Radioactive waste has been stored underground for decades. However, there is concern that this waste can escape and migrate into groundwater and sediments. • Indigenous bacterial population can interact with radionuclides via different mechanisms, causing immobilization of the metals. • Biomineralization can occur aerobically, making this process a possible remediation strategy for radionuclides in contaminated ground water and oxygenated subsurface zones.

6. Potentiometric Titrations Metrohm 718 STAT Titrino

7. pKa H3O+ + A- HA + H2O The propensity of a compound to donate a proton is measured as its acid ionization constant, or Ka. These Ka values cover a wide range of 1010 for the strongest acids such as sulphuric acid to 10-50 for the weakest acids such as methane. Theory taken from: http://web.chem.ucla.edu/~harding/tutorials/acids_and_bases/pKa_table.html

8. pKa A more convenient scale of acidity is pKa which is the negative logarithm of the Ka: Thus, a Ka of 1010 becomes a pKa of -10, and a Ka of 10-50 becomes a pKa of 50. More generally, more negative pKa values correspond to stronger acids and more positive pKa values correspond to weaker acids. Theory taken from: http://web.chem.ucla.edu/~harding/tutorials/acids_and_bases/pKa_table.html

9. pKa The exact pKa of an acid is a function of molecular structure (i.e., functional groups) and must be determined experimentally: • Titration data • (b) Plot of experimental buffering capacity (calculated as ∂[H+]consumed/∂pH) against pH. Theory taken from: http://web.chem.ucla.edu/~harding/tutorials/acids_and_bases/pKa_table.html

10. Potentiometric data of Sphingomonas sp.

11. Potentiometric data of Sphingomonas sp.

12. Potentiometric data of Sphingomonas sp.

13. Potentiometric data of Sphingomonas sp.

14. Functional groups on the cell surface pKa values are similar to data found in literature for Gram-negative bacteria.

15. Functional groups on the cell surface Site concentrations seem to be higher to data found in literature for Gram-negative bacteria.

16. Functional groups on the cell surface • pK = 4.27 can be assigned to carboxyl groups (pKa of carboxyl groups vary from 2 to 6). • pK = 7.03 can be assigned to phosphoryl groups (pKa of phosphoryl groups vary from 5.6 to 7.2). • pK = 9.22 can be assigned to hydroxyl and/or amine groups (pKa of phenolic (hydroxyl) groups vary from 8 to 12, and pKa of amine groups vary from 8.6 to 10.0). • pKa values are very similar to data found in literature for Gram-negative bacteria, although site concentrations seem to be higher. • pHzpc suggests that Sphingomonas sp is negatively charged at pH > 4.4. Therefore, positive ions (such as UO22+) could be attracted by its surface if pH > 4.4.

17. Functional groups on the cell surface Haas et al (2001) showed that, in presence of U(VI) solution, bacterial sorption is accounted for by using two separate adsorption reactions forming the surface complexes >COO–UO2+and >PO4H–UO2(OH)2. Therefore, phosphate and carboxyl groups are expected to be implicated in the binding of this radionuclide. (Haas, J.R.; Dichristina, T.J.; Wade, R. Thermodynamics of U(VI) sorption onto Shewanella putrefaciens. Chem. Geol. 2001, 180, 33-54)

18. Infrared Spectroscopy Perkin Elmer Spectrum One FTIR Spectrometer Perkin Elmer Spotlight Micro-FTIR Imaging System

19. Infrared Spectroscopy In a molecule, the atoms are not held rigidly apart. Instead they can move, as if they are attached by a spring: If the bond is subjected to infrared radiation (between 300 and 4000 cm-1), it will absorb the energy, and the bond will move. Weaker bonds require less energy, as if the bonds are springs of different strengths. Theory and animations taken from: http://www.ir-spektroskopie.de/ http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/irspec1.htm http://sis.bris.ac.uk/%7Esd9319/spec/IR.htm http://www.chem.uic.edu/web1/OCOL-II/WIN/SPEC/IR/IRF.HTM

20. Infrared Spectroscopy Absorption of infrared radiation by a typical organic molecule results in the excitation of vibrational, rotational and bending modes. Modes of vibration for H2O: • Types of Bending: • Rocking • Scissoring • Wagging • Twisting Symmetric stretching (us) Asymmetric stretching (uas) Symmetric bending (scissoring) (ds) Theory and animations taken from: http://www.ir-spektroskopie.de/ http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/irspec1.htm http://sis.bris.ac.uk/%7Esd9319/spec/IR.htm http://www.chem.uic.edu/web1/OCOL-II/WIN/SPEC/IR/IRF.HTM

21. Infrared Spectroscopy If there are more atoms, there will be more bonds, and therefore more modes of vibrations. This will produce a more complicated spectrum. Modes of vibration for >CH2: Theory and animations taken from: http://www.ir-spektroskopie.de/ http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/irspec1.htm http://sis.bris.ac.uk/%7Esd9319/spec/IR.htm http://www.chem.uic.edu/web1/OCOL-II/WIN/SPEC/IR/IRF.HTM

22. Infrared Spectroscopy For simple systems, we can consider the atoms as point masses, linked by a 'spring' having a force constant k and following Hooke's Law. To calculate the frequency of light absorbed, we use the Hooke’s Law as follows: where: k = force constant of the bond (dyne/cm) c = speed of light (cm/s) m1 and m2 are the masses of the two atoms (Kg) Theory and animations taken from: http://www.ir-spektroskopie.de/ http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/irspec1.htm http://sis.bris.ac.uk/%7Esd9319/spec/IR.htm http://www.chem.uic.edu/web1/OCOL-II/WIN/SPEC/IR/IRF.HTM

23. FTIR spectrum • C=O of amides associated with proteins 1639 cm-1 • C-O-C and n C-O of polysaccharides and some contributions from n PO2- and P(OH)2 in phosphates 1000 – 800 cm-1 • N-H and n C-N in amides and nas C O of carboxylates 1540 cm-1 • C=O of carboxylic and ester functional groups from membrane lipids and fatty acids 1735cm-1 nas P=O of general phosphoryl groups 1240 cm-1 nsC O of carboxylate anion 1398 cm-1 Band assignments made according to: Conley (1972), Naumann (1991), Wade (1995), Schmitt et al (1998),Jiang et al (2004), Yee et al (2004), Dittrich et al (2005) and Ojeda et al (2008).

24. Raman spectra before and after uranyl exposure, and the spectrum of the uranyl nitrate solution used, showing the peak characteristic of UO22+

25. X-Ray Photoelectron Spectroscopy Kratos Axis 165 Ultra Photoelectron Spectrometer

26. X-Ray Photoelectron Spectroscopy • Elemental identification and chemical state of element • Relative composition of the constituents in the surface region • Valence band structure Taken from: http://www.uwo.ca/ssw/services//xps/xpsgradcourse/resources/xps_grad_course.pdf

27. Evidence from X-ray spectroscopy

28. Evidence from X-ray spectroscopy

30. X-ray absorption spectroscopy Theory taken from: Jalilehvand, F. (2000) Structure of hydrated ions and cyanide complexes by X-ray absorption spectroscopy. PhD thesis, Royal Institute of Technology, Stockholm, Sweden.

31. X-ray absorption spectroscopy a:Errors in coordination numbers are ±25%, and standard deviations are given in parentheses. b: errors in distance are ±0.02 Å. c: Debye-Waller factor. d: value fixed for calculation

32. X-ray absorption spectroscopy a:Errors in coordination numbers are ±25%, and standard deviations are given in parentheses. b: errors in distance are ±0.02 Å. c: Debye-Waller factor. d: value fixed for calculation EXAFS analysis suggests that Sphingomonas cells precipitated uranium as meta-autunite mineral like phase.

33. Conclusions • In situ remediation strategies, particularly those mediated by microorganisms are attractive alternatives for the cleaning-up of radionuclide contaminated sites, as they are generally more cost-effective and less invasive that other options. • In aerobic conditions, U is usually present as the highly soluble uranyl ion (UO22+). In the case of bacterially mediated U precipitation in oxygenated zones, proposed strategies could be focused on precipitation of U as complexes of phosphate. • At this stage of investigation, the origin of the phosphates which precipitate U is still unknown. However, one possible source of the organic phosphate is that arising from the lysis of the dead cells. • In uranium contaminated sites, the lysed dead cells will liberate to the environment significant quantities of biopolymers such as polypeptides, phosphorylated peptides or nucleic acids, which are able precipitate uranium.

34. Further work: Infrared and Raman micro-imaging Absorbance 0.725 0.562 0.399 0.175 0.072 0.000 a b 50mm d e c g f (a) Optical image of stainless steel and Aquabacterium commune biofilm. (b) Full range (4000 to 700 cm-1) false-colour image of the sample scannd using micro-FTIR spectroscopy (c) Amide I and II. (d) C=O, C-O-H, C-H. (e) COO-. (f) P-O-H (g) –OH. Ojeda et al (2009) Analytical Chemistry. 2009, 81, 6467–6473.

35. Further work: Time lapse fluorescence imaging