Transport of Bacterial Endospores in Silica Sand

1. Transport of Bacterial Endospores in Silica Sand Sibylle Tesar, Fulbright Scholar Dr. Barbara Williams, Faculty Dr. Robin Nimmer, Res. Supp. Sci. Angelina Cernick, Undergraduate Kristina Beaulieau, NSF REU Department of Biological and Agricultural Engineering University of Idaho

2. Research Goals – transport mechanisms / endospores Background – transport mechanisms / endospores Research Questions – preliminary Methods – sporulation / saturated column tests / breakthrough curves / depth distribution data Preliminary Results Preliminary Conclusions Future Work – B. cereus, other microbes Outline

3. Mechanistic Goal: Contribute to the lively debate of attachment versus straining Microbe-specific goal: Bacterial endospore Practical Applications Drinking water protection - groundwater Shallow recharge Septic drainfield setbacks Surface water filtration Riverbank or riverbed filtration Research Goals – Spore Transport in Porous Media

4. Research Goals – transport mechanisms / endospores Background – transport mechanisms / endospores Research Questions – preliminary Methods – sporulation / saturated column tests / breakthrough curves / depth distribution data Preliminary Results Preliminary Conclusions Future Work – B. cereus, other microbes Outline

5. Attachment – adhesion – sorption Function of collision, collector efficiency, sticking efficiency Mechanical filtration – complete retention of particles that are larger than all of the soil pores (formation of filter cake) Straining – physical trapping in geometric corners Particles can be smaller than smallest pore openings Requires grain-grain contact Only occurs in some fraction of soil pore space, transport occurs elsewhere Terminology – Mechanisms for Retention Bradford et al, WRR, 2006

6. Strained versus Mechanically Filtered dp/d50 .005

7. Clean-bed Filtration Theory Depends on mechanism of attachment / detachment Deviation from Clean Bed Filtration Theory Unfavorable attachment condition; neg-neg Fine sand and large colloids (dp/d50 .005) Background

8. Attachment w/ porous media charge variability – Johnson and Elimelech, 1995 Attachment w/ heterogeneity in surface charge characteristics of colloids – Li et al, 2004 Attachment w/ deposition of colloids in a secondary energy minimum – Tufenkji et al. 2003, Redman et al., 2004 All of the above – Tufenkji and Elimelech, 2005 Attachment w/ straining – Foppen et al, 2005, Bradford et al, 2006a, b Explanations for Deviation from CFT

9. Theory (cont.) Aqueous Phase Colloid Mass Balance Equation- Bradford et al., 2003 Where: θw = volumetric water content [-] t = time [T] C = colloid concentration in the aqueous phase [N L-3] JT = total colloid flux [N L-2 T-1] EattSW = colloid attachment mass transfer between solid/water phases [N L-3 T-1] EstrSW = colloid straining mass transfer between solid/water phases [N L-3 T-1]

10. Endospore-forming bacteria have two viable modes Vegetative cell (growing) Endospore (dormant) – formed as survival mechanism Endospores have the potential to be more mobile than their vegetative cell counterparts smaller size potentially less adhesion Research Goals – Endospore Transport

11. Formed as a survival mechanism Cryptobiotic – no sign of life - dormant mode Bacterial Endospores http://www.textbookofbacteriology.net/

12. Differences between endospores and vegetative cells in Bacillus species http://www.textbookofbacteriology.net

13. Differences between endospores and vegetative cells in Bacillus species

14. In terms of physical passage through the pore space… …the spore has a “shorter” aspect ratio than the vegetative cell. • B. cereus spore properties: • Food poisoning pathogen • Length: 1-2 mm, Width: 0.5-0.75 mm • Hydrophobic • Isoelectric point: pH ~3

15. Research Goals – transport mechanisms / endospores Background – transport mechanisms / endospores Research Questions – preliminary Methods – sporulation / saturated column tests / breakthrough curves / depth distribution data Preliminary Results Preliminary Conclusions Future Work – B. cereus, other microbes Outline

16. Do spores obey CFT, exhibiting more retention in higher ionic strength solution or does spore transport deviate from CFT theory as do other negatively charged particles (unfavorable attachment)? Future: Do vegetative cells and endospores have a different charge? Future: Do vegetative cells exhibit more attachment than endospores? Preliminary Research Questions

17. Research Goals – transport mechanisms / endospores Background – transport mechanisms / endospores Research Questions – preliminary Methods – sporulation / saturated column tests / breakthrough curves / depth distribution data Preliminary Results Preliminary Conclusions Future Work – B. cereus, other microbes Outline

18. Materials: Sand Properties Saturated conductivity: Ksat = 1.8x10-4 m/sec Dry bulk density: rb = 1.65 g/cm3 Porosity: n = 0.34 dp/d50 .0017

19. Method: Constant Head, Sand Column

20. Breakthrough (C/Co) of B. cereus spores as a function of ionic strength Artificial groundwater DDI

21. Column Dissection

22. Column Dissection • Drain column to field capacity, in the flow direction. • Dissect into seven 2 cm increments • STR 1:Gently place sand, allowing bridging and loose packing, in a funnel that has been plugged with Scotchbritetm pad • STR 2: Wash off the strained bacteria by pouring the solution (the solution used in that particular experiment) over the sand into a graduated cylinder • ATT: To remove the attached bacteria, place a known amount of 2% Tweentm 80 solution into a beaker containing the sand. Stir then sonicate. • Used optical density (OD) measurements in addition to plate counting to enumerate. (Tween and sonication proven not to affect germination efficiency)

23. Depth Distribution Data “Strained” spores in AGW run

24. Depth Distribution Data “Attached” spores in AGW run

25. Depth Distribution Data Strained and attached fractions combined

26. Research Goals – transport mechanisms / endospores Background – transport mechanisms / endospores Research Questions – preliminary Methods – sporulation / saturated column tests / breakthrough curves / depth distribution data Preliminary Results Preliminary Conclusions Future Work – B. cereus, other microbes Outline

27. Breakthrough curvedata are consistent with CFT – higher ionic strength, more retention Depth distribution data show deviation from CFT – not exponential with depth Preliminary Conclusions

28. Research Goals – transport mechanisms / endospores Background – transport mechanisms / endospores Research Questions – preliminary Methods – sporulation / saturated column tests / breakthrough curves / depth distribution data Preliminary Results Preliminary Conclusions Future Work – B. cereus, other microbes Outline

29. Compare attachment/straining of spores versus vegetative cells Column experiments Micromodels and photographs Wet AFM Compare zeta potential pH and more ionic strength effects Different endospore bacteria, such as S. pasteurii, for biomineralization Future Work

30. Dr. Ron Crawford, Director, Environmental Biotechnology Institute, UI Nick Benardini, PhD Candidate, MMBB Elizabeth Scherling, MS, BAE Dr. Markus Tuller, PSES David Christian, Research Support Sci. Acknowledgements

31. Fulbright Scholars Program USDA Hatch UI URO Seed Grant Program NSF REU program Funding Acknowledgements

32. Bradford, S.A., J. Šimůnek, M. Bettahar, M. vanGenuchten, and S.R. Yates. 2003. Modeling colloid attachment, straining, and exclusion in saturated porous media. Environmental Science and Technology 37: 2242-2250. Bradford, S.A., J. Šimůnek, M. Bettahar, M.Th. vanGenuchten, and S.R. Yates. 2006a. Significance of straining in colloid deposition: evidence and implications. Water Resources Research, 42:doi:10.1029/2005WR004791. Bradford, S.A., J. Šimůnek, and S.L. Walker. 2006b. Transport and straining of E. coli 0157:H7 in saturated porous media. Water Resources Research (in review). Li, X., TD. Scheibe, and W.P. Johnson. 2004. Apparent decreases in colloid deposition rate coefficient with distance of transport under unfavorable deposition conditions: a general phenomenon. Environ. Sci. Technol., 38: 5616-5625. Redman, J.A., S.L. Walker, and M. Elimelech. 2004. Bacterial adhesion and transport in porous media: Role of the secondary energy minimum, Environ. Sci. Technol., 38:1777-1785. Tufenkji, N., J.A. Redman, and M. Elimelech. 2003. Interpreting deposition patterns of microbial particles in laboratory-scale column experiments, Environ. Sci. Technol., 37: 616-623. Tufenkji, N., Elimelech, M. 2005. Breakdown of colloid filtration theory: Role of the secondary energy minimum and surface charge heterogeneities. Langmuir 21: 841-852. References