chemical and physical interactions in bacterial adhesion and transport

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Outline. Background and ImportanceSelection and Preparation of Bacterial StrainsKinetics of Bacterial Adhesion/DepositionDLVO Interpretation of Bacterial AdhesionProposed Adhesion MechanismConcluding Remarks. Environmental Relevance. Transport of microbial pathogens in the subsurface environmentBioremediationBiofouling in membrane processes for water quality control applications.

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chemical and physical interactions in bacterial adhesion and transport

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1. Chemical and Physical Interactions in Bacterial Adhesion and Transport Sharon L. Walker, Jeremy A. Redman, and Menachem Elimelech Department of Chemical Engineering Environmental Engineering Program Yale University

3. Environmental Relevance Transport of microbial pathogens in the subsurface environment Bioremediation Biofouling in membrane processes for water quality control applications

5. Bacterial Adhesion in the Subsurface Aquatic Environment is Complex

6. Background: Bacterial Surface Characteristics Outer membrane structure of Gram negative cells Mention bacterial size/scale Walk people through schematic Gram + lack outer membrane and have a rigid inner layer made up of linear polymers of peptidoglycan (about 25nm thick) surrounding the phospholipid bilayer membrane. Gram + peptidoglycan layer has secondary polymers (ie teichoic acid) in framework that increases the negative charge density of the cell (from exposed carboxyl and phosphoryl groups) Gram - cells have inner and outer lipid bilayer membranes and less rigid peptidoglycn layer in between membranes. Peptidogylcan layer is only ~8nm thick LPS are chains of sugars protruding more than 30nm out from cell O-antigen is chain of sugars in 4 to 5 sugar sequences (often repeated and branched) Mention proteins in membrane can effect surface charge (-) Characteristics of membrane components (hydrophobic/hydrophilic) Mention bacterial size/scale Walk people through schematic Gram + lack outer membrane and have a rigid inner layer made up of linear polymers of peptidoglycan (about 25nm thick) surrounding the phospholipid bilayer membrane. Gram + peptidoglycan layer has secondary polymers (ie teichoic acid) in framework that increases the negative charge density of the cell (from exposed carboxyl and phosphoryl groups) Gram - cells have inner and outer lipid bilayer membranes and less rigid peptidoglycn layer in between membranes. Peptidogylcan layer is only ~8nm thick LPS are chains of sugars protruding more than 30nm out from cell O-antigen is chain of sugars in 4 to 5 sugar sequences (often repeated and branched) Mention proteins in membrane can effect surface charge (-) Characteristics of membrane components (hydrophobic/hydrophilic)

7. Lipid A-KDO-core-O antigen KDO-detodeoxyoctonate Hep -heptose Glu-glucose Gal - galactose GluNac - N-acetylglucosame GlcN - glucosamine lipid A of LPS can be toxic O-antigen usually contains galactose, glucose, rhamnose and mannose (all 6-C sugars) - often dideoxysugars too (ie abequose, colitose, paratose or tyvelose) (4-5 membered sequences) O-antigen - often repeating sequences and branched Mention Gram + and - cells produce exopolymers (aka gycocalyx) which is a thick (25-1000nm) amorphous matrix of polysaccharides and polypeptidesLipid A-KDO-core-O antigen KDO-detodeoxyoctonate Hep -heptose Glu-glucose Gal - galactose GluNac - N-acetylglucosame GlcN - glucosamine lipid A of LPS can be toxic O-antigen usually contains galactose, glucose, rhamnose and mannose (all 6-C sugars) - often dideoxysugars too (ie abequose, colitose, paratose or tyvelose) (4-5 membered sequences) O-antigen - often repeating sequences and branched Mention Gram + and - cells produce exopolymers (aka gycocalyx) which is a thick (25-1000nm) amorphous matrix of polysaccharides and polypeptides

8. Relevant Research Questions What is the role of electrostatic double layer interactions in bacterial adhesion (deposition) under flow conditions? What is the role of cell surface LPS and the interplay with electrostatic interactions?

9. Selected Model Bacteria E. Coli K12 is well-characterized and therefore strains have been selected with representative lipopolysaccharide (LPS) portions on outer membrane. These mutants have LPS of differing lengths and molecular composition. E. Coli K12 is well-characterized and therefore strains have been selected with representative lipopolysaccharide (LPS) portions on outer membrane. These mutants have LPS of differing lengths and molecular composition.

10. Experimental Approach Packed-bed column and radial stagnation point flow (RSPF) system experiments ? macroscopic vs. microscopic scale analysis

12. Column Setup Packed-bed column SPF – a single collector grain Qualitative results to be discussed as a method of interpreting deposition/adhesion trends as seen in column experiments SPF – a single collector grain Qualitative results to be discussed as a method of interpreting deposition/adhesion trends as seen in column experiments

13. Circle indicates clean-bed filtration Describe btc and how kd is calculated Point out: ~97% eluted at 1mM (10^-3 M) ~1% eluted (or ~99% retained) at 100mM (10^-1M) Circle indicates clean-bed filtration Describe btc and how kd is calculated Point out: ~97% eluted at 1mM (10^-3 M) ~1% eluted (or ~99% retained) at 100mM (10^-1M)

14. Bacterial Adhesion Kinetics Point out axes – showing deposition rate across range of IS Note increased kd with increase in IS for intermediate length LPS D21g Increase in kd is over 2 orders of magnitude over range of IS Increase in deposition due to compression of the double layer at higher ISPoint out axes – showing deposition rate across range of IS Note increased kd with increase in IS for intermediate length LPS D21g Increase in kd is over 2 orders of magnitude over range of IS Increase in deposition due to compression of the double layer at higher IS

15. Bacterial Adhesion Kinetics Most truncated LPS form, D21f2g, also follows trend of increased kd with increased IS Which is trend that DLVO would suggestMost truncated LPS form, D21f2g, also follows trend of increased kd with increased IS Which is trend that DLVO would suggest

16. Bacterial Adhesion Kinetics QUESTION : do we remove 300mM data? Finally jm109g data is plotted Longest lps shows general trend as seen for other two strains… Not this increase with IS is CONSIDERABLY LESS PRONOUNCED at higher IS QUESTION : do we remove 300mM data? Finally jm109g data is plotted Longest lps shows general trend as seen for other two strains… Not this increase with IS is CONSIDERABLY LESS PRONOUNCED at higher IS

17. Radial Stagnation Point Flow System

18. Relevance of Stagnation Point Flow System Flow field near collector surface can be decomposed into the forward stagnation region direct observation of deposition of particles when a transparent collector is used axisymmetric SPF (aka impinging jet or symmetrical radial stagnation flow) well characterized hydrodynamics fluid stream impinges on flat surface at a right angle and flows away radially in all directions particles are sufficiently small to be moving at the velocity of the carrying fluid(and not disturbing the flow field) immediate vicinity of the collector surface, the region where deposition takes place Flow field near collector surface can be decomposed into the forward stagnation region direct observation of deposition of particles when a transparent collector is used axisymmetric SPF (aka impinging jet or symmetrical radial stagnation flow) well characterized hydrodynamics fluid stream impinges on flat surface at a right angle and flows away radially in all directions particles are sufficiently small to be moving at the velocity of the carrying fluid(and not disturbing the flow field) immediate vicinity of the collector surface, the region where deposition takes place

19. Determining Deposition Rate in RSPF

20. RSPF Deposition Kinetics Refer to kRSPF as bacterial transfer rate Increase in transfer rate with increasing IS – same trend as seen in the column experiment Note below 312.6 there is “o” dep over the time scale of the experimentRefer to kRSPF as bacterial transfer rate Increase in transfer rate with increasing IS – same trend as seen in the column experiment Note below 312.6 there is “o” dep over the time scale of the experiment

23. QUESTION: remove data for column at 300mM? Note: In circled region is data for conditions at which we show data points for RSPF . Below these IS dep occurs in column, but not in SPF Evidence for electrostatics: For both systems increase in dep. Rate with increase in IS – follows DLVO prediction (click) all 3 strains “collapse” at highest IS suggesting electrostatics plays a dominate role at these higher IS particularl For both systems it is observed that the relative dep. Rate does not correspond with LPS length – rather to the relative zeta potential. Increased negative zeta potential there is decrease in deposition – suggesting electrostatic repulsion (click) 4.All three strains’ dep. Drops off at low IS in the RSPF – even under conditions when dep. Occurred in column…why? Segue – Lets consider the mechanisms involved and turn to traditional DLVO theory for insight QUESTION: remove data for column at 300mM? Note: In circled region is data for conditions at which we show data points for RSPF . Below these IS dep occurs in column, but not in SPF Evidence for electrostatics: For both systems increase in dep. Rate with increase in IS – follows DLVO prediction (click) all 3 strains “collapse” at highest IS suggesting electrostatics plays a dominate role at these higher IS particularl For both systems it is observed that the relative dep. Rate does not correspond with LPS length – rather to the relative zeta potential. Increased negative zeta potential there is decrease in deposition – suggesting electrostatic repulsion (click) 4.All three strains’ dep. Drops off at low IS in the RSPF – even under conditions when dep. Occurred in column…why? Segue – Lets consider the mechanisms involved and turn to traditional DLVO theory for insight

24. Interpretation using DLVO Theory Calculate Total Interaction Energy Profile:

25. DLVO Total Interaction Profiles

26. Depth of the Secondary Minimum

27. Depth of the Secondary Minimum

28. This parameter incorporates the factors controlling the height of the DLVO energy barrier as well as the depth of the secondary energy minimum: (4) Here, yp and yc are the surface potentials of the bacterial cells and quartz collectors, respectively, k is the inverse Debye length characterizing the range of electrostatic double layer interactions, A is the Hamaker constant of the interacting media, ?0 is the dielectric permittivity of a vacuum, and ?r is the relative dielectric permittivity of water. The NDLVO was determined for each of the ionic strength conditions and cell types used in the packed column experiments using the zeta potentials for both the cells and quartz grains in place of surface potentials This parameter incorporates the factors controlling the height of the DLVO energy barrier as well as the depth of the secondary energy minimum: (4) Here, yp and yc are the surface potentials of the bacterial cells and quartz collectors, respectively, k is the inverse Debye length characterizing the range of electrostatic double layer interactions, A is the Hamaker constant of the interacting media, ?0 is the dielectric permittivity of a vacuum, and ?r is the relative dielectric permittivity of water. The NDLVO was determined for each of the ionic strength conditions and cell types used in the packed column experiments using the zeta potentials for both the cells and quartz grains in place of surface potentials

29. Supporting Evidence for Secondary Minimum Deposition Elution Experiments Changes in ionic strength alter DLVO profiles Elimination of the secondary minimum will result in release of deposited bacteria

30. Elution from Secondary Minima

31. ~ 90% removed in this experiment with D21f2g at 31.6mM ~72% of originally deposited cells were released All cell types across range of IS considered resulted in the majority of cells being released~ 90% removed in this experiment with D21f2g at 31.6mM ~72% of originally deposited cells were released All cell types across range of IS considered resulted in the majority of cells being released

32. Supporting Evidence for Secondary Minimum Deposition Elution Experiments Changes in ionic strength alter DLVO profiles Radial Stagnation Point Flow (RSPF) RSPF can provide further evidence for secondary min deposition by considering the mechanisms involved for cell adhesion in this system.RSPF can provide further evidence for secondary min deposition by considering the mechanisms involved for cell adhesion in this system.

33. Once again we can observe how deposition “drops off” at low IS – even when dep. Occurs in column Why? Hydrodynamic influences We therefore postulate/suggest …Once again we can observe how deposition “drops off” at low IS – even when dep. Occurs in column Why? Hydrodynamic influences We therefore postulate/suggest …

35. Secondary Minimum Deposition in Stagnation Point Flow System

36. Comparison Between RSPF and Packed Column DO not mention the quartz having a rear stag point cells “translate” along quartz surface while held in secondary energy min Cells depositing in a secondary min in RSPF cannot be enumerated because they are swept out of viewing area - Only primary deposition would be captured by our visualization system for RSPF Cells in secondary min captured in local stag regions and rear SP – all captured in the enumeration process DO not mention the quartz having a rear stag point cells “translate” along quartz surface while held in secondary energy min Cells depositing in a secondary min in RSPF cannot be enumerated because they are swept out of viewing area - Only primary deposition would be captured by our visualization system for RSPF Cells in secondary min captured in local stag regions and rear SP – all captured in the enumeration process

37. In order to further evaluate the mechanisms of cell adhesion we need to compare directly the dep kinetics of the column and RSPF. This can be done by comparing the attachment efficiencies. This is calculated by normalizing the dep rate by dept rate achieved under chemically favorable, transport-limited conditions Aminosilane imparts a net positive charge on quartz Interactions between positively charged aminosilane and negatively charged bacteria are chemically fav and result in transport-limited conditions Theoretical deposition rates under favorable, transport-limited conditions cannot be calculated accurately due to the non-spherical shape of the bacteria and the irregular geometry of the quartz grains. Therefore, values for the favorable deposition rate in the column (kd,fav) and the RSPF system (kRSPF,fav) were determined experimentally for each cell type. Favorable, non-repulsive conditions were achieved in the column by using quartz grains modified with aminosilane, which imparts a net positive zeta potential. The average kd,fav values for the D21g and D21f2g strains were virtually identical (3.5×10-2 s-1), while the average kd,fav for JM109g was 6.4×10-2 s-1. Similarly, for the RSPF system, favorable (non-repulsive) electrostatic conditions were created by using aminosilane modified cover slips. The resulting average favorable transfer rate coefficients, kRSPF,fav, for D21g, D21f2g, and JM109g were 0.245, 0.109, and 0.034 mm/s, respectively. In order to further evaluate the mechanisms of cell adhesion we need to compare directly the dep kinetics of the column and RSPF. This can be done by comparing the attachment efficiencies. This is calculated by normalizing the dep rate by dept rate achieved under chemically favorable, transport-limited conditions Aminosilane imparts a net positive charge on quartz Interactions between positively charged aminosilane and negatively charged bacteria are chemically fav and result in transport-limited conditions Theoretical deposition rates under favorable, transport-limited conditions cannot be calculated accurately due to the non-spherical shape of the bacteria and the irregular geometry of the quartz grains. Therefore, values for the favorable deposition rate in the column (kd,fav) and the RSPF system (kRSPF,fav) were determined experimentally for each cell type. Favorable, non-repulsive conditions were achieved in the column by using quartz grains modified with aminosilane, which imparts a net positive zeta potential. The average kd,fav values for the D21g and D21f2g strains were virtually identical (3.5×10-2 s-1), while the average kd,fav for JM109g was 6.4×10-2 s-1. Similarly, for the RSPF system, favorable (non-repulsive) electrostatic conditions were created by using aminosilane modified cover slips. The resulting average favorable transfer rate coefficients, kRSPF,fav, for D21g, D21f2g, and JM109g were 0.245, 0.109, and 0.034 mm/s, respectively.

38. The dashed line indicates a slope of unity, on which experimental data points would lie if the adhesion efficiency in the two systems were identical. The majority of the data points lie above the dashed line, indicating differences in the attachment efficiency between the two deposition systems, and different deposition mechanisms. - deviation arises from deposition that is enumerated is occurring in the primary minimum in the RSPF system compared to deposition in both secondary and primary minima in the packed column The hydrodynamics in the two experimental deposition systems are different and so are the bacterial capture mechanisms. Figure shows that the attachment efficiencies for D21g and D21f2g lie above the dashed line, indicating acol > aRSPF. Fewer cells are captured in the RSPF system, because quantifiable deposition of D21g and D21f2g occurs only in primary minima. It must be noted that D21f2g does approach the dashed line to a greater extent than D21g. This suggests greater deposition of D21f2g in primary minima than D21g, which may be attributable to heterogeneities on the cell surface ? most likely due to membrane bound proteins ? that reduce electrostatic repulsion with the quartz because of the extremely short LPS molecule. JM109g lie along the dashed line – suggesting JM109g adhesion involves interactions not accounted for in classical DLVO theory The dashed line indicates a slope of unity, on which experimental data points would lie if the adhesion efficiency in the two systems were identical. The majority of the data points lie above the dashed line, indicating differences in the attachment efficiency between the two deposition systems, and different deposition mechanisms. - deviation arises from deposition that is enumerated is occurring in the primary minimum in the RSPF system compared to deposition in both secondary and primary minima in the packed column The hydrodynamics in the two experimental deposition systems are different and so are the bacterial capture mechanisms. Figure shows that the attachment efficiencies for D21g and D21f2g lie above the dashed line, indicating acol > aRSPF. Fewer cells are captured in the RSPF system, because quantifiable deposition of D21g and D21f2g occurs only in primary minima. It must be noted that D21f2g does approach the dashed line to a greater extent than D21g. This suggests greater deposition of D21f2g in primary minima than D21g, which may be attributable to heterogeneities on the cell surface ? most likely due to membrane bound proteins ? that reduce electrostatic repulsion with the quartz because of the extremely short LPS molecule. JM109g lie along the dashed line – suggesting JM109g adhesion involves interactions not accounted for in classical DLVO theory

41. Conclusions so far . . . . Bacterial deposition kinetics follow trends predicted by DLVO theory . . . Secondary minimum deposition controls adhesion/transport in porous media Is it so simple?

42. Influence of Growth Phase

43. Origin of Increased Adhesion? Changes in: Cell viability? Cell size? Extracellular polymeric substances (EPS) production? Electrokinetic properties? Other physiological changes?

44. Young and Old Cells Have Similar Electrokinetic Properties

45. Hydrophobicity: Microbial Adhesion to Hydrocarbon (MATH) Test

46. Cell Surface Charge Density

47. Comparison of Deposition Systems

48. Other Complications and Challenges?

49. Acknowledgments National Science Foundation – CRAEMS (Collaborative Research Activities in Environmental Molecular Sciences) Grant CHE-0089156 Yale Institute for Biospheric Studies National Water Research Institute

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