Transport of Bacterial Endospores in Silica Sand

Transport of Bacterial Endospores in Silica Sand

Sibylle Tesar, Fulbright ScholarDr. Barbara Williams, FacultyDr. Robin Nimmer, Res. Supp. Sci.Angelina Cernick, UndergraduateKristina Beaulieau, NSF REU

Department of Biologicaland Agricultural Engineering

University of Idaho

Outline

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

Research Goals – Spore Transport in Porous Media

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

Outline






Terminology – Mechanisms for Retention

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 elsewhereBradford et al, WRR, 2006

Strained versus Mechanically Filtered

dp/d50 .005

Background

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)

Explanations for Deviation from CFT

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

Theory (cont.)

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]

strSW

attSWT

w EEJt

C

Aqueous Phase Colloid Mass Balance Equation- Bradford et al., 2003

Research Goals – Endospore Transport

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

Bacterial Endospores

Formed as a survival mechanism Cryptobiotic – no sign of life - dormant mode

http://www.textbookofbacteriology.net/

Differences between endospores and vegetative cells in Bacillus species

Property Vegetative Cells Endospores

Surface coats

Gram-positive murein cell wall polymer; S-layer

Thick spore coat and unique core wall; no S-layer

Cytoplasmic water activity

High Very low

Macromolecular synthesis

Present Absent

Heat resistance Low High

Radiation resistance

Low High

Chemical resistance

Low High

Sensitivity to dyes and stains

Sensitive Resistant

http://www.textbookofbacteriology.net

Differences between endospores and vegetative cells in Bacillus species

Property Vegetative Cells Endospores

Surface coats

Gram-positive murein cell wall polymer; S-layer

Thick spore coat and unique core wall; no S-layer

Cytoplasmic water activity

High Very low

Macromolecular synthesis

Present Absent

Heat resistance Low High

Radiation resistance

Low High

Chemical resistance

Low High

Sensitivity to dyes and stains

Sensitive Resistant

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 m, Width: 0.5-0.75 m

– Hydrophobic

– Isoelectric point: pH ~3

Outline






Preliminary Research Questions

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?

Outline






Materials: Sand Properties

Saturated conductivity: Ksat = 1.8x10-4 m/sec

Dry bulk density:

b = 1.65 g/cm3

Porosity:

n = 0.34

dp/d50 .00170

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 20 40 60 80 100

Cumulative %

Par

ticl

e D

iam

eter

(m

m)

Method: Constant Head, Sand Column

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

C/Co Breakthough of B. cereus spores at different solution chemistries

-20%

0%

20%

40%

60%

80%

100%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Dimensionless Time pore volumes

rela

tive

bre

akth

rou

gh

%

Artificial Groundwater

DDI run

start bacteria stop bacteria stop bacteria

Artificial groundwater

DDI

Column Dissection

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)

Depth Distribution Data

"strained" bacteria in AGW run

1,E+05

1,E+06

1,E+07

1,E+08

1,E+09

1,E+10

section1

section2

section3

section4

section5

section6

section7

Bac

teri

a to

tal

in s

ecti

on

“Strained” spores in AGW run

Depth Distribution Data

"attached" bacteria in GW run

1,E+08

1,E+09

1,E+10

section1

section2

section3

section4

section5

section6

section7

Ba

cte

ria

to

tal i

n s

ec

tio

n

“Attached” spores in AGW run

Depth Distribution DataStrained and attached fractions combined

0.00E+00

2.00E+09

4.00E+09

6.00E+09

8.00E+09

1.00E+10

1.20E+10

1.40E+10

1 2 3 4 5 6 7

Strained

Attached

Outline





Preliminary Results

Preliminary Conclusions Future Work – B. cereus, other microbes

Preliminary Conclusions

Breakthrough curve data are consistent with CFT – higher ionic strength, more retention

Depth distribution data show deviation from CFT – not exponential with depth

Outline






Future Work

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

Acknowledgements

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.

Funding Acknowledgements

Fulbright Scholars Program USDA Hatch UI URO Seed Grant Program NSF REU program

References

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.

Documents

Transport of Bacterial Endospores in Silica Sand