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
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
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
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
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
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
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
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
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
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
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.