1. PRECIPITATION OF CALCITE BY INDIGENOUS MICROORGANISMS TO
STRENGTHEN SOILS
Malcolm Burbank, Ph.D.
Postdoctoral Fellow
University of Idaho

2. The problem – Soil Liquefaction
• Liquefaction is a soil phenomenon that occurs during
earthquakes in which saturated soils act like liquids and
can no longer support structures and buildings.
• Before the earthquake, water pressure in the soil is low
and soil particles are in contact with each other. Water
pressure increases due to shaking or rapid loading of
forces and pushes the soil particles apart, allowing them
to move relative to one another.
• Soils transition from a solid to a liquid phase.
• This reduces the stiffness and shear strength of the soil
and can cause structures built on the soil to fail.

3. Current Technologies to Mitigate Seismic-Induced Liquefaction
of Developed Sites
• Permeation: Grout is injected
into the soil at low pressure
and fills the voids
• Compaction Grouting: A
viscous grout is injected into a
compactable soil. The grout
acts as a radial, hydraulic jack
and physically displaces the
soil particles.
• Jet Grouting: Breaks up the
soil structure completely and
performs deep soil mixing to
create a homogeneous soil,
which in turn solidifies.
http://www.geotechnical.com

4. Injection of Sporosarcina pasteurii
• Inject model ureolytic microorganism, S.
pasteurii, into soil followed by injections of a
solution to promote the precipitation of calcite
• Process is being investigated in several
laboratories but there are some obstacles that
must be overcome before this is a reasonably
viable treatment solution

5. Chou, W.C., Seagren, E.A., Aydilek A.H., and Burbank, M., Weaver, T., Lewis, R., Crawford, R., Williams, B.
Maugel, T.K. (2009). American Society for (2012b). Journal of Geotechnical and Geoenvironmental
Microbiology Microbe Library Engineering

6. Challenges of Transport of Bacteria into Soil
– Cell surface properties
– Ionic strength of the carrier solution
– van der Waals forces
– Pore space geometry
– Straining
– Flow rate
– Public perception

7. Once in the soil
• Biotic stresses
– Predation
– Competition
• Abiotic stresses
– pH
– Osmotic pressure
– Temperature
http://www.bam.gov/sub_diseases/images/ip_microbes.jpg
The reality is that bacteria introduced into soil tend to rapidly
decline in number and rarely grow after being introduced.

8. Our approach - Stimulate Indigenous Ureolytic Bacteria to Induce
Calcite Precipitation
• Advantages
– Ureolytic bacteria are already there
– Bacteria are evenly distributed = more evenly
distributed calcite
– No need to grow large volumes of bacteria in the
lab
– No need to autoclave equipment or media for
field applications
– Lower energy demand
– Lower overall costs

9. Indigenous Ureolytic Microorganisms
• Common in many types of soil. In one study,
ureolytic bacteria comprised between 17-30% of the
cultivable aerophilic, micro-aerophilic, and
anaerobic microorganisms*
• Urea plays a crucial role in microbial nitrogen
metabolism for many microorganisms
• Production of urease is stringently regulated in
many microorganisms by the availability of nitrogen
but constitutively expressed in others
* Lloyd AB, Shaeffe MJ (1973). Plant Soils

10. Urease Regulation
• Constitutive expression: Urease is made
regardless of nitrogen concentration
•S. pasteurii
•S. ureae
• Inducible: Urease is expressed only in the
presence of urea
•Proteus mirabilis
• Formost of the characterized ureolytic soil
bacteria, urease is negatively regulated by the
presence of ammonia or other nitrogen compounds
but de-repressed in nitrogen-poor conditions

11. Enrichments
• Favor ureolytic bacteria which constitutively or
inducibly produce urease
• Can thrive in high pH
• Can thrive in high [CaCl2]
• Can thrive on an inexpensive carbon source with
very little added micronutrients

12. Indigenous Experiments (overview)
1. Determine the effects of carbon concentration on the
number of ureolytic microorganisms and on the
amount of calcite precipitated in column experiments
2. Determine the effects of two concentrations of CaCl2 on
pH and on the amount of calcite precipitated in a
column experiments
3. Test multiple soil types in column experiments
4. Do a large scale test and quantify the change in shear
strength of treated soil using cone penetration testing
5. Identify some of the microorganisms involved and
characterize how each regulate urease

13. Effects of Carbon Concentrations on ureC* gene copy number
and Calcite %
• Soil samples were enriched in
columns with a solution
containing either 1% molasses
and 170 mM sodium acetate [H]
or 0.1% molasses and 50 mM
sodium acetate [L], urea and
calcium chloride
• Samples were then treated with
a biomineralization solution
containing either 170 mM
sodium acetate [H] or 50 mM
sodium acetate [L], urea and
calcium chloride
* ureC codes for a functional unit of the urease enzyme and is highly
conserved among most known ureolytic bacteria

14. Enrichment of ureolytic bacteria - ureC gene copy number
Sample Initial ureC#/gm treated soil
Depth ureC#/gm
(cm) soil
1 enrichment
Untreated Low High
& 3 treatments
Carbon Carbon
30 4.49 x 106 2.37 x 109 3.81 x 109
60 2.74 x 107 1.01 x 108 2.27 x 108
90 1.53 x 106 3.38 x 109 5.64 x 109
150 4.99 x 106 5 x 109 6.11 x 109
L= soil treated with 50 mM Na-acetate
Burbank, M., Weaver, T., Green, T. Williams, B., Crawford, R. H= Soil treated with 250 mM Na-acetate
(2011). Geomicrobiology Journal, 28(4):301-312

15. Effects of [C] on Calcite %
s f ] n e
3.5
5
3
% Calcite (wt/wt)
)
2.5
5
2
e
1.5
5
1
0.5
5
0
30 L
0 30 H
0 60 L
0 60 H
0 90 L
0 90 H
0 150 L
0 150 H
0
Depth (cm)
h )
H = 1 pretreatment (enrichment) with 1% molasses and 170 mM sodium acetate, urea and
calcium chloride. 3 treatments with 170 mM sodium acetate
L = 1 pretreatment (enrichment) with 0.1% molasses and 50 mM sodium acetate, urea and
calcium chloride. 3 treatments with 50 mM sodium acetate
* Pretreatments (enrichments) were followed by three treatments with a biominerlization
solution.

16. Effects of [Ca2+] on pH
Treatment # Time (h) Average pH
50 mM CaCl2 250 mM CaCl2
Enrichment 36 9.5 7.4
2 48 8.6 7.6
3 48 8.9 8.5
4 24 9.2 8.1
6 24 9.2 7.9
7 24 9.3 7.7

17. % wt/wt

18. Soil from 150 cm
No urea 50 mM 250 mM CaCl2
control CaCl2
4.5 % calcite
Calcite 3.9% calcite
undetectable

19. Test of 6 other soil types
Soil Type # Treatments % Calcite (wt/wt)
Mined silica 3 2.5
Mined alluvial 3 2.3
Tidal #1 3 3.7
Tidal #2 3 4.5
Palouse loess 10 19.1
High organic * 10 11
•ureC copy number was below the threshold of detection by qPCR before enrichments.
•1.6 x 109 copies of ureC/gm soil were detected following the 2nd treatment

20. Field Test Overview
• Chemicals were mixed off-site in 55 gallon
barrels
• 250 liters of water from the Snake River was
pumped to the barrels then delivered by
gravity into a ring infiltrometer
• Originally, we modeled the experiment to
treat saturated soil.
• One enrichment treatment was followed by 10
biomineralization treatments. The experiment
lasted ~5 weeks.

22. Comparison of CPT and Calcite Precipitation

23. Soil Microcosm Experiment
• A 240-liter microcosm
measuring 76 cm x 102 cm x 31
cm (h x l x w) was constructed
from aluminum (30” x 40” x
12”)
• Box was filled with 56 cm of
compacted sand
• For each treatment, 99 liters of
solution was gravity fed
through the soil from the
bottom of the microcosm.
Approx 17 cm of solution was
allowed to pool on the top of
the soil
• 6.5 volumes of treatment
solution was delivered before
the soil clogged

24. At 46 cm there was a 12.5
fold increase in tip
resistance after 5
treatments and 34.1 fold
increase after 6.5
treatments (7.14 Mpa =
149,122 psf or 1035 psi)
Burbank, M., Weaver, T., Lewis, R., Crawford, R.,
Williams, B. (2012b). Journal of Geotechnical and
Geoenvironmental Engineering

25. Urease activity
• Ureolytic bacteria from enrichments were
isolated into pure culture
• Each bacterium was cultured in nutrient
broth(NB) alone*, and in NB supplemented with
100 mM (NH4)2SO4 or with 100 mM urea.
• 16s analysis for identification
• Cells were lysed by sonication and a crude
extract was isolated by centrifugation.
* Three isolates did not grow in NB alone

26. Isolation of ureolytic bacteria from soils used in our studies
Identification Soil source Soil type
Sporosarcina WB1 Willipa Bay, WA Tidal
Sporosarcina WB5 Willipa Bay, WA Tidal
Sporosarcina WB6 Willipa Bay, WA Tidal
S. pasteurii WB7 Willipa Bay, WA Tidal
Sporosarcina R-31323 Snake River, ID Sand
P. vermicola Spokane, WA Decomposed leaf litter
A. Tibet-ITa1 Spokane, WA Decomposed leaf litter
L. sphaericus Lane Mountain, WA Mined quartz silica
L. sphaericus Snake River, ID Mined alluvial
B. stationis Moscow, ID Loess
Burbank, M., Weaver, T., Williams, B., Crawford, R.
(2012a). Geomicrobiology Journal

27. Urease activity
Burbank, M., Weaver, T., Williams, B., Crawford, R. (2012a).
Geomicrobiology Journal

28. Current Research
• Small scale field experiment for Avista
– Shallow foundation experiment
– 3”x 6” sample column tests with loess soil
for triaxial shear testing
• Analysis of ions from MICP treated soil
to track the fate of MICP byproducts

29. Transmission line after wind storm

31. Measurement of MICP Byproducts
• Soil was collected from 30 cm, 60 cm
and 90 cm deep in each hole and
outside of each hole before each
treatment
• Ions were extracted into nanopure H2O
and analyzed for cations and anions on
a Dionex HPLC Ion Exchange system

32. Initial [Ion] and following 5 treatments
Na+ Cl- NH4+ NO3- Ca+
Untreated <1 ppm 48 ppm <5 ppm 15 ppm <5 ppm
soil
Final inside 1430 ppm 1970 ppm 5100 ppm 84 ppm 170 ppm
unlined
Final 255 ppm 40 ppm 189 ppm 50 ppm < 5ppm
outside
Total input 1250 ppm 9627 ppm 12,800 ppm 5404 ppm
Standard deviation ~10%

33. 2-D Microcosm

34. Other Applications
• Coprecipiation of divalent cations and
radionuclides from contaminated water
and soil
• Reduction of hydraulic conductivity to
alter the flow of groundwater
• Formation of grout curtains to shield
groundwater from contaminated
plumes

35. Funding
Sponsored research grant # KHK004
NSF Grant #0700918

36. The University of Idaho
Thank you!