PRECIPITATION OF CALCITE BY INDIGENOUS MICROORGANISMS TO STRENGTHEN SOILS

Malcolm Burbank Dissertation Defense Department of Microbiology, Molecular Biology and Biochemistry

Major Professor: Dr. Ronald Crawford

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.

Examples of liquefactioninduced damage:

1964 Niigata, Japan M = 7.5 earthquake - 36 dead, 385 injured 3,534 houses destroyed, 11,000 houses damaged 1971 San Fernando, Calif. M = 6.6 earthquake -over $500 million in property damage and 65 deaths. 1989 Loma Prieta Calif. M = 6.9 earthquake 63 deaths 3,757 injuries $ 5.9 billion dollars in property damage 1999 Chi-Chi, Taiwan M = 7.6 earthquake. 2400 killed, 11,000 injuries. ~20 billion dollars in damage. 2010 Haiti M=7.0
http://fiveblocks.wordpress.com/2008/06/0 8/photos-that-work/

http://pubs.usgs.gov/of/2002/of02-296/of02-296_2liq-sg.pdf

Former Alameda Naval Complex

Oakland Coliseum International Airport

Current Technologies to Mitigate SeismicInduced 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

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

Chemistry
Urea is hydrolyzed by the enzyme urease CO(NH2)2 + 2H2O 2NH4+ + CO32-

Ca2+ + CO32- CaCO3 (calcium carbonate)

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

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.

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

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 and Sheaffe 1973)

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
For most of the characterized ureolytic soil bacteria,

urease is negatively regulated by the presence of ammonia or other nitrogen compounds but derepressed in nitrogen-poor conditions

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

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

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

Effects of [Carbon] on ureC gene copy number

Sample Depth (cm) Initial ureC#/gm soil ureC#/gm treated soil

Untreated Low Carbon 30 60 90 150 4.49 x 106 2.74 x 107 1.53 x 106 4.99 x 106 2.37 x 109 1.01 x 108 3.38 x 109 5 x 109

High Carbon 3.81 x 109 2.27 x 108 5.64 x 109 6.11 x 109

1 enrichment &3 treatments

* All sequenced ureolytic strains have one copy of ureC per genome

Effects of [C] on Calcite %

3.5 3

% Calcite (wt/wt)

2.5 2 1.5 1 0.5 0 30 L 30 H 60 L 60 H 90 L 90 H 150 L 150 H Depth (cm)

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.

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

Effects of [CaCl2] on calcite

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

4.5 3.5 2.1

% calcite

3.6
2.4

3.9

46 cm H 46 cm L 90 cm H 90 cm L 150 cm H 150 cm L Depth & concentration % wt/wt

L=50 mM [CaCl2] H=250 mM [CaCl2] 1 pretreatment, 7 biomineralization treatments In addition to CaCl2, solutions contained urea and sodium acetate

Soil from 150 cm

No urea control Calcite undetectable

50 mM CaCl2 3.9% calcite

250 mM CaCl2 4.5 % calcite

Test of 6 other soil types

Soil Type Mined silica Mined alluvial Tidal #1 Tidal #2 Palouse loess High organic * # Treatments 3 3 3 3 10 10 % Calcite (wt/wt) 2.5 2.3 3.7 4.5 19.1 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

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.

http://pubs.usgs.gov/fs/2003/fs028-03/ http://pubs.usgs.gov/

Comparison of CPT and Calcite Precipitation

Tip Resistance, Mpa
0 0
CPT 1 - Untreated

% Calcite Precipitation by Mass

6 8
0.0%
0 20 40

1.0%

2.0%

3.0%

20 40

CPT 4 - Treated

60

Depth, cm

60 80 100 120

80 100 120 140 160

140 160

180 200

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

Tip Resistance, MPa

0
0

At 46 cm there is a 12.5 fold increase after 5 treatments and 34.1 fold increase after 6.5 treatments (7.14 Mpa = 149,122 psf or 1035 psi)

10

20

Depth, cm

30

40

50

60 0 2 4 6 8 10 12

Calcite Precipitation, %

Untreated 6.5 Treat 6.5 Treat (Calcite Precip)

5 Treat 5 Treat (Calcite Precip)

Excavation

Urease activity
Ureolytic bacteria from enrichments were isolated into pure culture Each bacteria 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

Isolation of ureolytic bacteria

Identification Soil source Soil type

Sporosarcina WB1 Sporosarcina WB5 Sporosarcina WB6 Bacillus WB7 Sporosarcina R-31323 Providencia vermicola Arthrobacter Tibet-ITa1 Lysininbacillus sphaericus Lysininbacillus sphaericus Brevibacterium stationis

Willipa Bay, WA Willipa Bay, WA Willipa Bay, WA Willipa Bay, WA Snake River, ID Spokane, WA Spokane, WA Lane Mountain, WA Snake River, ID Moscow, ID

Tidal Tidal Tidal Tidal Sand High organic High organic Mined quartz silica Mined alluvial Loess

Urease activity
2500 NB Specific activity (m/min/mg protein)

2000

NH3+
Urea

1500

1000

500

0 E. coli WB1 WB5 WB6 WB7 LM4 Isolate S1 L8 G1 G8 I1 S. past.

Summary
Calcite can be precipitated by indigenous bacteria in quantities sufficient to significantly strengthen soil The process works in a range of soil types Precipitation experiments were successful both on a laboratory scale and in in situ field experiments and simulations The use of indigenous ureolytic bacteria results in less clogging and more even distribution of calcite Soil bacteria capable of constitutive regulation of urease are more prevalent and more widely distributed than previously thought The cost are comparable to existing methods and likely less expensive than the use of exogenous bacteria

Publications and Patent

Publications
Precipitation of calcite by indigenous microorganisms to strengthen liquefiable soils. Accepted by Geomicrobiology Journal. Cone penetration tests on soil in which calcite was precipitated by indigenous bacteria in a soil microcosm. Pending submission to the Journal of Geotechnical and Geoenviromental Engineering. Urease regulation of ureolytic bacteria isolated from six soils in which calcite was precipitated by indigenous bacteria. Pending submission to Geomicrobiology Journal. Bio-induced Calcite, Iron, and Manganese Precipitation for Geotechnical Engineering Applications (Second Author). Geofrontiers conference paper accepted.

Patent
In situ precipitation of CaCO3 by indigenous microorganisms to improve mechanical properties of a geomaterial. Patent filed in July 2010

Acknowledgements
Committee Members Dr. Ronald Crawford Dr. Lee Fortunato Dr. Thomas Weaver Dr. Barbara Williams Funding NSF Grant 0700918 Environmental Biotechnology Institute Members

Tonia Green Dr. Martina Ederer Stephanie Smith Dr. Lee Deobald
Department of Civil Engineering Ryan Lewis Aaron Lewis Office of Technology Transfer

Dr. Gene Merrill Dr. Karen Stevenson

Urease specific activity and fold change

E. coli WB1 WB5 WB6 WB7 LM4 S1 Media NB NH4 Urea 7 9 8 507 507 390 300 662 462 NG NG 407 446 200 116 228 NG 123 744 754 236 85 63 206 502 430 2444 783 872 51 1072 680 1016 NG 871 1273 L8 G1 G8 I1 S. pasteurii

Isolate Fold change from NB to NH4 NB to urea NH4 to urea

WB1

WB5

WB6

WB7

LM4

S1

L8

G1

G8

I1

0.0 -0.2 -0.2

1.2 0.5 -0.3 2.5 2.3

0.9 0.9 0.0

-0.5 1.0 2.7

-0.6 -0.7 -0.4

-0.5 8.2 15.9

-0.1 2.0 2.3 1.0

Approximate costs
Unimproved sites Improved sites * *3 treatments

Bio-Induced cementation can be used under existing structures and in situations where other methods may not suitable, such as sites with buried utilities