PRECIPITATION OF CALCITE BY INDIGENOUS MICROORGANISMS TO STRENGTHEN SOILS

Malcolm Burbank, Ph.D.Postdoctoral FellowUniversity of Idaho

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.

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

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

Burbank, M., Weaver, T., Lewis, R., Crawford, R., Williams, B. (2012b). Journal of Geotechnical and Geoenvironmental Engineering

Chou, W.C., Seagren, E.A., Aydilek A.H., and Maugel, T.K. (2009). American Society for Microbiology Microbe Library

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

Once in the soil

• Biotic stresses– Predation– Competition

• Abiotic stresses

– pH– Osmotic pressure– Temperature

The reality is that bacteria introduced into soil tend to rapidly decline in number and rarely grow after being introduced.

http://www.bam.gov/sub_diseases/images/ip_microbes.jpg

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 AB, Shaeffe MJ (1973). Plant Soils

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 de-repressed 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 experiments4. 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

Enrichment of ureolytic bacteria - ureC gene copy number

Sample Depth (cm)

Initial ureC#/gm

soil

ureC#/gm treated soil

Untreated Low Carbon

High 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

1 enrichment & 3 treatments

Burbank, M., Weaver, T., Green, T. Williams, B., Crawford, R. (2011). Geomicrobiology Journal, 28(4):301-312

L= soil treated with 50 mM Na-acetateH= Soil treated with 250 mM Na-acetate

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 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

% wt/wt

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 # 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

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.

Comparison of CPT and Calcite Precipitation

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

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

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

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

Urease activity

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

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

Transmission line after wind storm

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

Initial [Ion] and following 5 treatments

Na+ Cl- NH4+ NO3

- Ca+

Untreated soil

<1 ppm 48 ppm <5 ppm 15 ppm <5 ppm

Final inside unlined

1430 ppm 1970 ppm 5100 ppm 84 ppm 170 ppm

Final outside

255 ppm 40 ppm 189 ppm 50 ppm < 5ppm

Total input 1250 ppm 9627 ppm 12,800 ppm 5404 ppm

Standard deviation ~10%

2-D Microcosm

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

Funding

NSF Grant #0700918

Sponsored research grant # KHK004

The University of Idaho

Thank you!