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Precipitation of Calcite by Indigenous Microorganisms

to Strengthen Liquefiable Soils
a b a c
Malcolm B. Burbank , Thomas J. Weaver , Tonia L. Green , Barbara C. Williams &
a d
Ronald L. Crawford
a
Environmental Biotechnology Institute, University of Idaho, Moscow, Idaho, USA
b
U.S. Nuclear Regulatory Commission, Washington, District of Columbia, USA
c
Department of Biological and Agricultural Engineering, University of Idaho, Moscow, Idaho,
USA
d
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho,
Moscow, Idaho, USA
Published online: 27 May 2011.

To cite this article: Malcolm B. Burbank , Thomas J. Weaver , Tonia L. Green , Barbara C. Williams & Ronald L. Crawford
(2011) Precipitation of Calcite by Indigenous Microorganisms to Strengthen Liquefiable Soils, Geomicrobiology Journal, 28:4,
301-312, DOI: 10.1080/01490451.2010.499929

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 Geomicrobiology Journal, 28:301–312, 2011
Copyright © Taylor & Francis Group, LLC
ISSN: 0149-0451 print / 1521-0529 online
DOI: 10.1080/01490451.2010.499929

Precipitation of Calcite by Indigenous Microorganisms

to Strengthen Liquefiable Soils

Malcolm B. Burbank,1 Thomas J. Weaver,2 Tonia L. Green,1

Barbara C. Williams,3 and Ronald L. Crawford1,4
1
Environmental Biotechnology Institute, University of Idaho, Moscow, Idaho, USA
2
U.S. Nuclear Regulatory Commission, Washington, District of Columbia, USA
3
Department of Biological and Agricultural Engineering, University of Idaho, Moscow,
Idaho, USA
4
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow,
Idaho, USA
Downloaded by [University of Connecticut] at 15:08 20 August 2013

Enrichments for indigenous microorganisms capable of hy-
drolyzing urea in the presence of CaCl2 were performed on po-
tentially liquefiable saturated soils in both the laboratory and in
situ. Following enrichment, treatment of soils with nutrients, CaCl2
and urea resulted in significant in situ precipitation of calcite, even
at depth, by indigenous microorganisms. The biomineralized soils
showed properties that indicate calcite precipitation increased their
resistance to seismic-induced liquefaction.
Keywords bio-soil, calcite, biomineralization, liquefaction,
bio-stimulation
INTRODUCTION
Biologically induced precipitation of CaCO3 in soils is of par-
ticular interest to engineers and microbiologists as a method to
alter soil characteristics. Biologically induced soil modification
can be used to decrease permeability, decrease compressibility,
increase strength and alter volumetric behavior during shearing
(DeJong et al. 2009). DeJong et al. (2006) suggest bio-induced
soil modification may be effective in mitigating seismic-induced
liquefaction. Seismic-induced liquefaction generally occurs in
Received 26 February 2010; accepted 7 June 2010.
We would like to thank Dr. Thomas Williams, University of Idaho,
College of Science-Materials Characterization Laboratory for his XRD
analysis of our samples. We would like to thank Joseph Marshal and
Greg Watson of the U.S. Corps of Engineers for granting us a special
events permit to carry out the field experiment. We would also like
to thank Dr. Peter Robertson and Ron Boggess of Gregg Drilling and
Testing Inc. for providing us with the miniature cone penetrometer.
This work was funded by a grant from the National Science Foundation
(070091).
Address correspondence to Thomas J. Weaver, U.S. Nuclear Reg-
ulatory Commission, Washington, DC 20555-0001, USA. E-mail:
thomas.weaver@nrc.gov
relatively loose granular soil deposits that are below the ground-
water table and thus saturated. As seismic waves cyclically shear
the soil, the soil porewater pressure increases and substantially
decreases the soil shear strength. Liquefaction resulting from
earthquakes has resulted in significant failures to man-made
structures.
Existing technology for stabilizing soils to mitigate lique-
faction include cementation (e.g., permeation grouting), den-
sification (e.g., vibro-replacement, deep dynamic compaction,
or compaction grouting), drainage, and thermal stabilization.
Although many of these approaches have proven successful
(Mitchell et al. 1995), they are largely limited to undeveloped
sites.
Injecting urease positive microorganisms such as
Sporosarcina pasteurii into soil along with CaCl2 and
urea is another approach that is currently being investigated
by us and others (Stocks-Fisher et al. 1999; Whiffen 2004;
Fritzges, 2005; DeJong et al. 2006; Rebata-Landa 2007;
Whiffen et al. 2007). Common obstacles to this approach are
clogging of the soil pore spaces near the injection point and
uneven distribution of bacteria and calcite, both of which tend
to be more concentrated near the injection point (Stocks-Fisher
et al. 1999; Whiffen et al. 2007).
These obstacles are most likely a result of difficulties with
uniform transport of bacteria and attachment of bacteria to soil
surfaces. Many factors affect the transport of bacteria through
and attachment to soil grains including the properties of the
cell surface, ionic strength of the carrier solution, flow rate, van
der Waals forces, pore space geometry within the soil matrix
(Torkzaban et al. 2008; Travors et al. 1990; Gannon et al. 1991;
Sanin, 2004) and straining (Ryan and Elimelech, 1996; Diáz
et al. 2009).
Once in the soil, introduced bacteria face challenges such
as reduction in population from predation and competition as
well as stress from abiotic factors such as pH, osmotic pressure,

301
 302 M. B. BURBANK ET AL.
temperature and availability of suitable nutrients (Van Elsas et al.
1991; Evans et al. 1993). Bacteria introduced into natural soils
tend to rapidly decline in numbers (Van Elsas et al. 1986; van
Veen et al. 1997) and rarely grow after being introduced (van
Veen et al. 1997).
These challenges may be overcome through the use or ma-
nipulation of indigenous bacteria. Microorganisms that are able
to hydrolyze urea to carbon dioxide and ammonia are widely
distributed in soils and, in one study, comprised between 17–
30% of the cultivable aerophilic, micro-aerophilic, and anaer-
obic microorganisms (Lloyd and Sheaffe 1973). In fact, urea
plays a crucial role in microbial nitrogen metabolism for many
microorganisms (Bremner and Mulvaney 1978). Production of
urease is stringently regulated in many microorganisms by the
availability of nitrogen but constitutively expressed in others.
For example, in Sporosarcina pasteurii and Sporosarcina
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ureae urease is expressed regardless of ammonia or nitrogen
compound concentrations; however, in Proteus mirabilis, ure-
ase is only made in the presence of urea (Morsdorf and Kalt-
wasser 1989; Mobley et al. 1995). Many other microbial ure-
ases are repressed by the presence of ammonia or other nitrogen
compounds, including urea, but de-repressed in nitrogen-poor
conditions (Mobley and Hausinger 1989; McCarty et al. 1991;
Mobley et al. 1995).
Microbially induced CaCO3 precipitation is a straightfor-
ward process in which one mole of urea, (NH2 )2 CO, is hy-
drolyzed to two moles of NH+ 2−
4 and one mole of CO3 per mole
of urea by the enzyme urease (urea amidohydrolase; EC 3.5.1.5)
as seen in the following simplified reaction:
CO(NH2 )2 + 2H2 O → 2NH+
4 + CO3
2−
The generation of NH+ 4 increases local pH and, in the presence
of calcium ions and the availability of nucleation sites (Hammes
and Verstraete 2002), the reaction continues spontaneously to
form calcium carbonate.
Ca2+ + CO2−
3 → CaCO3
Supersaturated solutions of calcium carbonate first precipi-
tate as unstable amorphous calcium carbonate (Swada 1997),
which is spontaneously converted to vaterite (Rivadeneyra et al.
1991; Hammes et al. 2003; Rodriguez-Navarro et al. 2003, 2007;
Zammarreno et al. 2009), a metastable polymorph of calcium
carbonate. Conversion from the thermodynamically unstable va-
terite (solubility product: log Ksp = −7.91 at 25◦ C) to the most
stable and least soluble polymorph of calcite (solubility prod-
uct: log Ksp = −8.48) occurs in the presence of water (Kralj
et al. 1997; Spanos and Koutsoukos 1998), and the conversion
happens over a short period of time, with almost all vaterite
converted to calcite in 24 hours (Nissenbaum et al. 2008).
In soil, microbial-induced calcite bridges adjacent soil parti-
cles, cementing soil grains together (DeJong et al. 2006) to form
a cemented sand or sandstone (Saxena and Lastrico 1978). It is
this change within the soil matrix that strengthens and stiffens
the soil. Formation of free fine (non-cementing) particles may
also alter the soil volume change characteristics and increase
the liquefaction resistance of soils.
In work reported here, we have demonstrated through lab-
oratory experiments and insitu field tests that it is possible to
manipulate indigenous soil microbial communities in natural
soils such that urease-positive bacteria are greatly enriched and
can hydrolyze urea in the presence of divalent calcium ions and
thereby cause the precipitation of calcite within the pores of
liquefiable soils. Changes in soil properties were observed that
indicate that the potential for seismic induced liquefaction was
reduced as a result of biomineralization.
MATERIALS AND METHODS
Soil and Water Samples
Potentially liquefiable soil samples were collected from the
shore of the Snake River, approximately 8000 m downstream of
the Lower Granite Dam, Washington, USA using a stainless steel
hand auger. Samples were collected from five depths (30–150
cm). Collected samples were placed directly into sterile 30 ml
Becton Dickenson syringes, with approximately 5% by volume
of co-sampled porewater retained in the headspace to preclude
air contact (and change in redox status), and placed on ice for
transport to the laboratory. All samples were collected from the
saturated zone and initial pH and temperature measurements
were taken using an Oakton pH 6 Acorn series meter. Samples
were stored at 4◦ C prior to use and were treated within 14 h
of collection. Water from the Snake River was collected in a
sterile bottle from an area adjacent to the place where the soil
samples were collected and stored on ice for transport and at
4◦ C between uses.
In an attempt to maintain the natural conditions of the soil
(e.g., reduce the geochemical effects due to sampling), all sam-
ples were collected from the saturated zone of our study site and
were kept saturated before and during treatments.
Experiments were conducted on soil samples collected from
depths of approximately 30 cm, 60 cm, 90 cm, and 150 cm
with soil grain size characteristics d10 = 91 µm (i.e., 10% of
the sand grains are smaller than this diameter) d30 = 170 µm,
d50 = 199 µm, d60 = 210 µm, and 2.55% fines. The pH of the
soil averaged 6.7.
Soil Characteristics
Soil grain size was determined by passing the dried soil
through sieves. The percentage by weight of soil retained in each
sieve was used to calculate the average grain size distribution of
the soil. Sieves with the following opening size were used: 2.00
mm, 0.841 mm, 0.420 mm, 0.297 mm, 0.210 mm, 0.149 mm,
and 75 µm.
Enrichments
In all cases where urea was part of a solution, 0.2 µm filter-
sterilized urea, pH 7.0 was added after the remaining ingredients
were cooled to prevent heat-induced hydrolysis of the urea. All
 PRECIPITATION OF CALCITE BY INDIGENOUS MICROORGANISMS 303

other solutions were sterilized by autoclaving at 121◦ C, 15 psi
for 25 minutes. Details on the enrichment solutions for investiga-
tion of effects of carbon concentration and CaCl2 concentration
are provided next.
Laboratory-scale experiments were carried out in new, ster-
ile 30 ml syringes (Becton Dickenson) used as columns. All
columns were sealed on the bottom to prevent leakage but re-
mained exposed to air on the top. ScotchBrite scour pads were
cut to completely cover the inside bottom of the syringe to act
as a filter to prevent soil loss from the syringe.
To determine the effects of concentrations of molasses and
sodium acetate as carbon sources to enrich urease-positive mi-
croorganisms, soils packed in columns (retaining a 5% by vol-
ume headspace) were pre-treated for seven days with either a
relatively high or low carbon source concentration. The high
concentration carbon source enrichment contained 1.0% mo-
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Following the enrichment period, soil columns were flushed
with approximately five pore volumes of 50 mM NaCl then filled
with approximately 1.05 pore volumes of pH 7 biomineraliza-
tion solution containing 333 mM urea, 170 mM CH3 COONa ·
3H2 O, 12.5 mM NH4 Cl, and 0.1g yeast extract per litercontain-
ing either 50 mM or 250 mM CaCl2 2H2 0. The columns were
incubated at approximately 21◦ C covered with aluminum foil
for the time indicated in the results section of this paper.
DNA Extraction and Purification
Soil DNA was isolated and purified with a Mo Bio
UltraCleanTM Soil DNA Isolation Kit according to the man-
ufacturer’s directions.
Quantitative Real-Time PCR (qPCR)

lasses (Grandma’s Molasses, unsulfured, Motts, USA) and 170

A series of quantitative PCR assays were performed us-
mM CH3 COONa · 3H2 O; whereas, the low concentration car-
ing metagenomic DNA extracted from soil samples to enu-
bon source enrichment contained 0.1% molasses and 50 mM
merate the urease gene copy number. The DNA extracted
CH3 COONa 3H2 O. Both the high and low carbon source con-
from samples was amplified with the primers specific to the
centration enrichments contained 250 mM CaCl2 · 2H2 O,250
highly conserved regions of the UreC protein. The follow-
mM urea, 12.5 mM NH4 Cl, and 0.1g Bacto yeast extract per
ing primers were used for the amplification ofa 317bp por-
liter of distilled water, pH 7.0. Both enrichments were followed
tion of ureC, 5 -AAGSTSCACGAGGACTGGGG-3 and 5 -
by three treatments with a biomineralization solution consisting
AGGTGGTGGCASACCATSAGCAT-3’ (Collier et al. 1999).
of 50 mM CH3 COONa · 3H2 O, 250 mM CaCl2 · 2H2 0, 333 mM
For all PCR reactions the StepOne Plus Real-Time PCR
urea, 12.5 mM NH4 Cl, and 0.1 g yeast extract per liter of dis-
system (Applied Biosystems, Foster City, CA) was used. Data
tilled water, pH 7.0.The biomineralization solution treatments
analysis was performed using StepOne Plus software. The cycle
were spaced 72 h apart.
at which the fluorescence of a certain target molecule number
The solutions were passed through the columns by gravity
exceeded the background fluorescence (threshold cycle - CT )
flow using 10 ml (approximately1.05 pore volumes) of solution
was determined from the dilution series of target DNA of pure
added at one time to the columns. The solution was allowed
culture containing defined amounts of target DNA sequences.
to flow out of the column until approximately a 5% solution
The CT values are inversely proportional to the logarithm of the
headspace remained to maintain the saturated condition, after
target molecule numbers. Each measurement was performed in
which the outlet was re-sealed. The columns were covered with
triplicate. PCR amplifications were performed in total volumes
aluminum foil between treatments to reduce evaporation and
of 25 µL consisting of 12.5 µL of Power SYBR green master
to limit the exposure of light to the soil, two conditions of the
mix (Applied Biosystems), 300 nM of each primer, and 1 µL of
natural setting. Negative controls were maintained in parallel.
DNA standard or sample.
Control columns were treated with sterile phosphate-buffered
Following the initial denaturation step at 95◦ C for 10 min,
saline containing (per liter of distilled water): 8 g NaCl, 0.2 g
all PCR amplifications were performed using 40 cycles of 95◦ C
KCL, 1.44 g Na2 HPO, pH 7.4 or with biomineralization solu-
for 1 min, 58◦ C for 1 min, and 72◦ C for 1 min. Fluorescence
tions from which urea or calcium chloride had been omitted.
readings were taken during the extension step at the 72◦ C in-
To determine the effects of CaCl2 concentrations on calcite
cubation. Melt curve analysis was performed following every
formation and soil pH, soils within columns were flushed with
run to confirm product specificity. The qPCR efficiency for the
10 ml (approximately 1.05 pore volumes) of enrichment me-
ureC copy number analysis was 80%.
dia solution containing 0.5% molasses, 333 mM urea, 170 mM
B. subtilis subsp. subtilis DSM 10 (NCIB 3610) chromoso-
CH3 COONa · 3H2 O, 12.5 mM NH4 Cl, and 0.1 g yeast extract
mal DNA was used to make the standard curve for quantification
per liter of distilled water containing either 50 mM or 250 mM
of all qPCR samples. This bacterium has one copy of ureC per
CaCl2 · 2H2 0. The 10 ml of solution was added at one time and
cell (Srivatsan et al. 2008).
allowed to gravity feed through the soil column until approx-
imately 5% pooled solution remained. Control columns were
treated with approximately 1.05 pore volumes of either the 250 Quantification of Calcium Carbonate
mM or 50 mM CaCl2 enrichment medium but with the urea Calcium carbonate reacts with acids, such as HCL or acetic
omitted. An additional set of control columns from each depth acid, and dissociates as shown in the following reaction:
were treated with approximately 1.05 pore volumes of water
collected from the Snake River. CaCO3 + 2H+1 → Ca+2 + H2 O + CO2
 304 M. B. BURBANK ET AL.
At the conclusion of each experiment, the soil was flushed
with approximately five pore volumes of 50 mM NaCl to remove
organics and poorly bound calcium carbonate. Then, approxi-
mately 5 mm of soil from the top of each column was removed
and discarded to remove carbonates that may have precipitated
in the 5% pooled solution and accumulated on the soil surface.
The remaining soil was collected from the column and dried
in an oven for 24 h at 121◦ C. The dry soil was weighed, washed
with approximately five pore volumes of 1 M HCL to dissolve
the calcium carbonate, rinsed with nanopure water, dried again
for 24 h and re-weighed to determine calcium carbonate content
as the loss of weight as compared to before the acid treatment.
Control columns were used to determine the average mass loss
in soils from each depth due to the flushing process. The average
loss of mass for soil in a control column from each depth was
subtracted from the final mass of the treated columns of the
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same depth. The adjusted difference in weight after the acid
wash was considered to be the weight of the calcium carbonate
in the sample.
X-Ray Diffraction (XRD)
X-ray diffraction scans were performed on a Siemens D5000
theta-theta goniometer XRD equipped with a Cu X-ray tube
and a solid-state (SiLI) wafer detector. Scans were performed at
40 kV and 30 mA tube power. Scan parameters: 2-theta range
from 2 to 80 degrees at 0.02 step-size and 2 s step-time. The
standard used to identify calcite in the samples was PDF 00-
005-0586, a synthetic form of pure calcite. A focused scan over
the 104 (hkl) calcite peak was performed using a step-time of
20 sec.
In situ Test
A field test was conducted in-situ approximately 8000 meters
downstream of the Lower Granite Dam, Washington, USA and
approximately 100 m west of the existing shoreline of the Snake
River. This site was located near the soil source for the laboratory
tests.
Site Preparation
Prior to enrichment and biomineralization treatments, soil
near the ground surface was removed to a depth of approxi-
mately 0.3 m where the soil was moist. A 91.44 cm diameter, 61
cm tall ring infiltrometer was pushed approximately 20 cm into
the ground. The ring infiltrometer provided a means to contain
the ponded treatment solutions over a circular area. Between
treatments, the infiltrometer was covered with a plastic sheet to
reduce the evaporation rate and prevent rain from entering the
ring.
Enrichment for Urease Positive Microorganisms
Soil at the field test site was first treated with an enrichment
medium designed to select for a bacterial population capable of
using urea as its primary nitrogen source and thrive in high soil
pH and high CaCl2 concentrations. Approximately 250 liters
of an enrichment solution consisting of 250 mM CaCl2 (bulk
anhydrous technical grade), 170 mM sodium acetate (bulk, tech-
nical grade), 0.5% unsulfured molasses (Grandma’s Molasses,
Motts), 12.2 mM NH4 Cl (bulk, technical grade), 333 mM urea
(McGregor Fertilizer, Moscow, ID), 0.1% yeast extract (Fisher
Biotech) was delivered from plastic, food grade barrels into the
ring infiltrometer.
Water was pumped from the Snake River using a Kawasaki
FA130D pump. The water was pumped through standard garden
hoses to barrels containing the ingredients for the enrichment
and biomineralization solutions for each 250-liter treatment.
The inlet hose to the pump was covered with a fine mesh nylon
screen to limit the amount of debris that was pumped to the test
site.
Treatment with Biomineralization Solution
Approximately 96 h after treating the soil with the enrich-
ment solution, 250 liters of a biomineralization solution contain-
ing 250 mM CaCl2 (bulk anhydrous technical grade), 170 mM
sodium acetate (bulk, technical grade), 12.2 mM NH4 Cl (bulk,
technical grade), 333 mM urea (McGregor Fertilizer, Moscow,
ID), 0.1% yeast extract (Fisher Biotech) was delivered into the
ring infiltrometer in the same manner as the enrichment solu-
tion. An additional nine treatments with biomineralization was
delivered into the ring at approximately 2–3 day intervals. After
the final treatment, the site was left undisturbed for ∼72 h before
cone penetration tests were performed and soil was excavated
from the site for quantifying the amount of precipitated calcite.
Cone Penetration Testing
Cone penetration testing was performed at the field site and
consisted of pushing a conical tipped rod into the ground at ap-
proximately 2.5 cm/s. The conical tipped rod was instrumented
with strain gages to measure the force applied to the conical tip
section and along a sleeve located directly behind the cone tip.
The miniature cone penetrometer used for testing was provided
by Gregg Drilling & Testing (Signal Hill, CA).
The miniature cone had a diameter of 1.6 cm. A National
Instruments SCXI data acquisition system was used to acquire
data at approximately 0.5 cm depth increments. Cone penetra-
tion tests extended from the soil surface inside the ring infiltrom-
eter to a depth of approximately 1.35 m. Water from the Snake
River was pumped directly into the ring infiltrometer prior to
cone penetration testing in an effort to saturate the soil within
and below the ring prior to cone penetration testing.
Soil Collection
Following cone penetration testing, soil inside and below the
ring infiltrometer was excavated to obtain soil samples for ad-
ditional testing in the laboratory. Soil samples were collected
at approximately 30 cm intervals directly into sterile 50 ml
 PRECIPITATION OF CALCITE BY INDIGENOUS MICROORGANISMS
Eppendorf tubes and stored on ice for transport to the labora-
tory. The percentage of CaCO3 in the collected samples was
determined as described above.
Water Collection from Snake River and Metagenomic
DNA Isolation
Water was collected into sterile bottles from the Snake River
in the area from which water was pumped for the in situ experi-
ment. The water was stored on ice for transport to the laboratory
then stored at 4◦ C until metagenomic DNA could be isolated.
Water (50 ml) was pre-filtered through Whatman #1 (11 µm re-
tention) filter paper and then processed for DNA isolation using
a Mo-Bio Rapid Water Kit using the manufacturer’s protocol.
Water Collection from a Pristine Artesian Well and
Metagenomic DNA Isolation
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Water from an artesian well in Latah County, ID was col-
lected into a sterile glass container directly from a tap that was
connected to a submerged well pump by plastic pipe. This nat-
ural artesian well has supplied water to a homestead for more
than 100 years and is not treated with disinfection agents. A
description of the well and a microbiological characterization
of the well water’s microbial community was described by Ball
and Crawford (2006).
Metagenomic DNA was isolated using a Mo-Bio Raid Water
Kit without pre-filtering and using the manufacturer’s protocol.
RESULTS AND DISCUSSION
The poorly graded sand described here was enriched for in-
digenous microorganisms that hydrolyze urea in laboratory ex-
periments and an in situ field test. The overall objectives of the
research were to demonstrate that indigenous microorganisms
could be cultivated sufficiently to produce urease for the hydrol-
ysis of urea and that calcite could be precipitated in sufficient
quantity to modify the geophysical properties of the treated soil.
A significant benefit for altering the geophysical soil properties
is the potential for decreasing or eliminating seismic induced
liquefaction.
Laboratory tests were performed to assess effects of carbon
concentration on the quantity of ureolytic microorganisms that
are cultivated and the associated impact on calcite precipitation.
Another set of experiments were conducted to assess the effects
of CaCl2 concentration on urease production and associated
impacts on calcite precipitation.
Based on successful results from laboratory tests, an in situ
field test was performed to demonstrate that the processes de-
veloped in the laboratory could be applied in the field. Results
from the laboratory and in situ field test are provided next, along
with our interpretation of the results.
Selection and Concentration of Carbon Sources
In this study, molasses and sodium acetate (CH3 COONa)
in the presence of high concentrations of urea were tested as
305
carbon sources for the initial enrichment of indigenous ureolytic
microorganisms and to determine the effects of enrichment on
soil microbial ureolytic communities and, ultimately, on the
percentage of calcite precipitated within the soil matrix after
treating the soil with a biomineralization-promoting solution.
Molasses is an inexpensive carbon source composed primar-
ily of sucrose, glucose and fructose and has been used for the
enrichment of soil and marine microorganisms for the purpose
of bioremediation (Boopathy et al. 1998; Fujita et al. 2007).
Acetate is a simple organic substrate that can be used by most
aerobic bacteria as a carbon and energy source.
In earlier experiments (data not shown), enrichments for soil
urease-positive microorganisms using molasses as a sole car-
bon source and urea as the primary nitrogen source yielded
more biomass than enrichments using sodium acetate as the
sole carbon source; however, enrichments using sodium acetate
as the sole carbon source with urea as the sole nitrogen source
yielded a substantially higher pH in the columns.
These results are consistent with the findings of Whiffin
(2004), who compared inexpensive carbon sources and looked
at the effects those carbon sources had on biomass and urease
activity. Based on these observations, two concentrations of mo-
lasses combined with two concentrations of sodium acetate were
tested to determine the effect that carbon concentration during
the enrichment phase had on the microbial ureolytic community
and precipitation of calcium carbonate in a potentially liquefi-
able soil.
Pre-treating soil samples for 7 days with an enrichment solu-
tion containing the relatively high or low carbon concentration
did not have a significant effect on the increase in pH of the
soil during the succeeding treatment phases, with the average
pH in the four soils treated with the higher carbon concen-
tration reaching 8.5 and the average in the same soils treated
with a lower carbon concentration reaching 9.2 after the first
biomineralization treatment. The average pH in the soils after
the second treatment was 9.2 in both sets of columns. After three
treatments, the pHs were 9.11 and 9.15, respectively.
There was only a small effect of carbon concentration on
the average percentage of CaCO3 precipitated in the two sets
of columns. In columns enriched with a higher carbon con-
centration for 7 days followed by three treatments spaced 72
hours apart, the average percentage of CaCO3 precipitation was
2.8%, versus 2.2% in the columns initially enriched with a lower
concentration of molasses and acetate. These results are sum-
marized in Table 1.
Changes in Urea-Utilizing Microbial Populations
To track the changes in ureolytic microbial populations over
the treatment period, qPCR analyses for copy numbers of ureC,
a gene encoding a subunit of urease, were performed on DNA
isolated from soil samples prior to and after the biomineral-
ization treatments performed on the soils used to optimize car-
bon sources. The soils treated with an enrichment solution and
 306 M. B. BURBANK ET AL.

TABLE 1 TABLE 3
Effects of carbon concentration on CaCO3 precipitation Effects of CaCl2 concentrations on pH in laboratory
percentage in laboratory
Average pH
% Weight Calcite (wt/wt)
Treatment # Time (h) 50 mM CaCl2 250 mM CaCl2
Sample Depth (cm) L H
Enrichment 36 9.5 7.4
30 2.2 3.2 2 48 8.6 7.6
60 2.2 2.8 3 48 8.9 8.5
90 2.1 2.5 4 24 9.2 8.1
150 2.2 2.7 6 24 9.2 7.9
7 24 9.3 7.7
L indicates enrichment solution containing 0.1% molasses and 50
mM CH3 COONa. H indicates enrichment solution containing 0.5%
molasses and 170 mM CH3 COONa. Snake River in Washington. Soil was collected into 30 ml sy-
ringe columns and kept saturated with the water it was collected
biomineralization solution contained 2–3 orders of magnitude with and placed on ice or at 4◦ C until treatments began.
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more copies of ureC than did untreated soils (Table 2). Soil samples were initially treated with enrichment media
solution of either a 50 mM or 250 mM CaCl2 for approximately
72 h. Following the enrichment period and between each treat-
Effects of CaCl2 Concentration on pH and Calcite ment columns were flushed with several pore volumes of 0.9%
Concentration NaCl to return the pH to approximately 7. The soil samples
The role of calcium on urease activity, pH and carbonate were treated a total of 7 times with either the 50 mM or 250 mM
precipitation (Zamarreno et al. 2009) has been investigated using CaCl2 biomineralization solution.
single strains of bacteria in pure culture. Whiffen (2004) found It should be noted that some fine particulate CaCO3 prob-
that urease activity in S. pasteurii was inhibited in the presence ably was lost in the effluent during thepH adjustment, which
of up to 2 M Ca(NO3 )2 . At concentrations of Ca(NO3 )2 2 M and may have lowered the final measured concentrations of calcite
higher, urease activity was undetectable. Hammes et al. (2003) precipitated in the soils.
looked at the effects of urease activity of soil isolates with 30 Though soils treated with a 250 mM CaCl2 biomineralization
mM CaCl2 and found that 4 of 12 isolates showed a 4- to 10- solution consistently had a lower pH than did soils treated with
fold increase in urease activity in the presence of calcium, while a biomineralization solution containing 50 mM CaCl2 (Table 3),
calcium had no effect or a slightly negative effect on urease the measured calcite concentration was higher in the samples
activity in the remaining isolates. treated with the higher calcium concentration (Figure 1).
To test the effects of calcium concentrations on pH and on To rule out the possibility that the precipitation of calcium
CaCO3 precipitation in soil with an indigenous bacterial popu- carbonate was chemically induced, autoclaved Snake River al-
lation, soil was collected from a saturated zone at approximately luvial soil (killed control) was treated with the same solutions
46 cm, 90 cm and 150 cm below ground surface adjacent to the and in the same manner as the live columns. No precipitation of
calcium carbonate was observed by XRD analysis in the killed
TABLE 2 controls.
Changes in ureC copy number/gram soil before and after
biomineralization treatments
ureC#/gm treated soil
Sample Initial ureC#/gm
Depth (cm) untreated soil L H
30 4.49 × 106 2.37 × 109 3.81 × 109
60 2.74 × 107 1.01 × 108 2.27 × 108
90 1.53 × 106 3.38 × 109 5.64 × 109
150 4.99 × 106 5 × 109 6.11 × 109
L indicates samples pretreated with an enrichment solution con-
taining 0.1% molasses and 50 mM acetate and treated with a biomin-
eralization solution containing 50 mM acetate. H indicates samples
pretreated with anenrichment solution containing 1% molasses and
250 mM acetate and a biomineralization solution containing 250 mM FIG. 1. Effect of CaCl2 concentrations on calcite % (wt/wt) in laboratory. L
acetate. indicates 50 mM CaCl2 . H indicates 250 mM CaCl2 .
 PRECIPITATION OF CALCITE BY INDIGENOUS MICROORGANISMS 307

cemented samples were considerably more resistant to crushing

than were the uncemented controls.
To rule out the possibility that calcite was already present in
the soil or was being precipitated by some process other than
those caused by the microbial hydrolysis of urea, soil samples
from each depth were treated with an enrichment and biomin-
eralization solutions from which ureaand/or calcium chloride
had been omitted or with water collected from the Snake River.
CaCO3 was only detected by XRD in soil samples that were
treated with a biomineralization solution containing urea and
calcium chloride (Figures 3a, 3b).
In addition to the alluvial soil reported here, six distinctly dif-
ferent soil types were successfully treated, including an alluvial
sand, a mined silica sand, two tidal soils collected from Willipa
Bay, Washington, Palouse loess, and an organic silty soil sam-
ple that was collected from beneath a pile of pine needles that
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had accumulated for more than 30 years. Calcite precipitation

in these samples ranged from 2.3 to 19.1% calcite (Table 4).
ureC was detectable in all samples tested for this study except
the soil collected from beneath the pine needles (high organic).
After two treatments over 6 days, ureC copies increased from
less than the qPCR detection limit of 1.5 × 103 per gram of soil
to 1.6 × 109 copies per gram of soil. Because of this finding,
we believe that a small-scale bench test on candidate soils is the
best method to determine if calcite precipitation by indigenous
bacteria will work in a specific type of soil.
In experiments in which columns were dissected and the per-
centage of calcite was measured at depths, precipitation through-
out the column was significant (about 4%) and relatively evenly
distributed in all layers, though concentrations of calcite were
often slightly higher (∼0.5%) in the center of the column.

In situ Treatment of Natural Soils

An in situ field experiment was conducted in alluvial soil at a
field site approximately 8000 meters downstream of the Lower
Granite Dam, Washington State. Following the treatment period
(1 enrichment and 9 biomineralization treatments), cone pene-
tration resistance was measured in treated and untreated soil
using a miniature cone penetrometer. The cone penetration test
(CPT) is a common method used by civil/geotechnical engineers

TABLE 4
Additional soil types tested
Soil Type # Treatments % Calcite (wt/wt)
FIG. 2. 150 cm samples. A) Treated with 50 mM CaCl2 biomineralization
solution. B) Treated with 250 mM CaCl2 biomineralization solution. C) Treated Mined Silica 3 2.5
with 50 mM CaCl2 biomineralization solution, but no urea. Mined Alluvial 3 2.3
Tidal #1 3 3.7
Tida1 #2 3 4.5
Substantial cementation occurred in samples treated with a
High Organic 10 11
biomineralization solution containing urea and CaCl2 (Figures
Palouse Loess 10 19.1
2a, 2b), while no cementation occurred in the samples treated
with a biomineralization solution containing CaCl2 but no urea The first treatment was with an enrichment solution containing 0.5%
(Figure 2c) or in samples treated with just river water. The molasses and 170 mM sodium acetate.
 308 M. B. BURBANK ET AL.
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FIG. 3a. XRD scan of 150 cm H soil sample. The arrow marks the 104 calcite peak.

in assessing soil type, soil engineering properties, and soil resis-
tance to liquefaction (Robertson and Campanella 1983a; Rober-
ston and Campanella 1983b; Robertson 1990; Youd et al. 2001).
A review of the cone penetration testing shows an approximate
two- to three-fold increase in cone penetration resistance in the
treated soil compared to untreated soil at depths of approxi-
mately 100 to 130 cm (Figure 4). These test results are con-
sistent with cone penetration testing in weakly cemented sand
(sand mixed with 1 to 2% cement) at low confining pressures as
reported by Puppala et al. (1995).
Treated soil was collected from the surface and at approx-
imately 30 cm intervals from the treatment area down to 180
cm and the percentage of CaCO3 was measured. The results
are shown in Figure 5 and Figure 6. The quantity of CaCO3
by weight was approximately 1% from the ground surface to a
depth of 90 cm. Below 90 cm, approximately 1.8 to 2.4% cal-
cite was precipitated in the soil. The zone with approximately
1.8 to 2.4% calcite is where the cone tip resistance increased
significantly.
These results indicate that 1% calcite precipitation had a
negligible effect on soil properties. The fact that the calcite pre-
cipitation increased with depth is noteworthy, especially since
other investigators have documented clogging of the soil pore
spaces and more calcite near the injection point (Stocks-Fisher
et al. 1999; Whiffen et al. 2007). This finding may be the result
of fine particles of calcite, or calcite precipitated on soil fines

FIG. 3b. XRD scan of 150 cm no-urea control with no calcite peak.
 PRECIPITATION OF CALCITE BY INDIGENOUS MICROORGANISMS
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FIG. 4. Cone tip depth and tip resistance.
(e.g., clays, silica fines, or humic macromolecules) being trans-
ported downward by the kinetic energy of the infiltrating water,
a process called eluviation. In soil science, the term “eluviation”
is used to describe the transport of finely-grained soil material
from upper layers of soil to lower levels by downward trans-
port of water through the porosity of intervening soil horizons;
accumulation of this material at a lower level is called “illuvi-
ation.” An alternative interpretation is a biogeochemical factor
favoring more urea hydrolysis in the deeper soil profile than the
overlying one, given the abrupt increase in calcite at 100 cm.
Although correlations developed for estimating liquefaction
resistance (cyclic resistance ratio) of sands (Youd et al. 2001) are
not applicable to cemented sands, some literature does indicate
that liquefaction resistance did increase for the cementation lev-
els observed in our field test. As stated previously, the increase
in cone tip resistance for the in situ field test with 1.8 to 2.4%
calcite precipitation is consistent with CPT results from Pup-
pala et al. (1995) for soil with 1 to 2% cement mixed with
sand. Work by Clough et al. (1989), showed an increase in the
cyclic resistance ratio of 16 to 45% for sand with 1% cement
added and approximately 80 to 100% for sand with 2% cement
added.
The higher increase in liquefaction resistance is associated
with higher cyclic shear stresses (e.g., larger earthquakes). Thus,
the field tests indicate that an increase in liquefaction resistance
was likely. Cyclic shear test results of biologically treated soils
are needed to determine the quantity of calcite required to suf-
309
FIG. 5. Percentage of CaCO3 (wt/wt) precipitated within and beneath the ring
infiltrometer.
ficiently increase soil liquefaction resistance. The quantity of
calcite required to mitigate liquefaction may vary depending on
grain size, grain shape, gradation, and initial density of the sand
(Ismail et al. 2002).
Whiffin et al. (2007) presented test results showing that pre-
cipitation of calcite greater than 3.5% was sufficient to signifi-
cantly improve the strength of soils, though less calcite may be
required in soils that have a greater percentage of fines, greater
density, or small soil grains, since there is likely to be a decrease
in pore space and thus less space would need to be filled in order
to bridge soil particles. In light of results from our field test, it
may be possible to significantly increase liquefaction resistance
at calcite levels less than 3.5% for some sands.
Although we were successful in inducing calcite precipitation
with indigenous microorganisms in the field test, the quantity
of calcite precipitation in the field was lower than quantities
from laboratory tests. The lower field quantities are largely at-
tributed to using a lower quality CaCl2 for the field testing
than was used in the laboratory tests. Additional laboratory
scale tests with the lower grade CaCl2 showed consistent results
with the field testing (e.g. lower quantities of calcite precipita-
tion). Thus, we believe that if analytic grade CaCl2 were used
for the field test, higher levels of calcite would have precip-
itated resulting in even greater increases to soil strength and
stiffness.
For field applications, it may not be feasible to use distilled
water to mix enrichment and biomineralization solutions and
the use of chlorinated municipal water could negatively impact
microbial numbers. We collected water from the Snake River
 310 M. B. BURBANK ET AL.
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FIG. 6. Cone tip resistance and percentage of calcite at depth.
(our water source for the field experiment) and compared ureC
copy numbers in that water to water that was collected from
a pristine artesian well. We found 1 × 106 ureC copies/ml in
the Snake River samples and 1 × 1.14 × 105 ureC copies/ml in
the well water. We believe that most water sources, other than
chlorinated or distilled water, will contain ureolytic bacteria
that will supplement the high numbers of indigenous ureolytic
bacteria already present in most soils.
CONCLUSIONS
A series of laboratory tests and an in situ field test were
conducted to determine if potentially liquefiable soils could be
enriched to increase the quantity of ureolytic microorganisms
for inducing calcite precipitation and cementing soil particles
together for increased resistance to liquefaction. The labora-
tory tests investigated the impacts of carbon concentration on
ureolytic microbial growth and resulting levels of calcite precip-
itation and the effects of CaCl2 concentration on pH associated
with urease activity and the associated impact on calcite pre-
cipitation. An in situ field test was performed to determine if
processes which showed good results in the lab would work in
the field.
Based on results from the laboratory testing, we have found
that for the carbon concentrations tested, specifically the mo-
lasses concentrations, calcite precipitation was not significantly
impacted. Ureolytic microorganism growth for both high and
low concentrations of carbon were of the same order of magni-
tude. Test results did show a reduction in pH when high concen-
trations of CaCl2 were used in the biomineralization solution,
which may indicate that urease activity was decreased. How-
ever, greater quantities of calcite were precipitated at the higher
CaCl2 concentration. Thus, the potential reduction in urease
activity at these high CaCl2 concentrations is not a significant
concern for the consortia of indigenous bacteria in the soils we
tested.
Testing on soils of various mineralogy and grain size dis-
tribution showed that enrichment of ureolytic microorganisms
is feasible in many soils. We found that qPCR should not be
relied on to determine if a soil is a candidate for enriching the
soil for ureolytic microorganisms. We recommend conducting
small column treatment tests to determine if a soil is a good
candidate for enrichment.
Finally, the insitu field tests showed that the laboratory pro-
cesses for enrichment of ureolytic microorganisms can be ap-
plied in the field at large scale. The CPT data showed a sig-
nificant increase in tip resistance for calcite precipitation levels
as low as 1.8 to 2.4%. We anticipate that liquefaction resis-
tance may be increased significantly for some soils with calcite
precipitation on the order of 3.5% or less.
The approach of enriching the soil to increase the quantity of
urease-producing microorganisms overcomes several problems
encountered when introducing bacteria into soil for the purpose
of biomineralization, including uneven distribution of microor-
ganisms and precipitated calcite and predation of the bacteria.
For industrial applications, this method may be less expensive
than adding exogenous bacteria because there is no need to
grow large quantities of bacteria. As compared to traditional
methods to mitigate seismic induced liquefaction (e.g. densifi-
cation method), inducing calcite precipitation with indigenous
bacteria is likely to be less intrusive to property owners and less
toxic to the environment when compared to soil improvement
with grouts (Ivanov and Chu 2009).
For the soil samples treated in this study, an enrichment
medium containing 0.5% or greater of molasses, 170 mM ac-
etate, 0.1 g/liter yeast extract, 250 mM CaCl2 and 12.5 mM
NH4 Cl, and 333 mM urea followed by a biomineralization so-
lution containing the same ingredients but with the molasses
 PRECIPITATION OF CALCITE BY INDIGENOUS MICROORGANISMS
omitted gave good results. It should be possible, however, to
use alternate carbon sources for the enrichment and treatment
phase and a lower cost substitution for yeast extract (Whiffen
2004), as well as alternate calcium sources, such as Ca-acetate
or Ca(NO3 )2 . In practice, it is likely that it will be necessary to
optimize carbon source(s) and calcium concentrations, though
pH of the soil during the treatment seems to be a good indicator
of how much time is needed between treatments.
Based on this study, we are confident that, in permeable soils
with sufficient urease positive microorganisms, enrichment for
indigenous bacteria capable of hydrolyzing urea in the presence
of CaCl2 is possible, and this approach may be more efficient for
in situ biomineralization than introducing exogenous ureolytic
bacteria.
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