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A Study On Biological Soil Improvement Using New Environmental Bacteria

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DOI: 10.1680/jenge.18.00176

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Cite this article Research Article Keywords: calcium carbonate/
Bahmani M, Fatehi H, Noorzad A and Hamedi J Paper 1800176 environmental engineering/soil
Biological soil improvement using new environmental bacteria isolated from northern Iran. Received 02/11/2018; Accepted 14/06/2019 stabilisation
Environmental Geotechnics
https://doi.org/10.1680/jenge.18.00176 ICE Publishing: All rights reserved

Environmental Geotechnics

Biological soil improvement using new

environmental bacteria isolated from
northern Iran
Maysam Bahmani MSc Ali Noorzad PhD
Faculty of Civil, Water and Environmental Engineering, Shahid Beheshti Faculty of Civil, Water and Environmental Engineering, Shahid Beheshti
University, Tehran, Iran University, Tehran, Iran
Hadi Fatehi MSc Javad Hamedi PhD
Department of Civil Engineering, Isfahan University of Technology, Isfahan, Iran Department of Microbial Biotechnology, School of Biology, College of
(corresponding author: h.fatehi@cv.iut.ac.ir) Science, University of Tehran, Tehran, Iran

New indigenous bacteria, extracted from soil samples that had been collected from different parts of Iran, were introduced
to improve dune sand using microbially induced carbonate precipitation (MICP) as an environmental engineering method.
In order to isolate urease-producing bacteria from local alkaline soils, a series of standard tests was conducted to evaluate
the urease performance and potential of calcium carbonate (calcite) sedimentation. Based on their properties, the bacteria
with the best performance were identified by 16S ribosomal ribonucleic acid sequencing. To achieve a better
understanding of the performance of the selected isolates, Sporosarcina pasteurii was employed as the control sample. The
geotechnical behaviour of bio-mediated sand was studied through a series of geotechnical experiments, including
unconfined compressive strength (UCS), constant-head permeability and wind erosion tests. The influence of freeze–thaw
cycles and the durability of specimens on the UCS was also investigated. It is worth noting that sand MICP-treated by
Bacillus sp. UTMC 2623 gave a performance relatively equal to that of the control sample during all UCS tests. Scanning
electron microscopy images and X-ray diffraction were also used to observe calcite precipitation formation using Bacillus
sp. UTMC 2623, which indicated precipitation of calcite throughout the sand column.

Introduction 2005), improvement in the stiffness/strength of sandy soil (Chu et al.,

In recent years, soil improvement has been taken more seriously
into consideration because of population growth and scarcity of
land with appropriate ground conditions (Fatehi et al., 2019).
Although there are several soil improvement techniques, most of
these have adverse effects on the environment or need substantial
energy for the production of materials and/or installation (DeJong
et al., 2010). It should also be noted that some of these common
techniques are not practical in sites where buildings and
underground structures exist (Jahandari et al., 2019).
Microbiologically induced calcium carbonate (CaCO3) precipitation
(MICP) is an interdisciplinary and a biogeochemical process that
induces calcium carbonate precipitation within the soil matrix
(Mortensen et al., 2011), and it can lead to altering the engineering
properties of the soil. Calcium carbonate can be precipitated in three
polymorphic forms, which in the order of their usual stabilities are
calcite, aragonite and vaterite (Simkiss, 1964).
MICP has experienced an increased level of interest in recent
years for applications such as restoration of calcareous stone
materials (Castanier et al., 2000; Rodriguez-Navarro et al., 2003;
Stocks-Fischer et al., 1999; Tiano, 1995), bioremediation (Ferris
et al., 2004; Fujita et al., 2000; Warren et al., 2001), wastewater
treatment (Hammes et al., 2003), strengthening of concrete and
remediation of cracks (Achal et al., 2010; Bang et al., 2001;
Ramachandran et al., 2001), selective plugging for enhanced oil
recovery (Ferris et al., 1997; Gollapudi et al., 1995; Nemati et al.,
2014; Rong et al., 2012; van Paassen, 2009; Whiffin et al., 2007),
reductions in foundation settlement (DeJong et al., 2010; Mahawish
et al., 2016), soil permeability (Dennis and Turner, 1998; Seki et al.,
1998), liquefaction mitigation (DeJong et al., 2006; Montoya, 2012),
dust control and prevention of soil erosion (Meyer et al., 2011).
A serious environmental problem occurring in dry sandy soils or
anywhere with loose soil is wind erosion. Fugitive dust, as a
major consequence of wind erosion, inevitably covers roads and
agricultural crops, which leads to reducing the productivity of
farms. It can also exacerbate airways and cardiovascular diseases
(Alsanad, 2011). Regarding this matter, MICP methods have been
proven able to strengthen wind erosion resistance up to 0·16% for
soil loss (Maleki et al., 2016; Wang et al., 2018).
Furthermore, calcium carbonate precipitation induced by urease-
producing bacteria (UPB) is a potentially long-lasting process that
can be used to suppress dust from landfills, open-pit mines,
unpaved roads and construction sites. In particular, MICP has
been recently employed as a potentially dust-suppressive
technique when applied to surfaces of different soils, including
poorly graded sands, silts and clayey soils (Achal et al., 2011).
The MICP method often employs ureolytic bacteria for
hydrolysing urea to make calcium carbonate crystals in the
presence of an adequate amount of calcium (Ca2+) ions (Mujah
et al., 2017). Consequently, soil particles can be bound together

1
Environmental Geotechnics
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so that an increase in soil strength is obtained. The following
simplified chemical reactions can be observed (Hammes and
Verstraete, 2002)
I. COðNH2 Þ2 þ 2H2 O ⇒ 2NH4 þ þ CO3 2−
II. Ca2þ þ CO3 2− ⇒ CaCO3 ðsÞ
During the aforementioned process, urea is hydrolysed by the
urease enzyme to 2 mol of ammonium (NH4+) and 1 mol of
carbonate (CO32−) per 1 mol of urea. In the presence of calcium
ions, the combination of calcium with carbonate leads to calcite
precipitation.
Biocalcification has attracted many researchers to investigate
MICP for different geotechnical applications. By increasing the
calcium carbonate content, the shear strength of bio-cemented soil
samples can be increased in terms of both cohesion and friction
angle because of the calcite crystals that formed in the soil pore
spaces. In addition, MICP reduces hydraulic conductivity (Al
Qabany and Soga, 2013; Cheng and Shahin, 2016; Chou et al.,
2011), soil erosion susceptibility (Kandasami et al., 2016) and
porosity (Qian et al., 2010), while compressive strength (Harkes
et al., 2010; Ivanov et al., 2015; Mortensen et al., 2011; Whiffin
et al., 2007) and stiffness (Montoya and DeJong, 2015) are
enhanced since the bonds among soil grains are rearranged. Then,
the mechanical behaviour of treated soil is dramatically improved
(Bahmani et al., 2017; Chu et al., 2012; Mahawish et al., 2018).
The mechanical properties of bio-cemented soils are highly
dependent on the formation of calcium carbonate crystals during
the MICP process. In fact, various sizes, shapes and distribution
of calcite crystals can cause different geotechnical behaviours of
MICP-treated soils (Al Qabany and Soga, 2013; Miralles et al.,
2012). Temperature plays a key role in formation of calcium
carbonate crystals and in the urease activity of microorganisms
(Mujah et al., 2017). Variation of temperature from 20 to 50°C
leads to an increase in the production rate of calcium carbonate,
and temperatures higher than 60°C would be detrimental to calcite
crystal formation because of the destruction of microorganisms
(Nemati and Voordouw, 2003; Rebata-Landa, 2007). An alkaline
environment is favourable for calcite precipitation, which is the
result of the generation of hydroxyl ions (OH−) in the ammonium
ion production process. pH is a crucial factor for gaining
uniformly distributed calcite crystals throughout the soil matrix
and samples with a more solid strength (DeJong et al., 2010;
Ferris et al., 2004). Also, soil samples treated at a low degree of
saturation have exhibited higher strength compared with samples
cured at a high degree of saturation (Cheng and Cord-Ruwisch,
2012). In addition, it has been noticed that the concentration of
the cementation solution and bacteria could strongly affect
calcium carbonate crystallographic patterns (DeJong et al., 2010;
Okwadha and Li, 2010).
2
Biological soil improvement using new
environmental bacteria isolated from
northern Iran
Bahmani, Fatehi, Noorzad and Hamedi
Different indigenous and exogenous bacteria have been examined
in soil treatment so far. Four microbes (Staphylococcus
saprophyticus, Sporosarcina globispora, Bacillus lentus and
Sporosarcina sp.) displayed higher urease activities than
Sporosarcina pasteurii and higher amounts of calcium carbonate
precipitation (except S. globispora) (Kim and Youn, 2016). An
increase in unconfined compressive strength (UCS) by Idiomarina
insulisalsae (Cheng et al., 2017; Venda Oliveira et al., 2014);
remediation of liquefaction potentials of sandy soils using
indigenous bacteria and also Bacillus sphaericus LMG 22257 and
Bacillus sp. (Burbank et al., 2011, 2012; Shirakawa et al., 2011);
and reduction of the hydraulic conductivity of bio-cemented soil
employing Bacillus sp. (UPB) VS1 and B. sphaericus (MCP-11)
(DSM 23526) (Cheng et al., 2013; Chu et al., 2012) are some
examples of other bacterial applications for improving soil
properties.
In this study, by identifying and investigating the presence of
bacteria with urease potential in loose soils, an effort has been
made to isolate these indigenous bacteria and investigate the role
of these isolates in biological soil improvement and
biomineralisation. Also, exogenous bacteria added into some
original soils may not be able to survive because of opposing
actions by indigenous bacteria (Tsesarsky et al., 2016). Having
considered S. pasteurii as the most common bacteria in MICP
studies, the novelty of this study is the extraction of new
indigenous environmental isolates and examination of their
activities in biological soil treatment. The present research is one
of the studies in this field to apply these new isolates in
improving the geotechnical parameters of dune sand. Furthermore,
sand dust, as a global environmental issue, particularly in Iran,
brings about many problems for infrastructures, as well as
people’s health. Using environment-friendly sand improvement by
employing indigenous bacteria could not only improve soil
properties, but also prevent environmental impacts. However,
further research studies are certainly required to be performed for
the evaluation of more aspects of utilisation of these isolates and
optimisation of the conditions.
In this study, firstly, some indigenous bacteria were isolated from
several soil samples. Then, the bacteria with the highest urease
activities were identified and characterised. To this end, different
kinds of microbial tests, including a test for urease activity,
qualitative and quantitative assessment of calcium carbonate
precipitation and bacterial identification by 16S ribosomal
ribonucleic acid (rRNA) sequencing, were conducted.
With the aim of investigating the potential of selected bacteria to
improve geotechnical behaviour, these isolates were compared
with S. pasteurii. The compressive strength and durability of
treated sand was studied by UCS tests. In addition, permeability
and wind erosion tests were performed. Moreover, scanning
electron microscopy (SEM) images and X-ray diffraction (XRD)
analysis was employed to understand directly the effectiveness of
the selected isolate in calcite precipitation.
Environmental Geotechnics Biological soil improvement using new
environmental bacteria isolated from
northern Iran
Bahmani, Fatehi, Noorzad and Hamedi

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Materials and methods medium was used because it has a lower buffering capacity than urea
broth (Stuart’s), which makes it possible to detect a lower amount of
Sample preparation
pH due to urea decomposition.
For this purpose, soil samples were taken from different
ecosystems with alkaline soils in Iran. The specifications of these
To screen the urease activity of isolates quickly, Christensen urea
soil samples are presented in Table 1.
broth base (CM0053; Oxoid, Basingstoke, UK) medium was selected
to assess urease production qualitatively. Based on the instructions,
Soil specimens were collected from a depth of 25–30 cm using
the medium components contained the following: peptone (1·0 g/l),
sterile tools, placed inside sterile containers and stored in a fridge
glucose (1·0 g/l), disodium phosphate (1·2 g/l), sodium chloride
at 4°C after transferring to the laboratory.
(NaCl) (5 g/l), potassium dihydrogen phosphate (0·8 g/l), urea (20 g/l)
and meta-cresol purple or m-cresol purple (0·76 g/l). In this study,
Biological materials
instead of phenol red, m-cresol purple was used as the indicator. To
S. pasteurii PTCC 1645, purchased from the bacterial collection
test the production of urease, one loop of the isolate was inoculated
of the Industrial Scientific Research Center of Iran, was used as
in the medium in test tubes and incubated under aerobic conditions at
the control sample. S. pasteurii was cultivated under aerobic batch
37°C for 6 d. The urease activity was investigated visually through
conditions in a medium containing yeast extract, 7 g/l; urea,
the observation of colour changes. Finally, the samples with the
20 g/l; tryptone, 5 g/l; and meat peptone. 5 g/l. Before using
ability to produce a higher amount of urease (identified through a
S. pasteurii, the cultivation medium was sterilised at 121°C and
more rapid change in the medium colour from yellow to violet) were
the pH of the medium was adjusted to 9. The growth medium was
selected for further investigations.
inoculated with the S. pasteurii stock culture at 28°C in a shaker
(100 revolutions per min (rpm)) for approximately 48 h before
Calcium carbonate precipitation by ureolytic bacteria
harvesting. Then, the bacteria were stored suspended in the
Qualitative assay
growth medium in the fridge at 4°C prior to use.
To examine qualitatively the calcium carbonate precipitation of
the selected isolates, a solid culture medium including urea and a
Isolation of the soil bacteria
calcium source was used as the screening medium. The
For isolation of alkaliphilic bacteria, 0· 5 g of soil samples was
composition of the medium contained agar (17 g/l), sodium
mixed sterilised with 5 ml of physiological saline, after vortexing,
bicarbonate (NaHCO3) (2·12 g/l), ammonium chloride (NH4Cl)
dilution series of the suspension was prepared. Then, 10−1 and
(10 g/l), nutrient broth (3 g/l), urea (20 g/l) and calcium chloride
10−2 dilution series were transferred to plates containing solid
dihydrate (CaCl2·2H2O) (4·41 g/l). First, a 24 h pure culture of
Horikoshi medium. The plates were kept at 28°C for 24 h, 48 h
selected isolates was prepared, and then, bacteria were cultured in
and up to 5 d for slow-growing colonies. The obtained colonies
the plates containing the screening medium, through the streak-
were transferred to the Horikoshi medium plates for pure
plate method. These plates, with the aim of producing calcium
cultivation and were reincubated at 28°C.
carbonate crystals, were kept at a temperature of 28°C and
checked continuously with a stereomicroscope (SMZ10; Nikon,
Screening ureolytic bacteria
Tokyo, Japan) with ×10 magnification.
Generally, there are two paramount kinds of growth media for
measuring the urease activities of bacteria – namely, urea agar base
Quantitative assay
(Christensen agar base) and urea broth (Stuart’s) medium (urea, 2%;
Bacterial isolates that had formed calcium carbonate due to their
yeast extract, 0·01%; and phosphate buffer). Urea broth (Stuart’s) is a
metabolic activities in the solid culture medium were
medium with a high buffering capacity and limited nutrients.
quantitatively evaluated. For this purpose, the selected isolates
However, the high buffer capacity of the medium would mask slight
were cultured in four flasks containing 50 ml of broth and urea
urease activity (Christensen, 1946). Thus, in this study, Christensen’s
(pH = 8). Then, the flasks were heated up at 28°C and shaken at
150 rpm for bacterial growth and calcite production. After 48 h,
Table 1. The numbers of isolates, location, pH and soil type the growth and lamination of samples were observed by using a
Number of Nikon SMZ10 stereomicroscope. After confirming the growth of
Number Soil type pH Location
isolates bacteria, flasks materials were poured into 50 ml Falcon tubes and
1 Wet, alkaline, 8·41 N 36° 040 49·0″ 65 centrifuged three times with an acceleration of 6042g for 10 min.
near river E 53° 040 13·6″ The supernatants were filtered, and the residue contained calcium
2 Alkaline 8·2 N 35° 460 56·5″ 77 carbonate precipitation. After drying the biomass, the back-
E 52° 480 53·1″
titration (BT) technique was used to measure the amount of
3 Dry, saline and 8·43 N 31° 540 08·0″ 26
alkaline E 59° 520 03·0″ calcite precipitation. Due to its insolubility, calcium carbonate
4 Dry, saline and 8·25 N 35° 560 23·2″ 48 should be first dissolved in acid, and then the remaining acid
alkaline E 54° 000 33·2″ titrated with sodium hydroxide (NaOH). The amount of calcium
5 Dry, alkaline 8·17 N 35° 560 23·2″ 31 carbonate can be determined by obtaining the amount of titrating
E 54° 000 33·6″
sodium hydroxide and two reactions as follows

3
Environmental Geotechnics
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1. nðtotal acidÞ − nðtitrated acidÞ ¼ nðconsumed acidÞ
CaCO3 ðsÞ þ 2HClðlÞ → CaCl2 ðaqÞ þ CO2 ðgÞ
III. þ H2 OðlÞ
In this way, 50 ml of hydrochloric acid (HCl) was poured onto the
obtained sediment in a 250 ml flask. After dissolution and using
five drops of phenol red, the obtained solution was titrated with
0·25 M sodium hydroxide. The colour change of the solution
from yellow to pink was the indicator of the titration endpoint.
Bacterial identification by 16S rRNA sequencing
Brain heart infusion broth medium was used to culture the strains for
48 h at 28°C. Pellets were collected after centrifugation (18 516 g for
1 min). Based on the manufacturer’s instructions (TianGen Co.,
China), total genomic deoxyribonucleic acid (DNA) was extracted
with a genomic DNA purification kit for use in polymerase chain
reaction (PCR) amplification of 16S rRNA genes, DNA sequencing
and synthesis of DNA sequence analysis templates for PCR. 16S
ribosomal DNA from the isolated bacteria was amplified through
PCR with the primers 9F (50 -AGGAGTTTGATCATGGCTCAG-30 )
and 1541R (50 -AGGAGGTGATCCACCCGCA-30 ) at the following
conditions.
The mixture was subjected to denaturation at a temperature of 96°C
for 5 min. The PCR temperature conditions included 45 s
denaturation at 94°C in 28 cycles, 75 s primer extension at 72°C,
55 s annealing at 57°C and finally extension at 72°C for 10 min.
Also, the PCR solution consisted of 1 ml of each primer (20 mM),
2 ml of a deoxyribonucleotide triphosphate mixture (1·25 mM each),
1 ml of Taq polymerase and 2·5 ml of DNA. To reach the total
volume of 100 ml, sterile deionised water was added. The analysis
and visualisation of PCR products were conducted using agarose
(1%) gel electrophoresis and ethidium bromide, and possible reagent
contamination was detected by using a negative control. Finally, after
sequencing the PCR products, the detected bacteria were verified by
a comparison between the PCR products and the GenBank database
through a Basic Local Alignment Search Tool search at the National
Center for Biotechnology Information (NCBI, 2019). In addition,
standard methods were used for characterising the bacteria’s
morphological properties, such as the Gram stain reaction, endospore
stain tests and cell morphology analysis.
Growth of the selected isolates at different
temperatures
To investigate the best temperature range, the growth of the
selected isolates growth was studied at different temperatures (4,
15, 28, 32, 37 and 45°C). The Horikoshi medium was used for
this purpose. The medium components were autoclaved at 121°C
for 20 min. Then, an inoculation loop full of selected bacteria was
cultivated on the plates of the Horikoshi medium. These plates
were kept in incubators at different temperatures for 48 h. Also,
4
Biological soil improvement using new
environmental bacteria isolated from
northern Iran
Bahmani, Fatehi, Noorzad and Hamedi
the growth curve of isolates at the optimum temperature was
attained by recording the optical density for 80 h.
Soil type
Nowadays, poorly graded sand causes many problems in civil
engineering projects because of its low mechanical strength and
uniform soil gradation and in particular the lack of cohesion
between soil grains (Fatehi et al., 2018). It is also widely
distributed throughout the world, and effective methods of soil
improvement are needed to enhance it. The soil in this study was
silica sand taken from the exact location of the soil where
Bacillus sp. UTMC 2623 had been isolated. Sieve analysis was
carried out to determine the particle-size distribution. Figure 1
shows the particle-size distribution curve of the soil classified as a
poorly graded sand (SP) according to the Unified Soil
Classification System (USCS) (ASTM D 2487-06) (ASTM,
2006a). The coefficients of uniformity (Cu) and curvature (Cc)
were measured as 2·2 and 0·77, respectively. Table 2 gives the
detailed properties of the sand.
Preparation of sand columns for geotechnical tests
The internal diameter and height of all soil columns (poly(vinyl
chloride) tubes) were 6 and 12 cm, respectively. Other properties of
the columns are shown in Figure 2. To obtain a density of
19·1–19·4 kN/m3, the sand particles were subjected to continuous
vibration. Porous paper was placed at the top and bottom of the
samples as a filter. All specimens were made of poorly graded sand
(Table 2) prepared to a loose initial state at a void ratio of 0·67. After
installation of the sand column, water was flushed into the soil
column from top to bottom. The bacteria and cementation solution
were added into the soil column from the top (Figure 2). The steps of
adding bacteria and cementation solution were as follows.
■ In step 1, bacterial solution with a volume of Vv (the mean
volume of sand column pores) equalling 98·1 ml was added to
the sand column. Afterwards, the process was stopped and the
bacterial solution was kept for 12 h in order to allow bacteria
100
90
80
Finer by weight: %
70
60
50
40
30
20
10
0
0·01 0·1 1 10
Grain size: mm
Figure 1. Particle-size distribution curve of the sandy soil
Environmental Geotechnics Biological soil improvement using new
environmental bacteria isolated from
northern Iran
Bahmani, Fatehi, Noorzad and Hamedi

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Table 2. Basic properties of sandy soil

USCS pH Gs Cc Cu D60: mm D50: mm D30: mm D10: mm emax emin
SP 8·5 2·63 0·77 2·2 0·22 0·18 0·13 0·10 0·86 0·53

Inlet device in accordance with the ASTM D 2434-68(2006) standard

(ASTM, 2006b). It should be noted that the samples were saturated
Scouring before the permeability test by flushing 2 litres of tap water under a
pad filter 15 kPa back-pressure.
Container

Durability of specimens
To evaluate the durability of bio-cemented sand column
specimens, after a 5 d curing period, they were subjected to ten
Direction of flow

cycles of freeze–thaw (FT) actions. During each cycle, the

20 cm

samples were placed for 12 h at a temperature of −10°C followed

12 cm

Sand Paper
filter by 12 h thawing at a temperature of 30°C. All specimens were
immersed in water during the cycling FT testing. The UCS
experiments were performed before and after ten FT cycles on
treated specimens.

Outlet
Figure 2. Schematic diagram of the sand column
to be attached to the soil particles. Finally, the medium
solution was drained from the bottom of the sample.
■ In step 2, the cementation solution consisting of 1·85 M urea and
1 M calcium chloride (CaCl2) with a volume of Vv was added to
the sand column. Next, the process was stopped for 12 h, and the
solution was drained from the bottom of the sand column.
These steps were repeated three times. After the injection steps,
the samples were cured at 21°C for 5 d.
Microscopic observation
SEM images were employed to explore the microstructural
behaviour of bio-cemented sand samples and to characterise
MICP performance and the bonding behaviour between the soil
particles and cement agent. The images were taken from the
centre of treated sand columns by a Philips XL30 scanning
electron microscope. After flushing the samples with tap water,
they were dried at 60°C for 24 h. Then, the surfaces of the
samples were coated with a conductive metal (gold) to avoid
electron scattering. SEM images were used to study three states of
specimens as follows
■ sand treated with bacteria, culture medium and urea–calcium
chloride solution
■ sand treated with bacteria and culture medium
■ sand treated with culture medium and urea– calcium chloride
solution.

UCS test X-ray diffraction

UCS tests, according to ASTM D 2166/2166M-16 (ASTM, 2016),
were performed to quantify the strength of sand samples. Three tests
were repeated for each sample, 6 cm dia. and 12 cm high, and the
sample was loaded at a constant rate of 1·0 mm/min. Also, the
amount of calcite was measured through the BT technique for
samples treated by S. pasteurii and the isolated bacteria.
Permeability test
Hydraulic conductivity (k) is one of the parameter sets which is used
as the establishment of variety scholar evaluation and prediction such
as groundwater numerical modelling and streamflow records in
conjunction with geotechnical and agricultural purposes (Hamraz et
al., 2015; Sadeghi-Tabas et al., 2016, 2017a, 2017b). The
permeability test was performed on untreated and MICP-treated
sands using the constant-head permeability test with a rigid-side-wall
The mineralogical composition of the MICP-improved sand was
analysed through XRD using a D/Max-II diffractometer (Rigaku
Co., Japan); the XRD analysis was conducted to confirm the
presence of calcite as the MICP product. In this way, the bacterium
with the best performance on UCS tests was selected and cultured
in sterile solid growth medium containing urea (20 g/l), agar
(17 g/l), sodium bicarbonate (2·12 g/l), ammonium chloride (10 g/l),
nutrient broth (3 g/l) and calcium chloride dihydrate (4·41 g/l).
Afterwards, the bacteria were kept at 28°C for 24 h. Next, sterile
broth consisting of urea was heated at 28°C and shaken at 150 rpm.
Then, the bacterial growth was investigated after 48 h of
lamination. After confirming bacterial growth, the solution was
poured into flasks and centrifuged three times at room temperature
for 10 min. Finally, XRD analysis was performed on the residual
sediment to verify the calcium carbonate presence.

5
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Wind erosion test
Treated samples at different states – namely, those treated using
S. pasteurii, selected isolates, water and just bacterial solution
without bacterium – were studied in this test. A soil erosion test
was conducted on pie plates filled with sand that had passed
through sieve number 30 (0·6 mm). The used setup included
aluminium plates with a size of 40 cm × 60 cm and a depth of
3 cm. The aluminium ductwork had an output span of 42 cm ×
42 cm and a length of 140 cm (Figure 3). To simulate wind
conditions, since the average wind speed in a desert area is around
7 m/s (Alamdari et al., 2012), an industrial stand fan with the
ability to produce 8 ± 0·3 m/s wind was utilised. As shown in
Figure 3, the plates were placed 0·5 m away from the end of the
fan and exposed to airflow for approximately 10 min. Five turbulent
grids were placed at 10 cm intervals to obtain a laminar flow
(Kuttarmare et al., 2014). The characteristics of the cementation
liquids (with a volume of 500 ml) sprayed on the sand specimens
are presented in Table 3. After spraying of the cementation liquid,
treated samples were kept in the laboratory at room temperature
(20°C) for at least 24 h. The soil loss value was obtained by
measuring the difference between the weight of sand plates before
and after wind blowing. Each test was repeated three times, and the
average of the calculated soil loss was utilised as the value of
MICP effectiveness in increasing wind-induced resistance.
Results
Screening of ureolytic bacteria
Bacteria with a high urease activity changed the colour of the
Christiansen medium into purple, while lilac colour was the result
of bacteria with a moderate urease activity. The colour of the
control tube (without urea) also turned to purple after 6 d; thus,
140 cm
42 cm
42 cm
40 cm
10 cm 10 cm 10 cm 10 cm 10 cm
60 cm
Turbulent grids
Laminar flow
Figure 3. Schematic view of the wind erosion tunnel
6
Biological soil improvement using new
environmental bacteria isolated from
northern Iran
Bahmani, Fatehi, Noorzad and Hamedi
Table 3. Properties of samples in the wind erosion test
Sample
Cementation liquid
number
1 —
2 Water
3 Cementation solution + bacterial growth medium
4 Cementation solution + bacterial growth medium +
S. pasteurii
5 Cementation solution + bacterial growth medium +
UTMC 2622a
6 Cementation solution + bacterial growth medium +
UTMC 2623a
7 Cementation solution + bacterial growth medium +
UTMC 2624a
a
Bacillus sp. UTMC cells
this time was not considered in final measurements. The colour
change might be due to peptone decomposition in the culture,
which produced alkaline compounds, leading to an increase in the
medium pH. The urease activities of 112 isolates were recorded.
Among them, six isolates with the highest urease activities and
growth rates were selected to evaluate the amount of calcium
carbonate precipitation. Table 4 presents the results for these
selected isolates. After 24 h, the colour of specimens with
laboratory codes 12 and 63 changed to lilac, while four other
samples (codes 21, 42, 72 and 98) were observed to be in purple.
It was shown that the potential of these four samples in term of
urease activity was larger in comparison with that of the others.
Calcium carbonate precipitation by ureolytic bacteria
The plates consisting of solid bacterial growth medium with urea and
calcium chloride were controlled continuously, and the grown
Fan
Airflow direction
Turbulent flow
Environmental Geotechnics Biological soil improvement using new
environmental bacteria isolated from
northern Iran
Bahmani, Fatehi, Noorzad and Hamedi

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Table 4. The results of urease activity and biological characterisation of six selected isolates
Laboratory code UTMC code Microscopic shape Gram reaction Colour production time: h Colour Production of spores
12 2612 Rod + 24 Lilac −
21 2622 Rod + 24 Purple +
42 2628 Short rod + 24 Purple +
63 2631 Rod − 24 Lilac −
72 2623 Rod + 24 Purple +
98 2624 Rod + 24 Purple +

colonies were observed after 5 d through a stereomicroscope
(SMZ10; Nikon, Tokyo, Japan) with ×10 magnification. As
illustrated in Figure 4, calcite crystals formed on the plate of each
bacterium. Among these six selected isolates, the isolates Bacillus sp.
UTMC 2622, Bacillus sp. UTMC 2623 and Bacillus sp. UTMC
2624 were chosen due to their good growth on the medium. A
quantitative assay (BT assay) was also performed to measure the
amount of calcium carbonate precipitation. The isolates UTMC 2622,
UTMC 2623 and UTMC 2624 and S. pasteurii led to the formation
of 2·2, 3·0, 2·8 and 3·2 g/l calcium carbonate precipitation,
respectively, over 2 d (Table 5). Among selected isolates, UTMC
2623 had the greatest potential to produce calcite in comparison with
S. pasteurii. As expected, the samples in purple colour indicated a
higher amount of calcium carbonate due to their appropriate urease
activity; however, the amount of calcite varied among these
specimens. Figures 5(a) and 5(b) show calcite precipitation in dune
sand before and after treatment, respectively.
Bacterial identification by 16S rRNA sequencing
The extracted DNA of the selected isolates was investigated and
identified more accurately through 16S rRNA sequencing
(Table 6). The presence of a specific individual band is an
indication of a particular 16S rRNA sequence in the isolates. The
results were also compared with the NCBI database. The
morphological properties of selected isolates are also presented
in Table 6.
Growth of selected isolates at different temperatures
The growth rate of the selected isolates was also investigated at
various temperatures of 4, 15, 28, 32, 37 and 45°C. It is important
to know the optimum conditions, as the temperature has a
significant effect on bacterial growth. The results (Figure 6) show
that the optimal growth temperature range for all of the three
selected bacteria and S. pasteurii is around 28–37°C. Lower
temperatures decreased the growth rate dramatically. Specifically,
when the temperature was 4°C, the bacterial growth stopped
completely. Higher temperatures (more than 37°C) also led to a
reduction in the growth rate.
In Figure 7, the maximum optical density (OD600) is illustrated
for S. pasteurii and the selected isolates. The optical density at
any time largely reflects the growth rate and can be used for
obtaining the specific growth rate (Mytilinaios et al., 2012). As
shown in Figure 7, the growth of the selected isolates slightly
increased from 10 to 20 h. After that, there was a considerable rise
in the growth rate during the period of 20–50 h for all bacteria.

Table 5. Amount of calcite precipitation for S. pasteurii and three

isolated bacteria
S. UTMC UTMC UTMC
pasteurii 2622 2623 2624
a
Amount of 3·2 3·0 2·2 2·8
calcite: g/l

b c

Figure 4. Precipitated calcium carbonate crystals of calcium

carbonate sedimentation isolates on the medium after 5 d. a, b (a) (b)
and c indicate the isolates UTMC 2623, UTMC 2624 and UTMC
2622, respectively Figure 5. Soil particles (a) before and (b) after treatment

7
Environmental Geotechnics Biological soil improvement using new
environmental bacteria isolated from
northern Iran
Bahmani, Fatehi, Noorzad and Hamedi

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Table 6. Identification of selected isolates based on 16S rRNA sequencing

Laboratory UTMC Closest relative in Microscopic Gram Production of
Identity: % Microbe
code code NCBI shape reaction spores
21 2622 EU841500.1 98·91 Bacillus horikoshii Rod + +
72 2623 AM051268.2 97·01 Bacillus Rod + +
alkalisediminis
98 2624 KP409413.1 97·79 Bacillus clausii Rod + +

Bacillus sp. UTMC 2623 Bacillus sp. UTMC 2622 In fact, the optimal growth was obtained after a period of 40–50 h
was passed for each isolate. Then, the growth rate significantly
Growth rate (log cell number/ml)

Bacillus sp. UTMC 2624 S. pasteurii

12 decreased up to 80 h, although an increase was observed for the
10 isolate UTMC 2623 from 70 to 80 h.
8
Unconfined compressive strength
6
The stress–strain results from the UCS tests (Figure 8) present how
4 each selected isolate affects the calcite precipitation in the MICP
2 method and illustrate the behaviour of the specimens stabilised using
0 different bacteria. Thus, UCS tests were carried out on the sand
0 10 20 30 40 50 samples treated using the selected isolates and S. pasteurii. Pure dune
Temperature: °C
sand gave a low UCS of 18 kPa, which was a result of lack of
Figure 6. Variation in the growth rate of S. pasteurii and selected cohesion among soil grains, as well as the negligible amount of fine-
isolates with temperature graded soil and angular particles in dune sand. It was proved that
each isolate can be used for bio-cementation, as the UCS rose up to
480 kPa for sand treated with the isolate UTMC 2623, whereas the
Bacillus sp. UTMC 2623 Bacillus sp. UTMC 2622 compressive strength of sand treated with S. pasteurii was 509 kPa.
Bacillus sp. UTMC 2624 S. pasteurii Moreover, the isolates UTMC 2622 and UTMC 2624 also
0·8 demonstrated good performance in increasing the mechanical
Optical density at 600 nm

0·7 strength of sand by achieving UCS values of 420 and 347 kPa,
0·6 respectively. The compressive strength of MICP-treated soils varied
0·5
considerably from the lowest value of 150 kPa to the highest value of
0·4
0·3
34 MPa (Mujah et al., 2017). Therefore, making a comprehensive
0·2 comparison with other studies would be difficult due to the
0·1 differences in soil types, nutrients, bacterial cell concentration,
0 bacteria distribution throughout soil, pH, temperature, injection
0 20 40 60 80 100 method and so on (Khaleghi and Rowshanzamir, 2019). In a study
Time: h
by Whiffin et al. (2007), the reported UCS value for treated sand
Figure 7. Growth profiles of S. pasteurii and the selected isolates was 570 kPa, which seems to be in good agreement with the results
obtained in the current study’s experiments.

600
Bacillus
500 sp. UTMC 2624
Bacillus
400 sp. UTMC 2622
UCS: kPa

Bacillus
300 sp. UTMC 2623
200 S. pasteurii
100 Untreated sand

0
1·00

2·00

3·00

4·00

5·00

6·00

7·00

8·00
0
0·25
0·50
0·75

1·25
1·50
1·75

2·25
2.50
2·75

3·25
3·50
3·75

4·25
4·50
4·75

5·25
5·50
5·75

6·25
6·50
6·75

7·25
7·50
7·75

8·25
8·50
8·75

Strain: %

Figure 8. Stress–strain results from the UCS tests

8
Environmental Geotechnics Biological soil improvement using new
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northern Iran
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The amounts of calcite precipitation in sand bio-modified using
the selected isolates and S. pasteurii are presented in Figure 9.
Comparing the amount of precipitated calcite, it can be inferred
that UTMC 2623 was more effective for strengthening sand, as it
led to 1·83% calcium carbonate compared with 1·73 and 1·64%
of the two other isolates. The amount of calcite of S. pasteurii
was 2·08%. This higher amount of calcite is in line with the
results obtained from the UCS tests indicating that sand treated
with S. pasteurii had a higher strength.
Effect of MICP-treated sand on permeability
The permeability coefficient (k), which is the average of values
for the three specimens tested for each experimental condition,
was measured for both untreated sands and bio-cemented sands.
Figure 10 illustrates the results of the comparison between
untreated and treated sand specimens employing three isolates and
S. pasteurii. It is observed that the permeability decreased for
each MICP-treated sample, particularly for S. pasteurii. The
permeability of bio-cemented sample using UTMC 2623 was
measured as 0·058 mm/s (87·5% reduction), which is significantly
lower than that of untreated sand and demonstrates a good
performance compared with S. pasteurii. Two other isolates also
resulted in the reduction permeability: 84·2% for UTMC 2622
and 79·5% for UTMC 2624. The loss of permeability is due
to the occupation of soil pore spaces by calcium carbonate
crystals (Mujah et al., 2017). Treated samples with high
UCS (UTMC 2623) yielded a higher reduction in the permeability
coefficient in comparison with the samples with less compressive
resistance.
effect of ten FT cycles on bio-cemented sand samples. After ten
cycles, a reduction in the UCS for the sand treated by each
bacteria was observed. For the sand treated using UTMC 2623,
the compressive strength decreased from 493 kPa to 454 (around
7·91%), while the decrease was from 420 to 398 kPa (5·24%) and
from 386 to 388 kPa (6·05%) for sand treated using UTMC 2622
and UTMC 2624, respectively. The reduction amount for S.
pasteurii was from 509 to 427 kPa (16·11% reduction).
Microscopic observation and XRD analysis
To observe the influence of Bacillus sp. UTMC 2623 bacteria on
the sand microstructure, SEM images are presented in Figure 12.
Figures 12(a) and 12(b) show that the existence of both bacteria
and urea–calcium chloride medium is necessary for gaining
sufficient calcite precipitation, as the number of crystals is almost
zero in these images. In contrast, the formation of calcium
carbonate precipitate is clearly marked in Figures 12(c) and 12(d),
which are images of sand treated with Bacillus sp. UTMC 2623.
It has been proved again that the presence of all culture medium,
urea–calcium chloride solution and bacteria is required to obtain a
desirable amount of precipitation in a short period of time. Figure
12(e) shows the growth of the selected strain, which has a shape
similar to that of other Bacillus subtypes as expected.
0·6
0·464
0·5
0·4
k: mm/s

0·3
Effect of MICP treatment on sand FT durability
Freezing and thawing has led to the destruction of many porous 0·2
materials. The phase change of water adsorbed in the soil pores is
0·095
the most significant cause of deterioration of exposed porous 0·1 0·073 0·058 0·041
materials. This has been of great concern for researchers,
particularly in cold regions, for more than 200 years (Cheng et 0
Bacillus sp. Bacillus sp. Bacillus sp. S. pasteurii Untreated
al., 2013). Porous solids with high porosity have good durability UTMC 2622 UTMC 2623 UTMC 2624 sand
after freezing and thawing. The water content of the porous solid
is the most crucial factor influencing the intensity of mechanical Figure 10. Permeability coefficients of bio-cemented sand samples
damage, although a sand matrix with high permeability and
porosity could also increase the FT strength because it allows
rapid transfer of water (Litvan, 1980). Figure 11 presents the 700

600 493 509

454 427
2·5 2·08 420
500 398
Calcite precipitated: %

1·82 347 326

UCS: kPa

2·0 1·75 1·63 400

1·5 300
1·0 200
0·5 100
0 0
Bacillus sp. Bacillus sp. Bacillus sp. S. pasteurii UTMC 2622 UTMC 2623 UTMC 2624 S. pasteurii
UTMC 2622 UTMC 2623 UTMC 2624 Before After

Figure 9. Calcite precipitated in treated sands Figure 11. UCS of treated sands before and after ten FT cycles

9
Environmental Geotechnics Biological soil improvement using new
environmental bacteria isolated from
northern Iran
Bahmani, Fatehi, Noorzad and Hamedi

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SU3500 15·0 kV ×141 100 µm SU3500 15·0 kV ×66·0 1 mm

(a) (b)

Bacillus sp. UTMC 2623

SU3500 15·0 kV ×360 200 µm SU3500 15·0 kV ×380 200 µm SU3500 14·0 kV ×6050 10 µm

(c) (d) (e)

Figure 12. SEM images: (a) sand treated with bacteria and culture medium; (b) sand treated with culture medium and urea–calcium
chloride solution; (c, d) sand treated with bacteria, culture medium and urea–calcium chloride solution; (e) Bacillus sp. UTMC 2623

XRD analysis
In Figure 13, the results of XRD analysis for the dried deposition
of the isolated bacterium, UTMC 2623, are presented. The XRD
pattern indicates that the phase of calcium carbonate was calcite
(calcite Joint Committee on Powder Diffraction Standards (JCPDS)
card number 05-0586), and all the diffraction peaks of the product
are well indexed to calcite (JCPDS card number 05-0586) and
sodium chloride (JCPDS card number 05-0628). As seen in
Figure 13, there are no pinched shapes in the image because of the
presence of bacterial biomass and other compounds. The peak
obtained for the calcium carbonate precipitation at 2q degrees is 29,
which is highly similar to Harrington’s calcium carbonate peak.
However, because of the presence of impurities, there is about 0·2°
incompatibility with Harrington’s peak (Harrington, 1927). Also,
there is a peak indicating the existence of sodium chloride, which
was present in the culture medium.

Bacillus sp. UTMC 2623

3000
Counts

2000

1000

30 40 50 60
Position (2θ) (Cu): º

Peak list

00-001-0994; NaCl

01-072-1651; CaCO3

Figure 13. The results of XRD analysis of dried deposition of UTMC 2623 bacteria

10
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Table 7. The performance of untreated and treated dune sand
against wind erosion
Sample Soil
Sample type
number loss: %
1 Dune sand 43·00
2 Water-treated sand 36·12
3 Cementation solution + culture medium 32·96
4 Cementation solution + culture medium 1·62
+ S. pasteurii
5 Cementation solution + culture medium 2·24
+ UTMC 2622
6 Cementation solution + culture medium 1·87
+ UTMC 2623
7 Cementation solution + culture medium 1·94
+ UTMC 2624
Wind erosion test
Wind erosion test was performed to gain a better understanding of
the role of the selected isolates in soil erosion prevention. A series
of cementation liquids (according to Table 3) was employed to
stabilise dune sand against wind erosion. The results of this test
are presented in Table 7. As can be seen, samples 1, 2 and 3 were
quite vulnerable to wind erosion, as huge amounts of reduction in
the weight of the samples were observed after 10 min of blowing
wind. By stabilising dune sand through the MICP method using
the selected isolates and S. pasteurii, the wind erosion resistance
increased considerably compared with that of natural, control and
water-treated sand samples. Based on the wind erosion test results
the least amount of soil erosion, among selected isolates, related
to UTMC 2623 by 1·87% soil loss, which is quite comparable
with S. pasteurii by 1·62% soil loss. Wang et al. (2018)
investigated five sand specimens with different treatment cycles in
the aim of measuring the wind erosion resistance of dune sand.
They reported that the wind erosion rate of the water-treated sand
was as high as 10·23%, whereas it was less than 3% in 10 min for
the treated sand in a cycle of MICP treatment (Wang et al., 2018).
The lack of cohesion among sand grains and low shear strength
resulted in a high amount of eroded sand. Eventually, employing
the selected indigenous bacteria could be an effective solution for
preventing sand erosion.
Conclusion
The main objective of this study was to introduce new indigenous
environmental isolates in MICP. After collecting soil samples from
different locations in Iran and isolating the soil bacteria, the best
isolates with the highest urease activity and maximum calcite
precipitation were chosen for further investigation. The Christiansen
medium was employed to separate isolates with the highest urease
activities among 112 samples. In this stage, six bacteria with the
highest urease activities were selected. Finally, the best isolates that
were obtained were UTMC 2622, UTMC 2623 and UTMC 2624.
Bacterial identification was performed based on 16S rRNA
sequencing and comparing with the NCBI database. The observation
through a stereomicroscope obviously indicated calcite crystals near
Biological soil improvement using new
environmental bacteria isolated from
northern Iran
Bahmani, Fatehi, Noorzad and Hamedi
the colony of each bacteria. Having evaluated the optimal thermal
conditions, temperature between 28 and 37°C was the most
appropriate temperature range for growing the selected isolates.
Based on UCS tests and the amount of calcite, UTMC 2623 was
more effective in bio-cementation of sand in comparison with UTMC
2622 and UTMC 2624. Moreover, the UCS values of bio-modified
samples were reduced after 10 FT cycles. In the present research, the
effect of permeability was also studied. As expected, using the MICP
method for soil improvement led to a decrease in the permeability
coefficient of around 84% on average. A simple wind erosion test
was also performed to realise how this selected isolate can improve
the strength of dune sand against wind erosion. Consequently, bio-
cemented sand demonstrated significant improvement in soil erosion.
Microstructural analysis was carried out using SEM images and
XRD to demonstrate how the bacteria affect sand grains. It was
shown that, from SEM images, Bacillus sp. UTMC 2623 has
great potential in calcite precipitation. The XRD analysis was
carried out to help detect the presence of calcite, which proved the
existence of calcium carbonate and sodium chloride crystals.
Employing indigenous bacteria might be considered as an
alternative to using other external bacteria which are usually used
in the MICP process. Also, there might be some issues with
adding exogenous bacteria into the soil due to the adverse
reactions of indigenous bacteria. However, further research is
required to investigate the role of these bacteria in depth and to
optimise various effective factors, such as carbon source and
calcium concentration, as well as the economic aspects of
employing these bacteria in large-scale applications. Additionally,
because the UTMC 2623 isolate showed performance relatively
equal to that of S. pasteurii based on UCS, permeability,
durability and wind erosion tests, it could be used as a substitute
of S. pasteurii in soil treatment, particularly wherever this
bacterium exists.
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