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Ecological Engineering 89 (2016) 49–55

Contents lists available at ScienceDirect

Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Effect of injected bacterial suspension volume and relative density on


carbonate precipitation resulting from microbial treatment
Karim Rowshanbakht a , Mashaallah Khamehchiyan a,∗ , Reza H. Sajedi b ,
Mohammad Reza Nikudel a
a
Department of Engineering Geology, Faculty of Sciences, Tarbiat Modares University, Tehran, Iran
b
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Microbial-induced calcite precipitation (MICP) is a relatively green and sustainable soil improvement
Received 19 June 2015 technique in which by-products of a chemical reaction network managed and controlled by biological
Received in revised form activity alter the engineering properties of the soil. The present study carried out a range of laboratory
20 December 2015
tests to investigate the effect of reducing the injected volume of bacterial suspension and the relative
Accepted 22 January 2016
density (as a soil parameter) on bacterial retention, calcite precipitation, and improvement in the strength
and impermeability of sand specimens. The results show that reducing the volume of injected voids to
Keywords:
up to one third of the pore volume did not significantly affect the improvement performance. This is
Microbial-induced calcite precipitation
Bio-grout
important as it can improve the efficiency of the process and make it economical and more practical for
Biological improvement engineering applications. For similar microbial-induced calcite precipitation conditions and soil type, the
Relative density final strength of the improved columns increased and permeability decreased as the relative density of
the soil increased slightly as precipitated calcite decreased.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction monuments (Dick et al., 2006; Jimenez-Lopez et al., 2008), to


improve compressive strength of concrete (Ramachandran et al.,
Rehabilitation and expansion of civil infrastructures is required 2001; Jonkers et al., 2010), to improve concrete durability (De
to meet ever-growing societal needs, but is directly limited by the Muynck et al., 2008; Achal et al., 2011), for selective plugging at
availability of competent soil upon which they can be constructed enhanced oil recovery sites (Gollapudi et al., 1995; Nemati et al.,
(DeJong et al., 2010). Some products used for treatment of soil 2005), for wastewater treatment (Hammes et al., 2003), to improve
are not considered as environmentally friendly because of pollu- soil (Whiffin et al., 2007; Ivanov and Chu, 2008; DeJong et al.,
tion and toxins produced during manufacturing and application 2010), to increase durability of bricks (Sarda et al., 2009) and as
(Gurbuz et al., 2011). Among the range of treatment techniques, bio-concrete (D’Aquino Henriques, 2011). MICP has the potential
microbial-induced calcite precipitation (MICP) is a relatively green of increasing shear stiffness and decreasing hydraulic conductivity
and sustainable soil improvement technique (Ng et al., 2012). It by harnessing a natural microbiological process that precipitates
refers to a chemical reaction network that is managed and con- calcium carbonate (Martinez et al., 2013).
trolled within the soil through biological activity that produces Microbial urease hydrolyses urea to produce dissolved ammo-
by-products that alter the engineering properties of the soil (DeJong nium and inorganic carbon, and CO2 . The ammonia released into
et al., 2010). the surrounding material increases pH, leading to accumulation
MICP has been used to mitigate engineering problems, such as of insoluble CaCO3 in a calcium rich environment. Quantitatively,
for crack repair in concrete (Ramachandran et al., 2001; Nelson 1 mol of urea hydrolyses intracellularly to 2 mol of ammonium (Eqs.
and Launt, 1991; De Belie and De Muynck, 2009), to reduce per- (1) and (2))
meability (Nemati and Voordouw, 2003), for repair of calcareous
CO(NH2 )2 + 2H2 O →→ 2NH4 + + CO3 2− (1)
2+ 2−
Ca + CO3 →→ CaCO3 (2)

∗ Corresponding author at: Department of Engineering Geology, Faculty of Sci-


These reactions occur under the influence of natural environ-
ences, Tarbiat Modares University, Jalal Ale Ahmad Highway, P.O. Box: 14115-111
mental factors that control the activity of the urease. Subsurface
Tehran, Iran. bacterial populations can be put in place by injection (DeJong
E-mail address: Khamechm@modares.ac.ir (M. Khamehchiyan). et al., 2006) or stimulated by injection of nutrient materials (Fujita

http://dx.doi.org/10.1016/j.ecoleng.2016.01.010
0925-8574/© 2016 Elsevier B.V. All rights reserved.
50 K. Rowshanbakht et al. / Ecological Engineering 89 (2016) 49–55

injected. For example, van Paassen et al. (2010) injected 100 L of


bacterial suspension for a m3 specimen; for a 100 m3 specimen in
which 40 m3 was improved, a mere 5 m3 of bacterial suspension
was injected.
Although many studies exist on relative density (RD), these
studies do not cover the entire improvement process. Different
aspects of the effect of RD on improvement have been evaluated.
Cheng et al. (2014) compared the strength of improved loose and
dense samples in terms of their calcite content and concluded that
at the same calcite content, the looser sample showed less strength.
Chou (2007) studied loose and dense microbially-improved sam-
ple behaviour in California bearing ratio (CBR) and shear tests. They
found that improving the friction angle improved the strength more
in loose samples in shear tests but that dense samples recorded
more strength enhance in CBR tests.
Tsukamoto et al. (2013) used sand samples with RDs of 30%,
Fig. 1. Grain size distribution of material.
65% and 85% to study improvement in parameters during three
axial tests. They showed that more calcite precipitated at lower
et al., 2008). In the present study, augmentation was carried out in RDs, but that strength decreased. Kim et al. (2014) studied the effect
columns with one-dimensional flow. of RD after mixing bacteria and soil without injection. They found
Reagents and catalysts must be injected and transported to that maximum carbonate precipitation in sand occurred at a RD of
the location at which strengthening is required to induce MICP 60% and in silt at 90% compaction. Ng et al. (2012) tested residual
in subsurface soil. The two-phase injection method for bacterial soils having 85%, 90% and 95% compaction by mixing them with
retainment suggested by Whiffin et al. (2007) was used in the bacteria and injection of cementation fluid. They found that shear
current study. This process for homogeneous immobilization of strength increased, but permeability decreased, as relative density
bacteria first injects the bacterial suspension and then injects a increased.
fixation fluid (a solution with high salinity). This approach has It is evident that none of the numerous studies in the litera-
been frequently used (e.g., Harkes et al., 2010; Whiffin et al., 2007; ture consider the effect of relative density in a complete process.
Kaltenbacher et al., 2014; Martinez et al., 2013; van Wijngaarden Scatter can be observed in the results as an effect of soil properties
et al., 2012; Sarmast et al., 2014; van Paassen et al., 2010). or improvement conditions. The present study evaluated the effect
The use of a high injection volume is not desirable for grouting. of relative density after complete injection and improvement on
A disadvantage of this approach is the injection of 2 pore volumes bacterial fixation, calcite precipitation, strength, and permeability.
(PV) before injection of the main cementation liquid (Harkes et al., The effect of decreased bacterial suspension at different relative
2010). The volume of injection is not very important for laboratory densities is investigated.
research; thus, many laboratory studies use high-volume injection.
In most cases, 1 PV or more of bacterial suspension is injected and
a high volume of cementatious material (up to 30-fold) is flushed 2. Materials and methods
(Harkes et al., 2010; Whiffin et al., 2007; Shirakawa et al., 2011;
Tagliaferri et al., 2011; Tsukamoto et al., 2013; Mortensen et al., The present study investigated the effect of improvement of soil
2011; Ng et al., 2012). For improvement of larger bodies, there at different RD using void injection experiments on soil samples in
are restrictions that decrease the volume of bacterial suspension a 20-cm column.

Fig. 2. Columns: (a) during treatment; (b) improved; (c) setup schematic.
K. Rowshanbakht et al. / Ecological Engineering 89 (2016) 49–55 51

Table 1 constant at approximately 200 mL/h. At each injection step, micro-


Physical properties of material.
bial activity was monitored indirectly by measuring the ammonium
Soil class Cc Cu D50 (mm) emin emax Gs concentration in the effluent. The columns were finally rinsed with
SP 0.83 1.46 0.2 0.89 0.59 2.65 distilled water of about 1.5 PV and stored in the saturated condition
until the onset of the experiments. The result presented here are
the averages of three independent test series.
2.1. Characteristics of materials used for treatment
2.5. Monitoring methods and controlling tests
The soil type used in the experiments was determined using the
Unified Soil Classification System according to ASTM D2487-11. The
The biological, chemical, physical, and mechanical properties
soil type was poorly graded sandy silica with negligible calcite. Its
essential to performance of the MICP were measured during and
grain size distribution was determined according to ASTM D421-87
after treatment.
and D422-63, specific gravity according to ASTM D854-10, mini-
mum density according to ASTM D4254-00, and maximum density
according to ASTM D4253-00. Fig. 1 shows the grain size distribu- 2.5.1. Optical density
tion curve and Table 1 lists the physical properties of the soil. The Optical density (OD) was used as a biomass concentration
samples were prepared with RDs of 40%, 70% and 85%. indicator. OD was measured using a spectrophotometer at a wave-
length of 600 nm. Samples with high bacterial concentrations were
2.2. Microorganisms and culture medium diluted to an OD value of 0.2–0.8, where the relationship between
absorbance and particle concentration became linear (Harkes et al.,
Ureolytic bacteria Sporosarcina pasteurii (formerly Bacillus pas- 2010; Van Paassen, 2009). These values were multiplied by the
teurii) have generated much interest in this area (Whiffin et al., dilution factor to obtain the undiluted value.
2007; Ivanov and Chu, 2008; DeJong et al., 2006) and were pro-
vided by the Persian Type Culture Collection (PTCC1645). Different 2.5.2. Urease activity
mediums and conditions were tested to find the most suitable cul- Urease activity was measured immediately after sampling and
ture medium. The medium selected was tested with and without was determined using the conductivity method. Next, 1 mL of
NiCl2 and it was found that NiCl2 strongly increased the growth bacterial suspension was added to 9 mL of 1.11 M urea (reaction
rate. The organism was cultivated under aerobic batch conditions concentration = 1 M urea) and changes in the relative conductivity
in a medium containing 20 g/L yeast extract, 10 g/L NH4 Cl, and were recorded over 10 min at 20 ± 2 ◦ C. The urease activity was then
10 ␮mol/L NiCl2 at a pH of 8.5. The bacteria were first cultivated calculated taking dilution into account.
in the medium as a preculture to the growth phase and were sub-
sequently transferred aseptically to fresh medium. Growth was 2.5.3. Ammonium concentration
continued for about 18 h to ensure survival of the bacteria for Ammonium concentration was determined using the modified
improvement. The bacteria was harvested at the end of growth Nessler method (Greenberg et al., 1992). Using a volumetric flask,
phase (optical density of >2 at 600 nm) and stored at 4 ◦ C for 48 h the sample was diluted with deionized water to 0–0.5 mM. The 2 mL
prior to use. sample was mixed with 100 ␮L of Nessler reagent and allowed to
react for exactly 1 min. The sample was then read in a spectropho-
2.3. Column preparation tometer at 425 nm. Absorbance readings were calibrated using
several NH4 Cl standards measured under the same conditions.
Samples were prepared in columns 36 mm in diameter and
20 cm in height by dry tamping at three RDs (40%, 70%, and 85%). To
achieve a certain RD, the weight of the dry sand required to achieve 2.5.4. Tests on improved samples
the desired RD was calculated for emax , emin and Gs and the samples The constant head permeability test was used to determine the
were placed in columns by dry tamping. Each end of the column was permeability of samples before opening the columns. The head was
fitted with a scouring pad (Scotch-Brite) and approximately 1 cm about 100 cm.
of filter gravel was layered to the inside of the pad, next to the sand Two samples, one from the base of the improved column (near
(Fig. 2). The column was positioned vertically with an upward flow the injection point) and another from the top were prepared to
direction to avoid settling of the packing material and generation test for unconfined compressive strength (UCS). The samples were
of preferential flow paths. An upward flow direction saturated the prepared with a length-to-diameter ratio of about 2. The length of
columns during the test. each sample was about 7 cm and was capped with gypsum. The
final length was 8 cm. Tests were performed according to standard
ASTM D2166 methods.
2.4. Treatment procedure
The precipitated calcite in columns measured by Bernard cal-
cimetry. In this method, the percentage of calcite was calculated
Samples were prepared at RDs of 40%, 70%, and 85%. To inves-
based on the amount of gas released during the reaction of the
tigate the effect of reducing the void injection percentage (VIP)
calcite with dilute hydrochloric acid.
on improvement, three columns were prepared for each RD. The
Scanning electron microscopy (SEM) was performed on the sam-
columns saturated with autoclaved deionized water before treat-
ples prepared from the base and top of the improved columns.
ment. A three-phase injection scheme using peristaltic pumps was
X-ray fluorescence spectroscopy (XRF) was used to determine the
used (Harkes et al., 2010).
composition of precipitated material.
In the first phase, at each RD, bacterial suspension was
introduced at volumes equal to 1, 2/3, and 1/3 PV. This was imme-
diately followed by injection of 1 PV of fixation fluid (50 mM CaCl2 ) 3. Results and discussion
for each column. About 4 h after injection of the fixation fluid, the
cementation fluid containing 1 M equimolar CaCl2 and urea was Microbial improvement was determined based on its effect on
flushed through. All samples were injected with cementation fluid the biological and engineering parameters under different experi-
three times every 24 h. During all flushes, the flow rate was kept mental conditions.
52 K. Rowshanbakht et al. / Ecological Engineering 89 (2016) 49–55

Fig. 3. Efficiency of bacterial suspension fixation vs. RD and VIP: (a) at OD 600; (b) at urease activity.

Fig. 4. Bacterial concentration remaining in situ in column vs. RD and VIP: (a) at OD 600; (b) at urease activity.

3.1. Efficiency of bacterial suspension injection 3.3. Ammonium concentration

To evaluate the efficiency of the bacterial suspension injection, Changes in the ammonium concentration were investigated for
the OD and urease activity of the effluent were normalized (accord- each cementation fluid injection phase. Changes in the ammonium
ing to volume) and are represented as the percentage of initial value concentration at different densities and VIPs were analyzed, but a
pre-injection in Fig. 3. Both the OD and urease activity showed that significant relation was not found. The average ammonium concen-
increasing the VIP by bacterial suspension, decreased the efficiency tration at each injection steps is compared in Fig. 5. It is evident that
of bacterial retention significantly, although they slightly increased after the second injection stage, the ammonium concentration had
as the RD increased (Fig. 3(a) and (b)). It is clear that increasing the decreased significantly. The results of stoichiometry calculations
injection volume increased the loss of bacteria and urease activity indicate that a major portion of the urea injected in the first and
from the column. At higher RDs, increased compaction, a decrease second step participated in the reaction (85–95%). After the third
in porosity, pore volume, and pore throat size, and filtering of bacte- injection, this decreased to about 25%.
ria between the soil grains increased slightly.
3.4. Uniaxial compressive strength

3.2. Bacteria remaining and enzyme activity in column UCS tests were performed on specimens from the base and top
of each column and the results were analyzed. For both specimen
At the same VIP, the initial net volumes of injection differed for
different soil RDs because of the different volumes of voids. As a 40
basis for comparison, the in situ remaining bacterial concentration
and urease activity in each column was measured. These values
Ammonium concentration (g/L)

30
were then normalized by total injection volume. After normaliza-
tion, the values for the effluent are subtracted from the initial values
(pre-injection) to determine the properties of the retained bacte- 20
rial suspension. Overall, increases in VIP and RD (Fig. 4), increased
bacterial retention, but was not important at values above 2/3 PV. 10
The OD in Fig. 4(a) shows maximum values at 2/3 PV and a RD of
85%. Urease activity increased as the VIP increased, but an injection
0
greater than 2/3 PV had no significant effect on retention. Increas-
1st Inj. 2nd Inj. 3rd Inj.
ing the RD slightly increased the urease activity and OD of the
Injection Steps
remaining fluid (Fig. 4(b) and (d)). Increased adsorption was the
result of smaller voids and throats making filtration easier. Fig. 5. Average ammonium concentration at each injection step.
K. Rowshanbakht et al. / Ecological Engineering 89 (2016) 49–55 53

Fig. 6. Effect of VIP and RD on: (a, b) UCS; (c) permeability.

types, an overall increase in average ultimate strength of the sam- the uncompacted samples and CaCO3 crystals formed over a shorter
ples with an increase in the RD was evident (Fig. 6(a)). The increase distance to bridge the sand particles. These results are in agreement
in strength with the increase in RD was obtained by decreasing the with those of Tsukamoto et al. (2013) and Cheng et al. (2014).
calcite content under the same conditions (Fig. 7(a)). The sand par- The average ultimate strength of samples decreased as the
ticles in the compacted sand columns were closer together than in VIP increased (Fig. 6(b)). The ultimate strength of improved

600

400
UCS (kPa)

200

y = 55.824x - 93.211
R² = 0.2126
0
4 5 6 7 8 9
Calcite Content (%)
(a)
8 8
Specimen from base Specimen from base
Specimen from top Specimen from top
Calcite content (%)

Calcite content (%)

6 6

4 4

2 2

0 0
40% 70% 85% 33% 66% 100%
RD (%) IPV

(b) (c)
Fig. 7. (a) Calcite content vs. UCS; effect of RD (b) and VIP (c) on calcite content.
54 K. Rowshanbakht et al. / Ecological Engineering 89 (2016) 49–55

samples varies from 100 to 500 kPa (Fig. 7a). Maximum strength 3.6. Calcite content
was recorded in samples having a RD of 85%; one sample from
the base of the 1/3 PV injection column and one from the top of Small pieces of the specimens extracted near the entrance, mid-
the 2/3 PV injection column. It was observed that the samples dle, and outlet of the columns were prepared and their calcite
prepared from the top of the columns showed greater strength content determined. The results indicated local irregularities in cal-
than those prepared from the bottom, which were nearer the cite distribution, possibly related to sample size. To account for
injection point. This was true for the majority of samples; how- possible local irregularities, after testing the UCS, the entire sam-
ever, the calcite content of the samples from the base of the ple was powdered and a small portion used for calcimetery and the
column was greater than from those at the top. This could be results used for analysis.
in response to the more homogenous distribution of calcite at The calcite content was first plotted versus UCS as shown in
the top of the column. It appears that calcite forms simulta- Fig. 7(a). The calcite percentage varied from 4.5% to 8%. Although
neously during the injection of cementation fluid. Some of this an increasing trend was recorded for UCS, the R2 value (0.21) was
calcite moves with the pore fluid flow and precipitates away from not favourable. This was caused by the difference in RD, inhomo-
the bottom, is distributed more homogenously and, as a result geneous calcite distribution, and its position in the voids (covering
of filtration, greater precipitation in pore throats increase the the grains or precipitation on contact). The relation between pre-
strength. cipitated calcite and RD was obtained for samples prepared from
UCS test specimens by plotting the precipitated calcite at different
RDs (Fig. 7(b)). The results confirmed that the lower the RD, the
greater the precipitation of CaCO3 , which is in agreement with the
3.5. Permeability tests findings of Tsukamoto et al. (2013). This occurred because the low
RD increased the number of microbes and nutrients absorbed by
The results of the permeability tests are presented in Fig. 6(c) the test specimen as a result of the higher void volume.
and (d). Permeability decreased as RD and VIP increased (Fig. 6(c)). This relation was also be seen for VIP (Fig. 7(c)). In general, the
As RD increased, PV decreased and the pore throats become calcite content at the base of the column was greater than at the
smaller and less permeable. The relation between density and per- top. As the other parameters were held constant, this could be the
meability is in agreement with the results of Ng et al. (2012); result of a greater local concentration of bacteria in proximity to
however, some discrepancy was seen at an RD of 85%. This the injection point at the base of the column.
occurred as the pore throats became smaller, making it more
likely for clogging to occurs; this more easily creates preferen- 3.7. Scanning electron microscopy
tial flow paths during bacterial suspension and cementation fluid
injection. The decrease in permeability with the increasing in The results of SEM and XRF in Fig. 8 show calcite precipitation.
VIP could be the result of more local concentration of bacte- The results in Fig. 8(b) show the silica grains and calcite cement.
ria near the injection point, prompting increased local calcite This image demonstrates the increased tendency for precipitation
precipitation. at particles contacts (Fig. 8(a)). Calcite precipitation was compared

Fig. 8. (a) SEM of calcite concentration at grain contact; (b) XRF analysis; (c), base of column at RD = 40% and VIP = 66; (d) at top.
K. Rowshanbakht et al. / Ecological Engineering 89 (2016) 49–55 55

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