Construction and Building Materials 183 (2018) 202–214

Contents lists available at ScienceDirect

Construction and Building Materials

journal homepage: www.elsevier.com/locate/conbuildmat

Investigation into the optimal bacterial concentration for compressive

strength enhancement of microbial concrete
Sandip Mondal, Aparna (Dey) Ghosh ⇑
Department of Civil Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah 711103, West Bengal, India

h i g h l i g h t s

Significant calcite precipitation obtained without external calcium source.

Optimum compressive strength enhancement at intermediate cell concentration.

Crack healing and water absorption most efficient at highest cell concentration.

Reasons for the existence of the optimum concentrations provided.

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

Article history: The efficient crack healing capability of microbial concrete leads to an improvement in its various
Received 12 February 2018 mechanical properties such as compressive strength, water absorption and water permeability. Studies
Received in revised form 18 June 2018 on microbial concrete have reported that the enhancement of the compressive strength is maximum
Accepted 21 June 2018
at a particular bacterial concentration, which is not necessarily the highest amongst the considered levels
Available online 26 June 2018
of bacterial concentrations. So far, the reason for the existence of such an optimal bacterial concentration
for the increase in the compressive strength of concrete remains unexplored. In this paper, an attempt has
Keywords:
been made to establish the cause of the presence of this optimal bacterial concentration. Three different
Microbial concrete
Optimal concentration
bacterial concentrations of Bacillus subtilis have been used in this study, namely 103 cells/ml, 105 cells/ml
Compressive strength and 107 cells/ml of water. Results indicate that though the higher bacterial concentration of 107 cells/ml
Crack healing is more efficient for crack healing, the best performance in compressive strength enhancement is
Precipitation pattern achieved with the bacterial concentration of 105 cells/ml. It is seen that for a given bacterial type and
mortar mix, the different calcite precipitation patterns inside the mortar matrix at varying levels of bac-
terial concentrations constitute the reason for the existence of the optimal bacterial concentration for
compressive strength enhancement.
Ó 2018 Elsevier Ltd. All rights reserved.

1. Introduction Since healing of micro-cracks enhances the compressive

strength and other mechanical properties of concrete, some
The prime strategy of microbial concrete relies on microbial researchers have also been investigating the effect of the addition
induced calcite precipitation at the cracks, thereby precluding the of bacteria on the compressive strength of concrete. Generally,
deterioration of concrete, as well as of reinforcement, due to strength enhancement has been reported [13–16], though reduc-
ingression of harmful substances like chloride, sulphate, moisture, tion in strength has also been obtained [17]. A review of these
etc. Amongst the various aspects of microbial concrete, the thrust studies indicates that researchers who have dealt with strength
of research so far has chiefly been on the types of bacteria used enhancement have considered bacterial concentrations in the
for crack healing in concrete [1–7], on the survivability of bacteria range of 103–107 cells/ml, whereas researchers who have focused
inside the concrete matrix [8–10] and on the compatibility of bac- more on crack healing usually have used higher bacterial cell con-
teria with different types of cement replacing material in concrete, centrations (107–109 cells/ml). Various studies have also reported
namely flyash, cement kiln dust etc. [11,12]. the existence of an optimum bacterial concentration for maximiz-
ing the compressive strength of microbial concrete. Though it is
evident that higher concentration of bacteria leads to higher calcite
⇑ Corresponding author. precipitation [18], it is seen that the optimum concentration for
E-mail address: [email protected] (A.(Dey) Ghosh). compressive strength enhancement is not necessarily the highest

https://doi.org/10.1016/j.conbuildmat.2018.06.176
0950-0618/Ó 2018 Elsevier Ltd. All rights reserved.
 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214 203

considered cell concentration. Ghosh et al. [13] investigated seven Microscopy (SEM) is employed to examine the calcite precipitation
different bacterial concentrations of the Shewanella species at the inner concrete matrix. Finally, based on the results, a sche-
(10–107 cells/ml) and obtained the optimal bacterial concentration matic diagram of calcite precipitation patterns at different bacte-
to be 105 cells/ml, with a corresponding 25% increase in compres- rial concentrations inside the mortar matrix is provided to
sive strength. Chahal et al. [16] used three bacterial concentrations explain the existence of the optimal bacterial concentration for
(103, 105 and 107 cells/ml) and found that the maximum increment compressive strength enhancement.
of 22% in compressive strength of flyash concrete was also attained
at the cell concentration of 105 cells/ml of Sporoscarcina pasteurii.
2. Materials and test methods
Sarkar et al. [19] showed that even for the genetically modified
Bacillus subtilis, the maximum compressive strength was achieved 2.1. Materials
at the cell concentration of 105 cells/ml. Further, Andalib et al. [14]
used five cell concentrations (viz. 10  105–50  105 cfu/ml) of Bacillus Subtilis (MTCC 441) obtained from the Microbial Type Culture Collec-
tion and Gene Bank, India, is used in this study. The culture was grown in a nutrient
Bacillus magaterium and reported that the optimum concentration broth made with Beef Extract 1.0 gm/l, Yeast Extract 2.0 gm/l, Peptone 5.0 gm/l,
for strength enhancement was at 30  105 cfu/ml. Thus in all these NaCl 5.0 gm/l and distilled water (pH = 7.0). After 5–6 days of inoculation, about
works, the maximum compressive strength enhancement was 10 ll from the culture medium was taken on a Haemocytometer and counted under
attained at an intermediate value of the range of considered bacte- the microscope. This was followed by serial dilution to obtain the required bacterial
concentrations. The live bacterial cells obtained from the pre-culture were added to
rial concentrations. Apart from the above, in a single instance as
water at different cell concentrations, namely 103 cells/ml, 105 cells/ml and
reported in literature, the optimum concentration for compressive 107 cells/ml.
strength enhancement was obtained at the highest considered A total of 123 mortar samples were prepared by using Ordinary Portland
level of bacterial cell concentration. Kumari et al. [20] used three cement (OPC) of grade 43 and locally available river sand. Mortar cubes of dimen-
different cell concentrations (105, 105 and 107 cfu/ml) of Bacillus sion 70.6 mm  70.6 mm  70.6 mm were prepared for both control and bacterial
mortar samples. The cement to sand ratio was taken as 1:3 (by weight) and water
conhii and found that the maximum strength enhancement of to cement ratio was fixed at 0.4 (by weight). The samples were removed from the
49.18% occurred at the bacterial cell concentration of 107 cells/ml. moulds after 24 h and cured at room temperature (27 °C) in fresh water.
Several researchers who have focused on crack healing with a
single high bacterial concentration have also presented results on 2.2. Compressive strength test
the compressive strength of the microbial concrete samples. Khaliq
et al. [5] reported a 12% increase in the compressive strength by The compressive strength tests of the control and the bacterial mortar cubes
using lightweight aggregate with Bacillus subtilis at bacterial cell were performed at the age of 3, 7 and 28 days of curing, in a 2000 kN capacity com-
pression testing machine.
concentration of 3  108 cells/cm3, while Jonkers et al. [17] stated
a 10% decrease in the compressive strength at 6  108 cells/cm3
of bacterial spores. These results indicate that high cell concentra- 2.3. Water absorption test
tions have a negative impact on the enhancement of compressive
For water absorption test, initially the mortar cubes were oven dried at 105 °C
strength in microbial concrete. for 24 h and the dry weight measured ð¼ Woven dried Þ. The samples were then kept in
It is thus observed that the optimum bacterial concentration for a saturated condition in water at room temperature for 24 h and weighed again
the increase in compressive strength lies between 105–107 cells/ml ð¼ Wsaturated Þ. The water absorption was then calculated by using the following
for all considered bacteria, whereas enhanced crack healing occurs formula.
at higher bacterial cell concentrations of 108–109 cells/ml. Further, Wsaturated  Woven dried
the rate of calcite precipitation is dependent upon the type of bac- water absorptionð%Þ ¼  100 ð1Þ
Woven dried
teria and the concentration of the bacteria. However, the reason for
The water absorption tests of the control and the bacterial mortar samples were
the reduction of compressive strength at higher bacterial concen- performed after 3, 7 and 28 days of curing.
trations is not currently understood. It is true that the main pur-
pose of microbial concrete is crack healing, but it should be
2.4. Self-healing study at mortar surfaces
without adversely affecting the compressive strength. Therefore,
it is necessary to identify the reason for the existence of the opti- 2.4.1. Crack healing
mal concentration for compressive strength enhancement of To evaluate the self-healing efficiency at different bacterial concentrations,
microbial concrete, which can lead to an appropriate selection of crack healing in microbial concrete was analysed. At 28 days of curing, the mortar
samples were loaded on the compression testing machine. When visible cracks
bacterial concentration as per the requirement. An external cal-
appeared on the surface, the loading was stopped. Thereafter, the widths of the
cium source like calcium lactate, calcium oxide, calcium glutamate cracks were measured by the crack-measuring instrument. The widths of the cracks
etc. is often used to enhance the self-healing efficiency of microbial varied from 0.1 mm to 1.2 mm. Next, the cracked samples were submerged in
concrete [17,21,22]. However, various researchers have also water. For crack healing quantification, the crack widths were measured after 3, 7
and 28 days of curing. Further, Scanning Electron Microscopy (SEM) and Energy Dis-
obtained improvement in the mechanical properties of concrete
persive Spectroscopy (EDS) were carried out to examine the shape and morphology
by the addition of bacteria alone [13,23]. of the healing product. Additionally, X-ray Diffraction (XRD), with a Cu anode (40
The aim of the current study is to explore the reasons behind kV and 40 mA) and scanning from 10° to 80° (2h) at the rate of 0.02° s1 at room
the existence of an optimum bacterial concentration for the temperature, was employed to analyse the chemical composition of the healing
enhancement of compressive strength of microbial concrete. To product.

avoid the influence of any external effects on the precipitation of

bacteria as well as on the mechanical properties of concrete, no 2.4.2. Surface pore-healing
external calcium source has been used here. In this study, firstly, The effect of different bacterial concentrations on the calcite precipitation rate
on the concrete surface was evaluated by analysing the healing of the surface pores
the effect of three different bacterial concentrations on the com-
of the mortar samples. This was carried out by capturing the digital surface images
pressive strength and water absorption of mortar samples is exam- of the samples immediately after demoulding and again after submerging the sam-
ined. This is followed by an investigation of the calcite ples in water for 7 days and 28 days. The images were analysed by pixel analysis in
precipitation on the mortar cube surface, at different bacterial cell Matlab version R2015b. All pixels were represented by Red, Green and Blue (RGB)
concentrations, through crack healing analysis and surface pore- components where the values of the intensities range from 0 to 255. Thus, point
(0, 0, 0) stands for black and (255, 255, 255) stands for white. By analysing the pix-
healing analysis. Next, a water penetration test is performed to els in and around a pore as shown in the Fig. 1, it was observed that the values of the
comprehend the effect of bacterial concentration on moisture RGB components in the pore area ranges from (0, 0, 0) to (120, 120, 120). The latter
transport through the mortar cubes. Thereafter, Scanning Electron set of values was fixed after carrying out pixel analysis of a large number of surface
204 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214

Fig. 1. Magnified view of a surface pore with the corresponding RGB values of the pixels.

pores. In an attempt to quantify the total area of pores on the face of the mortar
cube, a ratio termed as the pore ratio, expressed as a percentage, is defined as
follows.

Pixels having RGB value from ð0; 0; 0Þ to ð120; 120; 120Þ

Pore ratioð%Þ ¼  100
Pixels having RGB value from ð0; 0; 0Þ to ð255; 255; 255Þ
ð2Þ

2.5. Water penetration depth test

To evaluate the effect of different bacterial concentrations on the property of

moisture transport within the mortar matrix, water penetration depths of the sam-
ples were measured. A small quantity of lac-dye was mixed with the water to make
coloured water that helped to visualise the water penetration depth. After 3, 7 and
28 days of curing the samples were oven-dried at 105 °C for 24 h. Subsequently, the
samples were submerged in coloured water for 30, 60 and 90 min. A constant water
head of 2 cm was maintained for all the samples. Next, the samples were split into Fig. 2. Compressive strength test results for control and microbial mortar.
two parts from the middle and the penetration depth was measured. The depth was
measured by the pixel colour analysis at different points on four sides and the aver-
age of all the values was taken as the water penetration depth of that sample. Fur-
ther, the weight of the absorbed water was calculated to corroborate the water
penetration depth results.
reduction at 107 cells/ml. The maximum enhancement in strength
is achieved at the cell concentration of 105 cells/ml at all ages.
The increase in compressive strength is measured by the difference
3. Test results in the compressive strength of microbial mortar and control mortar
w.r.t. that of control mortar. At 28 days of curing, the compressive
3.1. Compressive strength test results strength is increased by 15%, 27% and 19% for cell concentrations
103 cells/ml, 105 cells/ml and 107 cells/ml respectively, in compar-
The 3, 7 and 28 days compressive strength of the control and ison to the control specimen.
microbial mortar specimens are presented in Fig. 2. The results indi-
cate that the strength is increased by the incorporation of bacteria
in all the considered concentrations at all ages. The improvement in 3.2. Water absorption test results
compressive strength is due to the filling of pores inside the mortar
matrix by calcite precipitation by the bacteria. It is observed that The water absorption of the control and microbial mortars at
the compressive strength of mortar samples increases with increase 3, 7 and 28 days are indicated in Fig. 3. It is clear that the presence
in bacterial concentration up to 105 cells/ml and then there is a of bacteria in the mortar mix reduces the water absorption at all
 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214 205

3.3. Surface precipitation analysis of the mortar cubes

3.3.1. Crack healing analysis

The images of surface crack healing by the different bacterial
concentrations at different ages are presented in Fig. 4. It can be
clearly observed that the visible cracks on the surface of the mortar
cubes are healed by white precipitations. The healing efficiency of
the different bacterial concentrations is different (see Fig. 5). After

Fig. 3. Water absorption test results for control and microbial mortar.

the studied concentrations and ages. Moreover, the water

absorption decreases with increase in bacterial concentration,
thereby the maximum reduction is obtained at cell concentration
107 cells/ml at all ages of curing. The reduction in water absorption
is measured by the difference in the water absorption of microbial
mortar and control mortar w.r.t. that of control mortar. At 28 days
of curing, the water absorption is reduced by 13%, 23% and 27% for
cell concentrations 103 cells/ml, 105 cells/ml and 107 cells/ml Fig. 5. Rate of crack healing w.r.t. initial crack width at different bacterial
respectively, in comparison to the control specimen. concentrations.

Fig. 4. Progress of crack healing in microbial concrete at different bacterial concentrations.

206 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214

crack width up to 0.6 mm and 0.9 mm can be healed with the bacte-
rial concentration 103 cells/ml and 105 cells/ml respectively,
whereas by using 107 cells/ml the crack width is healed up to 1.2
mm. These results indicate that higher bacterial cell concentrations
lead to higher precipitation, thus the cracks healed at a faster rate at
107 cells/ml. However, it is perceived that the rate of crack healing
with time is decreased (see Fig. 5). At the initial stage, the healing
rate is faster at higher cell concentrations. Thereafter, the rate
decreases at higher cell concentration as compared to the lower cell
concentrations. This is due to the lower availability of space at the
crack portion that decelerates the growth of the calcite crystal due
to the enhanced precipitation that has already taken place at the ini-
tial stage. The precipitation patterns at a surface crack for different
Fig. 6. Calcite precipitation pattern at different bacterial concentrations inside the bacterial concentrations are visualised in Fig. 6.
crack (a) 103 cells/ml, (b) 105 cells/ml, (c) 107 cells/ml. Next, the results of SEM and EDS and XRD are presented. Fig. 7a
shows the SEM images of the morphology of the white precipita-
3 days of incubation, the crack width is almost completely healed at tion at the cracks. The element composition of the precipitation
107 cells/ml concentration. After 3 days of curing, the cracks are is obtained from the EDS analysis (see Fig. 7b) which indicate that
healed by 70% and 30% respectively at 105 cells/ml and 103 cells/ the precipitation is mainly composed of three elements namely,
ml. Further, after 7 days, the cracks are healed by 85% and 50% at Calcium (Ca), Carbon (C) and Oxygen (O). Again, XRD analysis of
105 cells/ml and 103 cells/ml respectively and after 28 days, the the precipitation is presented in Fig. 8, which shows increased
cracks are healed by 90% and 60% at 105 cells/ml and 103 cells/ml intensity at 2 theta (2h) values of 29.3°, 35.7° and 47.3° confirming
respectively. It is further noted that after 28 days, the maximum that the precipitation is calcite.

Fig. 7. Microstructure analysis of the precipitation at the cracks by Bacillus subtilis (a) SEM images, (b) EDS.

Fig. 8. XRD analysis of the precipitation.

 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214 207

3.3.2. Pore-healing analysis pixel ratio from 1 day to 7 days is 63% and from 7 days to 28 days
Fig. 9 presents the images of the mortar cube surfaces with dif- is 97%. This indicates that the surface pores are almost completely
ferent bacterial concentration at different ages. It is clearly visible filled up at this cell concentration.
that surface pores on the microbial mortar samples have filled up
at a faster rate as compared to the control samples. Higher cell con- 3.4. Water penetration depth test results
centrations have accelerated the rate of filling of pores. Thus, the
maximum pore filling is achieved at cell concentration 107 cells/ The photographs of the control and microbial mortar cross-
ml, which is also reflected from the pixel analysis (Table 1). It is sections at 30, 60 and 90 min taken at 3 days, 7 days and 28 days
noted that for bacterial concentration 107 cells/ml, the change in of curing are given in Figs. 10–12 respectively. The corresponding

Fig. 9. Progress of surface pore-healing in control and microbial concrete at different bacterial concentrations.

Table 1
Surface image pixel ratio for controlled and microbial mortar.

Sample Pixel ratio Change in pixel ratio (%)

1 day 7 days 28 days 1–7 days 7–28 days
Control 27.88 23.47 16.88 15.81 28.04
103 cells/ml 15.37 12.25 5.62 20.30 54.07
105 cells/ml 12.65 7.95 3.05 37.11 61.59
107 cells/ml 10.43 3.82 0.29 63.36 92.24
208 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214

Fig. 10. Water penetration depth at 3 days of curing.

values of average water penetration depths and weights of the Further, to understand the effect of bacterial precipitation on
samples are plotted in Fig. 13 and in Fig. 14 respectively. The the penetration of water inside the concrete matrix, the rates of
results indicate that microbial mortar samples have less water water penetration (see Fig. 13) and rates of increase in absorbed
penetration as compared to the control samples. Further, higher weight of water (see Fig. 14) are analysed at intervals of 30 min.
bacterial concentration leads to lower penetration. The same trend It is observed that at the initial 30 min of testing, the water pene-
of penetration is exhibited at all ages of testing. The reduction in tration velocity and the rates of the absorbed weight of water are
penetration depth by increasing bacterial concentration reveals decreased with the increase in bacterial concentration at all the
that the presence of greater number of bacterial cells leads to more testing ages. It is also noticed that the differences in the water pen-
amount of calcite precipitation and thus the total pore area is etration velocity and the rates of the absorbed weight of water for
reduced at the surface region. Thus, at cell concentration the different bacterial concentrations are maximum at the initial
107 cells/ml, the maximum reduction in penetration depth and 30 min of testing. This implies that at the initial age, greater
water absorption are achieved as compared to the other bacterial precipitation occurs at the surface region. At 3 days, in between
concentrations. At 28 days of curing, after 90 min of submergence, 30 min to 60 min of submergence, the water penetration velocity
as compared to the control specimen, the water penetration depth and the rate of the absorbed weight of water for the different
is reduced by 23%, 30% and 53% for cell concentrations 103 cells/ml, bacterial concentrations are almost identical. At 7 days and
105 cells/ml and 107 cells/ml respectively. 28 days, however, the rates decrease with increase in bacterial
 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214 209

Fig. 11. Water penetration depth at 7 days of curing.

concentration. This indicates that higher precipitation due to centrations. To this end, SEM was carried out on the mortar sam-
increase in bacterial concentration blocks the surface pores. ples at two different depths, 5 mm and 20 mm from the concrete
surface (see Fig. 15). Fig. 15(a)–(d) contain the SEM images at 5
4. Discussion mm depth; Fig. 15(a) is that of the controlled sample while
Fig. 15(b)–(d) are those of the microbial samples at bacterial
The results in the foregoing section indicate that while the max- concentrations of 103, 105 and 107 cells/ml respectively.
imum reduction in water absorption and maximum surface crack Fig. 15(a)–(d) clearly exhibit the lack of precipitation in the case
and pore healing take place at 107 cells/ml of bacterial concentra- of the controlled sample and an increase in calcite precipitation
tion, the maximum enhancement in compressive strength of the with higher bacterial concentration at the surface region of the
microbial concrete is obtained at 105 cells/ml. Further, the water microbial concrete samples, corroborating the earlier test results
penetration depth test also exhibits that after 30, 60 and 90 min in Section 3.3. Next, the images taken from the inner concrete
of incubation, the maximum reduction in water penetration is at matrix (at 20 mm depth) are examined. Fig. 15(e) is that of the con-
the bacterial concentration of 107 cells/ml. trolled sample while Fig. 15(f)–(h) are those of the microbial sam-
An explanation for the optimal cell concentration for compres- ples at bacterial concentrations of 103, 105 and 107 cells/ml
sive strength enhancement occurring at 105 cells/ml may be respectively. It is interesting to note that as compared to both
arrived at by examining the calcite precipitation at the surface 103 cells/ml as well as 107 cells/ml, greater precipitation has taken
and at the inner matrix of the mortar cubes at different cell con- place in the inner matrix at 105 cells/ml.
210 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214

Fig. 12. Water penetration depth at 28 days of curing.

Thus, it can be inferred that adding different bacterial concen- the mortar matrix for 105 cells/ml as compared to that for
trations leads to two different types of healing in concrete, namely, 107 cells/ml. Thus the overall filling of pores in the mortar matrix
surface healing and inner matrix healing. At the surface region, as is more in case of 105 cells/ml concentration than 107 cells/ml
the availability of water is equal for all the samples, the precipita- concentration, leading to maximum compressive strength
tion is only dependent upon the bacterial concentration. Since enhancement at 105 cells/ml concentration. At the low concentra-
greater number of bacterial cells can precipitate higher amount tion of 103 cells/ml, though precipitation takes place throughout
of calcite, the maximum amount of precipitation at the surface the mortar matrix, it is not sufficient to fill the pores substantially.
region of the mortar takes place at the highest cell concentration Further, reduction in the flow of water inside the mortar matrix at
of 107 cells/ml. Thus all the test results directly related to the sur- an early stage may also affect the hydration of cement, which can
face region of the mortar, such as surface crack and pore healing be a reason for the reduction of compressive strength at higher
and reduction in water penetration depth, exhibit better perfor- bacterial concentration.
mance at 107 cells/ml. However, as observed earlier in Section 3.4 The reasoning presented above indicates that the precipitation
from Fig. 13(a)–(c), the high calcite precipitation at 107 cells/ml pattern of CaCO3 inside the concrete matrix at different bacterial
almost blocks the surface pores, and that leads to lower availability concentrations is different. The schematic diagram of the precipita-
of water inside the mortar matrix. At 105 cells/ml, the surface pores tion patterns of CaCO3 at different bacterial concentrations is given
are not fully blocked due to comparatively lower precipitation at in Fig. 16.
the surface region of the mortar, which allows the flow of water Apart from the bacterial concentration, the precipitation
inside the mortar matrix. This results in higher precipitation within pattern is also dependent upon several factors such as the type
 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214 211

Fig. 14. Increase in absorbed weight of water w.r.t. time for control and microbial
mortar samples (a) 3 days, (b) 7 days, and (c) 28 days.

Fig. 13. Increase in water penetration depth w.r.t. time for control and microbial
mortar samples (a) 3 days, (b) 7 days, and (c) 28 days.

concrete matrix. As a result, enhancement of compressive strength

could be achieved at higher cell concentrations. This would explain
of bacteria, water to cement ratio and cement to sand ratio. As dif- why Kumari et al. [20] found the optimum bacterial concentration
ferent bacteria have different calcite precipitation rates, if the pre- for compressive strength and water absorption at 107 cells/ml.
cipitation rate of the considered bacteria is very low, even at higher Fig. 16 further reveals the possibility of a condition that could
cell concentrations the available precipitation would be unable to lead to the optimal cell concentration for compressive strength
block all the pores at the surface region, thus allowing water to and water absorption being achieved at the same but intermediate
penetrate inside the concrete matrix. Again, high water to cement value of bacterial cell concentration. Due to any or a combination
ratio and low cement to sand ratio, resulting in high porosity of the factors that govern the precipitation patterns as mentioned
[24,25], would lead to a condition where the bacteria would be above, a condition could arise where, in Fig. 16(c), the surface
unable to fill the surface pores completely even at high bacterial precipitation is insufficient to completely block the surface pores.
concentrations, thereby allowing the flow of water inside the As the inner concrete matrix in Fig. 16(c) has a higher porosity as
212 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214

Fig. 15. SEM images inside the concrete matrix at a depth of (a–d) 5 mm from the surface (e–h) 20 mm from the surface.

compared to Fig. 16(b), the water absorption in case of the higher which contains about 0.3–1.3% of free calcium oxide [26]. More-
cell concentration in Fig. 16(c) would be higher than in Fig. 16(b). over, the water to cement ratio of 0.4 considered for the test
The maximum reduction in water absorption would thus occur at implies that about 7.5 gm/l to 32.5 gm/l calcium oxide is already
the concentration that corresponds to minimum overall porosity, available in the cement for use by the bacteria for calcite precipita-
which also is the optimum concentration for maximum enhance- tion. Achal and Pan [22] have reported that the reaction between
ment of compressive strength. This indicates why Chahal et al. calcium hydroxide (formed by hydration of CaO and water) and
[16] found the optimum concentration for both compressive carbon dioxide leads to the precipitation of calcite. The autogenous
strength and water absorption at 105 cells/ml. healing of concrete occurs in the same manner, but due to lack of
The precipitation of CaCO3 both at the surface and in the inner binding material, the maximum portion of calcium hydroxide is
matrix of the microbial mortar samples as evinced from Fig. 15 fur- leached out from the concrete. Thus, the autogenous healing capac-
ther indicates that even in the absence of an external calcium ity of concrete is very low. Pei et al. [27] experimentally proved
source, free calcium oxide already present in the cement can serve that bacterial cell walls accelerate the carbonation of Ca(OH)2
as the calcium source for the bacteria for CaCO3 precipitation. leading to the precipitation of CaCO3 in aqueous medium. This
Externally supplied CaO has earlier been used as an effective explains why significant calcite precipitation takes place in
calcium source for calcite precipitation by bacteria in concrete microbial concrete samples even without providing an external
[22]. In the current study, ordinary Portland cement has been used, source of calcium.
 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214 213

Fig. 16. Schematic diagram of the precipitation pattern of calcite at different bacterial concentration inside the concrete matrix (a) 103 cells/ml, (b) 105 cells/ml, (c) 107 cells/ml.

5. Conclusions building materials, World J. Microbiol. Biotechnol. 29 (2013) 2397–2406,

https://doi.org/10.1007/s11274-013-1408-z.
[2] S.J. Park, Y.M. Park, W.Y. Chun, W.J. Kim, S.Y. Ghim, Calcite-forming bacteria for
This study explores the possible reasons for the existence of an compressive strength improvement in mortar, J. Microbiol. Biotechnol. 20
optimum bacterial concentration in concrete for compressive (2010) 782–788, https://doi.org/10.4014/jmb.0911.11015.
[3] Y.Ç. Ersßan, H. Verbruggen, I. De Graeve, W. Verstraete, N. De Belie, N. Boon,
strength enhancement. Based on the experimental results, the fol- Nitrate reducing CaCO3 precipitating bacteria survive in mortar and inhibit
lowing conclusions can be drawn. steel corrosion, Cem. Concr. Res. 83 (2016) 19–30, https://doi.org/10.1016/j.
cemconres.2016.01.009.
[4] K. Van Tittelboom, N. De Belie, W. De Muynck, W. Verstraete, Use of bacteria to
1. Higher cell concentrations lead to higher precipitation amount
repair cracks in concrete, Cem. Concr. Res. 40 (2010) 157–166, https://doi.org/
and rate and thus at higher cell concentration the crack healing 10.1016/j.cemconres.2009.08.025.
and surface pore-healing are more efficient as compared to the [5] W. Khaliq, M.B. Ehsan, Crack healing in concrete using various bio influenced
lower cell concentration. self-healing techniques, Constr. Build. Mater. 102 (2016) 349–357, https://doi.
org/10.1016/j.conbuildmat.2015.11.006.
2. Higher amount of precipitation at the surface region inhibits the [6] H.K. Kim, S.J. Park, J.I. Han, H.K. Lee, Microbially mediated calcium carbonate
penetration of water to the inner concrete matrix and thus the precipitation on normal and lightweight concrete, Constr. Build. Mater. 38
precipitation in the inner matrix is less. Lower penetration of (2013) 1073–1082, https://doi.org/10.1016/j.conbuildmat.2012.07.040.
[7] A. Nugroho, I. Satyarno, S. Subyakto, Bacteria as self-healing agent in mortar
water at the early stage can also affect the hydration of cement. cracks, J. Eng. Technol. Sci. 47 (2015) 279–295, https://doi.org/10.5614/j.eng.
Consequently, the compressive strength of concrete is impaired. technol.sci.2015.47.3.4.
3. The optimum bacterial concentration for the enhancement in [8] J. Wang, K. Van Tittelboom, N. De Belie, W. Verstraete, Use of silica gel or
polyurethane immobilized bacteria for self-healing concrete, Constr.
compressive strength of concrete is not necessarily a high value, Build. Mater. 26 (2012) 532–540, https://doi.org/10.1016/j.conbuildmat.2011.
though higher cell concentration leads to greater calcite precip- 06.054.
itation. For a particular water to cement ratio and cement to [9] J.Y. Wang, H. Soens, W. Verstraete, N. De Belie, Self-healing concrete by use of
microencapsulated bacterial spores, Cem. Concr. Res. 56 (2014) 139–152,
sand ratio, the difference in precipitation patterns within the
https://doi.org/10.1016/j.cemconres.2013.11.009.
concrete matrix at varying levels of bacterial concentrations is [10] J. Zhang, Y. Liu, T. Feng, M. Zhou, L. Zhao, A. Zhou, Z. Li, Immobilizing
responsible for the existence of an optimal concentration for bacteria in expanded perlite for the crack self-healing in concrete, Constr.
Build. Mater. 148 (2017) 610–617, https://doi.org/10.1016/j.conbuildmat.
strength enhancement. In this study, for the specific bacteria,
2017.05.021.
water to cement ratio and cement to sand ratio, the optimum [11] Kunal, R. Siddique, A. Rajor, Influence of bacterial treated cement kiln dust on
concentration for strength improvement was achieved at 105 the properties of concrete, Constr. Build. Mater. 52 (2014) 42–51, https://doi.
cells/ml. org/10.1016/j.conbuildmat.2013.11.034.
[12] N. Chahal, R. Siddique, Permeation properties of concrete made with fly ash
4. As noted in (2) above, the precipitation layer produced at the and silica fume: influence of ureolytic bacteria, Constr. Build. Mater. 49 (2013)
surface with higher cell concentration acts as a shield to the 161–174, https://doi.org/10.1016/j.conbuildmat.2013.08.023.
mortar, that can protect the inner matrix of concrete from the [13] P. Ghosh, S. Mandal, B.D. Chattopadhyay, S. Pal, Use of microorganism to
improve the strength of cement mortar, Cem. Concr. Res. 35 (2005) 1980–
penetration of water and harmful substances. Thus, the selec- 1983, https://doi.org/10.1016/j.cemconres.2005.03.005.
tion of bacterial concentration should be dependent upon the [14] R. Andalib, M.Z.A. Majid, M.W. Hussin, M. Ponraj, A. Keyvanfar, J. Mirza, H.S.
desired objective. A higher bacterial cell concentration would Lee, Optimum concentration of Bacillus megaterium for strengthening
structural concrete, Constr. Build. Mater. 118 (2016) 180–193, https://doi.
be more efficient where protection of concrete is more impor- org/10.1016/j.conbuildmat.2016.04.142.
tant than strength. In cases where compressive strength [15] M. Sarkar, N. Alam, B. Chaudhuri, B. Chattopadhyay, S. Mandal, Development of
enhancement is of greater significance, instead of directly an improved E. coli bacterial strain for green and sustainable concrete
technology, RSC Adv. 5 (2015) 32175–32182, https://doi.org/10.1039/
adopting a higher cell concentration, the optimum concentra-
C5RA02979A.
tion should be evaluated for provision in the design mix. [16] N. Chahal, R. Siddique, A. Rajor, Influence of bacteria on the compressive
strength, water absorption and rapid chloride permeability of fly ash concrete,
Constr. Build. Mater. 28 (2012) 351–356, https://doi.org/10.1016/
Declarations of interest j.conbuildmat.2011.07.042.
[17] H.M. Jonkers, A. Thijssen, G. Muyzer, O. Copuroglu, E. Schlangen, Application
None. of bacteria as self-healing agent for the development of sustainable
concrete, Ecol. Eng. 36 (2010) 230–235, https://doi.org/10.1016/j.
ecoleng.2008.12.036.
References [18] Y.C. Ersßan, N. De Belie, N. Boon, Microbially induced CaCO3 precipitation
through denitrification: an optimization study in minimal nutrient
[1] N.K. Dhami, M.S. Reddy, A. Mukherjee, Bacillus megaterium mediated environment, Biochem. Eng. J. 101 (2015) 108–118, https://doi.org/10.1016/j.
mineralization of calcium carbonate as biogenic surface treatment of green bej.2015.05.006.
214 S. Mondal, A.(Dey) Ghosh / Construction and Building Materials 183 (2018) 202–214

[19] M. Sarkar, D. Adak, A. Tamang, B. Chattopadhyay, S. Mandal, Genetically- [23] S. Ghosh, M. Biswas, B.D. Chattopadhyay, S. Mandal, Microbial activity on the
enriched microbe-facilitated self-healing concrete – a sustainable material for microstructure of bacteria modified mortar, Cem. Concr. Compos. 31 (2009)
a new generation of construction technology, RSC Adv. 5 (2015) 105363– 93–98, https://doi.org/10.1016/j.cemconcomp.2009.01.001.
105371, https://doi.org/10.1039/C5RA20858K. [24] Y. Kim, K. Lee, J. Bang, S. Kwon, Effect of W/C ratio on durability and porosity in
[20] C. Kumari, B. Das, R. Jayabalan, R. Davis, P. Sarkar, Effect of nonureolytic cement mortar with constant cement amount, Adv. Mater. Sci. Eng. 2014 (2014).
bacteria on engineering properties of cement mortar, J. Mater. Civ. Eng. (2017), [25] J. Bu, Z. Tian, S. Zheng, Z. Tang, Effect of sand content on strength and pore
https://doi.org/10.1061/(ASCE)MT.1943-5533.0001828. structure of cement mortar, J. Wuhan Univ. Technol. Mater. Sci. Ed. 32 (2017)
[21] J. Xu, W. Yao, Z. Jiang, Non-ureolytic bacterial carbonate precipitation as a 382–390, https://doi.org/10.1007/s11595-017-1607-9.
surface treatment strategy on cementitious materials, J. Mater. Civ. Eng. 26 [26] A.M.M. Neville, J.J.J. Brooks, Concr. Technol. 11 (2010) 442, https://doi.org/
(2014) 983–991, https://doi.org/10.1061/(ASCE)MT. 10.1016/0360-1323(76)90009-3.
[22] V. Achal, X. Pan, Influence of calcium sources on microbially induced calcium [27] R. Pei, J. Liu, S. Wang, M. Yang, Use of bacterial cell walls to improve the
carbonate precipitation by Bacillus sp. CR2, Appl. Biochem. Biotechnol. 173 mechanical performance of concrete, Cem. Concr. Compos. 39 (2013) 122–130,
(2014) 307–317, https://doi.org/10.1007/s12010-014-0842-1. https://doi.org/10.1016/j.cemconcomp.2013.03.024.