Cement and Concrete Composites 104 (2019) 103340

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Cement and Concrete Composites

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Bacterial self-healing of concrete and durability assessment T

*
Thanh Ha Nguyen, Elhem Ghorbel , Hanaa Fares, Annelise Cousture
University of Cergy Pontoise, 5 Mail Gay Lussac, Neuville, Oise, 95031, France

ARTICLE INFO ABSTRACT

Keywords: Bacterial self-healing is an innovative technology allowing repairing open micro-cracks in concrete by CaCO3
Bacterial self-healing precipitation. This bio-technology improves the durability of the structure. In this paper, peptone, yeast extract
Mechanical strengths and Bacillus Subtilis were added as microbial adjuvant in concrete mix design. This led to a decrease in porosity
Porosity resulting in an increase of strength, dynamic modulus as well as a reduction of water uptake, gas permeability
Gaz permeability
and chloride permeation. Scanning electron microscopy, energy dispersive spectroscopy and raman spectroscopy
Chloride ions migration
SEM observations
showed that the microbial precipitations in the crack were CaCO3. Moreover, the morphology of calcite crystals
was a needle-like, bouquet-like and rhombohedral-shaped. At 44 days, 400 μm crack surface width was com-
pletely filled. Hence, peptone, yeast extract and Bacillus Subtilis could be considered as a promising concrete
admixture in enhancing durability and mechanical properties of concrete. Furthermore it is established that
Eurocode 2 could be applied with confidence for predicting properties of bacterial concrete.

1. Introduction induced calcium carbonate precipitation via urea hydrolysis shows

Concrete is a building material that is used worldwide (approxi-
mately 6 million m3/year are manufactured) because of its first-rate
properties. However, it is a fragile material as it easily cracks due to
multiple reasons such as autogenous shrinkage, freeze-thaw reactions,
mechanical compressive and tensile forces. Micro-cracks do not sig-
nificantly affect the strength of concrete but lead to more fluid pene-
tration such as water and other chemical solutions (chlorides, sulfates
and acids) resulting in cement matrix degradation and consequently
corrosion of reinforcing embedded steel bars [1]. Presence of cracks not
only decrease the mechanical strength and durability of material but
also affect the structure safety. Recently, many different types of bac-
teria have been incorporated in the concrete formulation for crack re-
paration and durability improvement. The type of nutrients in the
culture media as well as of bacteria have a great effect on the urease
activity and therefore microbial calcite precipitation [2]. Most scientists
[3–5] focused on ureolysis processes of bacteria to improve CaCO3
precipitated capacity. It is established that almost bacteria are able to
induce calcium carbonate precipitation by producing urease enzyme.
Hence, adding bacteria in cement based materials can be cost-effective
and environment-friendly alternative method to improve the service life
of buildings [6–10]. F. Hammes and W. Verstraete [11] reported that
the quality of microbial precipitation (CaCO3) is determined by four key
factors such as: the concentration of dissolved inorganic carbon, pH,
calcium concentration and the presence of nucleation sites. Microbially
*
Corresponding author.
E-mail address: Elhem.Ghorbel@u-cergy.fr (E. Ghorbel).
potential to precipitate large quantities of calcite rapidly. Hence,
adding bacteria in concrete allows its microbial self-healing and con-
sequently the reduction of considerable sums of money spent on con-
crete structures maintenance as closing them for repairing becomes no
longer necessary.
The presence of bacteria appears as a promising, effective and eco-
friendly technology for self-healing of concrete structures.
Statistical percentage of cement based materials incorporating
bacteria during the last decade is given in Fig. 1. Many studies have
focused on the bacterial genus Bacillus (up to 84%) due to the following
reasons:
• They are commonly found in soil and are able to form spores under
unfavorable conditions [12].
• They dormant in the high alkaline environment of concrete (pH
value up to 13) for over 50 years old [13].
• They produce enough amount of urease enzyme resulting in calcium
carbonate precipitation through urea hydrolysis [3].
Bacteria were incorporated inside concrete and mortars through two
methods:
• In the first method namely “direct”, the bacteria and nutrients have
been introduced directly into the mixing process [6–9,14–22].
• In the second method called “indirect”, the bacteria with nutrients

https://doi.org/10.1016/j.cemconcomp.2019.103340
Received 8 March 2019; Received in revised form 28 May 2019; Accepted 29 May 2019
Available online 03 June 2019
0958-9465/ © 2019 Published by Elsevier Ltd.
T.H. Nguyen, et al. Cement and Concrete Composites 104 (2019) 103340

Fig. 1. The statistics about bacteria application in the cementitious material.

have been immobilized into other materials such as ceramsite [23],
polyurethane and glass tubes [24,25], lightweight aggregates
[26–32], graphite nano platelets [26], hydrogel [33] and zeolite
[28,34].
Fig. 2 shows the effects of different types of bacteria on the com-
pressive strength of concrete for both methods of bacteria incorpora-
tion. It could be observed that:
• Most bacterial species improved the concrete compressive strength.
However, some bacteria such as Bacillus sphaericus, Diaphorobacter
nitroreducens [28] and Bacillus pseudofirmus [16] decreased it up to
68%, 11% and 10%, respectively.
• The concentration of 10 5
cells/ml of mixing water has led to better
performance for concrete compressive strength. N. Chahal and R.
Siddique [19] have found that replacement cement by 10% of fly
ash and incorporation 105 cells/ml Sporosarcina Pasteurii enhanced
22% the compressive strength. In other studies, it has been estab-
lished that Bacillus alkalinitrilicus [35], Bacillus megaterium, Bacillus
flexus, Bacillus licheniformis [7], Bacillus aerius [21] and Shewanella
[8] have improved concrete compressive strength by 7.15%, 16%,
6.1%, 10.6%, 11% and 26% respectively.
The effects of bacteria on durability against chloride penetration

Fig. 2. Effect of different types of bacteria on the compressive strength.

2
T.H. Nguyen, et al.
Fig. 3. Effect of different types of bacteria on the chloride ions migration.
were investigated and bibliography results are summarized in Fig. 3.
The analysis of these results outlined that incorporating bacteria
whatever the adopted method has provided an enhancement of con-
crete resistance against ions chloride penetration. It appears that:
• For “direct” method: F. Nosouhian et al. [14] reported that the in-
corporation of Sporosarcina Pasteurii and Bacillus Subtilis enhanced
the resistance of concrete to chloride permeation. Indeed, the
average number of coulombs passed through the bacteria-containing
concrete was 11.7% less than that of control concrete. Bacillus
Sphaericus [10] enhanced 30% the chloride migration resistance of
mortar specimens. N. Chahal and R. Siddique observed that adding
105 cells/mL of Sporosarcina Pasteurii decreased of about 53% the
total charge passed through concrete at 28 days containing 10% fly
ash [19] and of about 41% for concrete elaborated with 10% silica
fume and tested at 91 days [20]. In another research, R. Siddique
et al. have found that 105 cells/mL of Bacillus aerius [21] improved
of about 22% the resistance of concrete to total charge passing
through concrete elaborated with 10% replacement of cement by
rice husk ash and tested at 56 days. These results highlighted that
the bacterial activity effectiveness can be, also, ascribed to the ce-
mentitious additions.
• For “indirect” method: N. Hosseini Balam et al. [29] have indicated
that the use of Sporosarcina Pasteurii have enhanced by 34% the
resistance of lightweight concrete against chloride ions penetration
at 91 days. S. Bhaskar et al. [34] have noticed that the mortars with
Sposarcina ureae showed an average decrease in chloride ions dif-
fusion of around 30% at 120 days, 45% at 180 days and 56% at 240
days. During the process of cell growth, calcite precipitated on the
surface of cells and in the cement matrix resulting in less porosity
and permeability of cement mortar.
The researchers have relied mainly on the standard ASTM C1202 to
assess the bacterial concrete resistance to chloride ions diffusion by
measuring the total charge passed in coulombs. Only W. De Muynck
et al. [10] have used the standard NT Build 492. The advantage of this
method is that it allows to measure the chloride penetration depth and
to calculate the chloride migration coefficient.
Few studies have focused on the effects of bacteria on the concrete
gas permeability which is the most common indicator of durability. We
can cite, for example, the results of W. De Muynck et al. [36,37] who
have found that Bacillus sphaericus has reduced by 32% the concrete
permeability due to the deposition of a calcite layer on the surface of
specimens at 28 days.
The results of literature have outlined some outputs that led to the
following aims of this investigation:
3
Cement and Concrete Composites 104 (2019) 103340
• Bacillus Subtilis is a member of Bacillus genus, converting urea to
ammonium carbonate more efficiently than any other species.
Although they are friendly and largely commercialised by several
companies, they don't have been sufficiently utilized in civil en-
gineering applications [14,26]. Therefore, Bacillus Subtilis are in-
corporated into this study to determine their influence on the crack
reparation and durability of concrete structures.
• In general, the culture medium of bacteria consisted in nutrients
supplemented with varied amount of urea
[9,10,14,17,19–21,24,25,28,29,31,34,36–38]. Only few studies
didn't add urea to the mixture [6,15,16,22,26]. In this work, pep-
tone and yeast extract are selected as nutrients for Bacillus Subtilis
growth on the basis of K. Wang et al. [39]. The culture medium is
chosen without urea. Indeed, the urease activity can occur without
urea as yeast contains abundant of amino acids, peptides, carbo-
hydrates, salts, vitamins and minerals which are necessary for mi-
crobial precipitation.
•
Direct method is less efficient than the indirect one. However, its
implementation in the incorporation of bacteria is easier than in-
direct method. Hence it has been selected in this work. The con-
centration of 105 cells/ml of water is adopted because it led to the
best increase in compressive strengths at 28 days according to lit-
erature [7,8,19,21,35].
• There is a lack of literature results dealing about the effects of in-
corporation bacteria on the gas permeability up to 210 days.
Therefore this property is investigated.
• NT Build 492 method is proposed in this study to evaluate more
deeply the effect of bacteria on chloride ions diffusion up to 210
days. Results reported in the literature [36,37] were related to the
effects of Bacillus sphaericus on concrete specimens at 28 days.
• Applicability of design code Eurocode 2 [40], EC2, to bacterial
concrete has checked in this investigation. This point has never been
treated in literature before.
2. Experimental program
2.1. Materials and mixtures
Portland cement (CEM II/A-L42.5) and limestone fillers (HP-OG)
with densities of 3.09 g/cm3 and 2.7 g/cm3 respectively were used.
To ensure the workability of concrete a superplasticizer, SP, (MC
Power-Flow 3140) was used.
Siliciousfine aggregates and two gradation ranges of coarse calcar-
eous aggregates were used to produce the concrete. The particles size
distribution of the aggregates was obtained by sieving according to NF
EN 933–1 [41] and the curves are plotted in Fig. 4.
Bacillus subtilis 5265 T cells were cultured and prepared aerobically
Fig. 4. Sieve size of the design mixtures.
T.H. Nguyen, et al. Cement and Concrete Composites 104 (2019) 103340

Table 1
Mix design proportions for 1 m3 of concrete.
Mixture no. Water (kg/ CEM II/A Filler (kg/ SN (kg/ GN1 (kg/ GN2 (kg/ SP (kg/ Peptone (kg/ Yeast Extract Bacillus Subtilis Slump (cm)
m3) (kg/m3) m3) m3) m3) m3) m3) m3) (kg/m3) (Cells/m3)

Control 182 294 57 757 259 795 2.1 0 0 0 18.5

Nutrient 182 294 57 757 259 795 2.1 1.82 0.91 0 20.5
Bacteria 182 294 57 757 259 795 2.1 1.82 0.91 1.82 × 1010 19.5

at 37 °C in 100 mL of Trypticase Soy (TS) medium then incubated for
24 h. Afterward, bacterial cells were collected and their concentration
was measured using thermo scientific GENESYS 20 apparatus.
The microbial adjuvant is constituted of Bacillus Subtilis cells with a
concentration of 108 cells/l of mixing water, 10 g/l of Peptone and 5 g/l
of Yeast extract.
Three concrete formulations have been designed. The first, named
“Control”, is formulated to achieve consistency class of S4 with a target
slump about 18 ± 2 cm and a compressive strength class of C35/40 in
accordance with standard NF 206–1 [42]. For the second, called “Nu-
trient”, only peptone and yeast extract were incorporated to mixing
water while for the third, called “Bacteria”, both nutrients and Bacillus
subtilis (with a concentration of 108 cells) were added to mixing water
during the concrete manufacturing. The components were introduced
into the mixer in this order: at first coarse aggregates, followed by fine
ones aggregates, fillers, mixing water in which adjuvants were in-
corporated (superplasticizer, nutrients and microbial adjuvant). The
specimens were removed from molds after 24 h and left in water cure at
23 °C. The mix proportions of three concrete are shown in Table 1.
2.2.3. Capillary water absorption
For capillary water absorption test was carried out, to compare the
penetration resistance between specimens with and without bacteria.
The cylindrical disc specimens (Φ10 × 5 cm) were dried in an oven at
105 ± 5 °C until a mass equilibrium of less than 0.1% at 24 h intervals.
After drying, all specimens were coated with epoxy on the sides to
ensure unidirectional absorption through the treated surface. The spe-
cimens were exposed to water at a depth of 1 ± 0.1 cm, with a treated
surface facing downwards. At regular time intervals (5 min, 30 min, 1 h,
2 h, 4 h, 6 h, 24 h and 2, 3, 6, 7 days), the specimens were taken out of
water and weighed after drying the surfaces with a wet towel. After
weighing, all specimens were re-submerged immediately into water.
The sorptivity coefficient, k (m/ s ) is determined using Eq. (3):
Q
=k t
S (3)
3
Where Q is the quantity of absorbed water [m ]; S is the cross section in
contact with water [m2] and t is the time [s]. By plotting Q/S against
the square root of time, the sorptivity coefficient, k0, is calculated from
5min to 24 h according to ASTM C1585-04 standard [44].

2.2. Experimental methods

2.2.1. Mechanical properties
The compressive strength and splitting tensile strengths tests on
concrete were carried out using a servohydraulic INSTRON machine
with a maximum capacity of 3500 KN and a loading rate of 0.5 and
0.05 MPa/s respectively. The dynamic modulus of elasticity was mea-
sured by an equipment namely E-Meter MK II. The principle is based on
the determination of resonant frequency testing method. For each
mixture, three specimens have been tested and the giving results are the
averages of obtained values.
2.2.2. Water absorption and porosity
Water porosity, n , and water absorption capacity, WA 48, were de-
termined using the vacuum saturation method according to the stan-
dard NF P 18–459 [43] on three cylinders with 15 cm diameter and
5 cm thickness. Specimens were placed under vacuum for 4 h at a
pressure of 25 mbar. Tap water was drawn into the vacuum chamber
progressively so that the samples were covered with approximately
20 mm of water after 15 min. Specimens were maintained immersed in
water under vacuum for 44 h then removed and weighed in water to
obtain their apparent mass (Mw). Next, they were wiped to surface dry
condition and weighed to obtain the saturated surface dry mass (MSSD).
Finally, they were completely dried in an oven at 105 ± 5 °C and
weighed (Mdry). The coefficient of water absorption, WA48, and the
porosity, n, were calculated using Eq. (1) and Eq. (2) respectively.
MSSD Mdry
WA 48 (%) = × 100
Mdry
MSSD Mdry
n (%) = × 100
MSSD Mw
Where MSSD is mass of samples at the saturated surface-dried state, Mdry
is the mass of samples at oven dried state and Mw is the mass of concrete
specimens in water.
2.2.4. Rapid chloride permeability
The rapid chloride permeability test was investigated by means of
the CTH rapid test according to NT Build 492 Nordtest method [45].
Cylindrical concrete specimens (diameter of 10 cm and thickness of
5 cm) were achieved at different ages. The test consisted in placing
samples in a vacuum container for vacuum treatment (4 h). These
samples were saturated with a NaOH solution (12 g/L) under vacuum
for 44 h after which they were inserted in chloride ions diffusion cells.
The liquids (NaOH 12 g/L and NaCl 112 g/L solutions) were filled in
two cells, namely anode and cathode, respectively. The attack by Cl−
ions are carried for 24 h by imposing a potential difference between two
electrodes. Thereafter, the specimens are cut into two by machine and
sprayed with silver nitrate (0.1 M). The non-steady-stade migration
coefficient is obtained through Eq. (4):
0.0239(273 + T ) L (273 + T ) LXd
Dnssm = Xd 0.0238
(U 2) t U 2 (4)
−12
Where Dnssm is the non-steady-state migration coefficient (x10 m2/
s), U is the absolute value of the applied voltage (V), T is the average
value of initial and final temperatures in the anolyte solution (°C), L is
the sample thickness (mm), Xd is the average value of chloride pene-
tration depth (mm), t is the test duration (h). The experimental device is
shown in Fig. 5.
2.2.5. Air permeability
Air permeability was measured by using a CEMBUREAU permea-
meter (Fig. 6) according to XP P 18–463 [46] and Darcy's Law. The
(1) measurement was carried out on the Φ15 × 5 cm discs in the central
portion of 15 × 30 cm cylinders (3 discs are cut from each sample). The
specimens were dried at a temperature of 100 ± 5 °C until the constant
mass. Before testing, three specimens were sealed with self-adhesive
(2)
aluminum in order to guarantee the uniaxial flow of gas. The principle
of the permeameter is based on the measurement of the flow of gas at
the inlet and at the outlet, after the pressure stabilization. The apparent
permeability, Ka, is calculated as given by Eq. (5):

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T.H. Nguyen, et al. Cement and Concrete Composites 104 (2019) 103340

Fig. 5. Experimental setup and measurement of the chloride ions migration.

Fig. 6. Gas permeability measurement system.

2. µ . Q.l.p2 Fig. 7. The experimental devices and crack measurements.

Ka =
S . (p12 p22 ) (5)
2 experimental devices and crack measurements are illustrated in Fig. 7.
Where p2 is the atmospheric pressure [N/m ], Q is the volumetric gas
flow rate [m3/s], L is the thickness of specimen [m], S is the cross
section of specimen [m2] and p1 is the applied oxygen pressure [N/m2].
2.2.7. SEM observations, EDS and Raman analysis
The permeability measurements were performed by choosing four dif-
Scanning Electron Microscope (SEM) coupled with Energy
ferent applied pressures: 2 bar, 3 bar, 4 bar and 5 bar. Besides, the in-
Dispersive Spectrometer (EDS) was used to analyze the crack fill of
trinsic permeability Kint is deduced from the apparent permeabilities
concrete. The observations were performed at 15 kV in vapor pressure
with a correction proposed by Klinkenberg (Eq. (6)):
mode (chamber pressure at 37 Pa) using a ZEISS Gemini SEM 300 with
P1 + P2 a backscattered electron detector (BSD) and a topographic mode in
Pmoy = and K a = Kint 1 + order to avoid metallization of samples and charge phenomena. The
2 Pmoy (6)
elemental composition was determined by EDS microanalyses with a
Coefficient of Klinkenberg, β, is a constant that depends mainly on Bruker AXS Quantax detector.
the porous structure of concrete. To improve the identification of formed products in the cracks,
Raman Spectroscopy was performed under a confocal microscope
Apyron with a 50x objective (ZEISS Epiplan: numerical aperture 0.7
2.2.6. Creation and observation of artificial cracks
and working distance 1.1 mm) and with a grating spectrometer 1200 g/
All specimens (Φ15 × 5 cm) were pre-cracked at 28 days. The
mm from WITec. The laser wavelength used to illuminate the sample
cracks were created throughout the complete thickness of specimens
was 532 nm. SEM, EDS and Confocal devices are given in Fig. 8.
using the INSTRON machine. The crack width was controlled such that
it lies between 80 and 400 μm. Before immersion in water at a tem-
perature of 30 ± 5 °C, the crack widths were marked and measured
with an optical microscopy Nikon SMZ800. Microscopic observations
were conducted periodically to quantify the self-healing process. The

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T.H. Nguyen, et al. Cement and Concrete Composites 104 (2019) 103340

Huang [54] observed that CaCO3 was precipitated nearby the crack
mouth. More generally, the reaction products of autogenous self-
healing were investigated and characterized using EDS, TGA and
FTIR techniques. The reported results from the literature showed
that the main precipitated products in cracks were calcite, portlan-
dite and C–S–H [49–51]. This is explained by the fact that not only
additional hydration of unhydrated cement is possible, but also the
leached portlandite from the bulk paste can be recrystallized. Hearn
[55], Suleiman and Nehdi [56] explained that the autogenous
healing with the presence of water fills cracks through hydration of
unhydrated cement particles or carbonation of dissolved calcium
hydroxide. Calcium hydroxide can react with carbon dioxide [49].
Fig. 8. SEM, EDS (a) and Confocal Apyron WITec (b) devices. These reactions can be seen from Eq. (7) and Eq. (8):
CaO + H2 O Ca(OH)2 (7)
3. Results and discussion
Ca(OH)2 + CO2 CaCO3 + H2 O (8)
3.1. Crack-healing capacity
• Concrete samples incorporating nutrients (Fig. 9b) were subjected to
a crack healing phenomenon more important than this observed for
The efficiency of crack healing of control, nutrient and bacteria-
control concrete and less than concrete with bacteria. The observed
based concrete at different predefined times of immersion in tap water
crack healing can be attributed to autogenous self-healing more than
is shown in Fig. 9. White products were noticed on the cracks. The
to bacterial self-healing. As a matter of fact, bacteria can be found in
characterization of these products was done through microstructural
all environments, for example, water, air, aggregates …. Hence, they
investigations. It can be concluded that:
subsist in all concrete components during the mix design. Therefore,

• The Control concrete samples were subjected to crack healing phe- •

bacterial crack healing can occur but it is slow.
The concrete samples incorporating microbial adjuvant (bac-
nomenon which can be attributed, essentially, to autogenous self-
teria + nutrients) showed significant self-healing and higher pre-
healing (Fig. 9a). However, this crack healing was slow and limited.
cipitation products (Fig. 9c). This product formation is due to both
These obtained results are consistent with those of the literature
mechanisms: autogenous and microbial self-healing. Microbial self-
[47–51]. Indeed, several authors studied the autogenous self-healing
healing phenomenon seems to be the most dominant one especially
mechanisms. Hence, Yang et al. [52] showed that the crack widths
at short water exposure duration. Jonkers et al. [16,35] observed
within cement-based materials must be controlled to below 150 μm.
similar phenomena for concrete adjuvanted with pseudofirmus, B.
In a recent study, Qian et al. [53] confirmed that the mechanism of
cohnii and B. alkalinitrilicus. They established that the observed
autogenous self-healing in concrete was the growth of calcites,
products are the result of calcium carbonate precipitation due to
CaCO3, from both faces towards the middle of crack. Moreover,

Fig. 9. Stereomicroscopic images showing the evolution of cracks after 23 days and 44 days of water immersion (a) Control; (b) Nutrient and (c) Bacteria.

6
T.H. Nguyen, et al.
bacterial activity.
The efficiency of crack healing at different predefined curing times
in Fig. 9 was attested that, after 23 days of immersion in water, sig-
nificant differences in crack closure were observed between the control
specimen and others. Indeed, the crack surfaces of bacteria-based
concrete were almost closed (approximately 60%) due to microbial
carbonate precipitation. At 44 days, 400 μm crack surface width was
completely filled by microbial carbonate precipitations.
These results are in agreement with those established by Burne and
Chen [57] and Okwadha and J. Li [58]. They revealed that the presence
of nutrients, urea and bacteria in concrete at a temperature of
30 ± 5 °C conducted to the formation of ureolysis. The reactions in-
volving microbial mineral precipitations are described by Eq. (9)-Eq.
(13) [59,60]. Hence, S. Bhaskar et al. [34] showed that a maximum
crack width of 70 μm was filled by Sporosarcina ureae while V. Wiktor
and H. M. Jonkers [35] reported that the presence of Bacillus alkalini-
trilicus led to the closure of 460 μm crack after 100 days of immersion in
tap water.
Bacterial urease
CO(NH2)2 + H2 O NH2 COOH + NH3
NH2 COOH + H2 O NH3 + H2 CO3
2NH3 + 2H2 O 2NH+4 + 2OH
2OH + H2 CO3 2CO32 + 2H2 O
Ca2 + + CO32 CaCO3
SEM analysis was conducted in order to investigate the morphology
of bacterial self-healing products (Fig. 10). SEM images exhibit a
homogeneous distribution of bacterial precipitation crystals that ap-
pears in the form of needles-like, bouquets-needles-like and rhombo-
hedral structure. According to H. Cheng et al. [61] and H. Guo et al.
[62], the concentration of CO(NH)2 increase promoted the bacterial
urease catalyst so the bouquets-like morphology were formed through
the linkage of needles-like rods. When the amount of ammonia is high,
the calcium carbonate tends to form a stable rhombohedral shape. Two
main morphologies (rhombohedral-shaped and needles-like) were ob-
served with Bacillus sphaericus [36,37] and Bacillus alkalinitrilicus [35].
EDS analysis, realised in the cracked and uncracked area (Fig. 11),
shows that the bacterial products contain three main elements: C, O and
Ca, confirming results reported by Wiktor and Jonkers [35]. According
to the authors, the above-described mineral precipitations were CaCO3
which are related to bacterial activity. The dense calcium carbonate
crystals were developed on the surface, near and inside the cracks. EDS
results attested that the microbial adjuvant induced microbial calcite
precipitation even if no urea has been added to the culture media. Other
elements (Al, Mg and Si) appear in EDS spectra that could be attributed
Fig. 10. SEM observation of the crack area (a) and self-healing products (b).
Cement and Concrete Composites 104 (2019) 103340
to the matrix and/or cement aggregates. The presence of these elements
was more visible outside the crack (Fig. 11a) than inside (Fig. 11b). It
can be observed that the main product in crack is CaCO3. In order to
confirm that the self-healing process by calcite precipitation is mainly
due to microbial-phenomenon, Raman Spectroscopy analysis was per-
formed in the cracked area. The Raman spectrum presented in Fig. 12
shows four prominent bands. The first two bands at 156 and 284 cm−1
were ascribed to Ca, the band at 715 cm−1 was due to symmetric CO3
deformation, and the last band at 1088 cm−1 was attributed to sym-
metric CO3 stretching. These results are in agreement with those of S.
Dedera et al. [63]. It can be concluded that the observed products are
microbial calcium carbonate crystals and confirmed that peptone added
to yeast extract without urea was a good source of nutrients for Bacillus
Subtilis and its urease activity.
3.2. Water absorption and porosity
The water porosity depends on three parameters: the porosity of
paste, the porosity of aggregates and the quality of interface paste/
aggregates. In the present study, the two first factors are constant. The
(9)
evaluation of water porosity depends only on microbiologically induced
(10) calcium carbonate precipitation in the cracks that may appear at the
interfaces aggregates/paste or/and inside the paste. Water absorption
(11) and porosity are illustrated in Fig. 13 where it can be seen that:
(12) • Water absorption (Fig. 13a) as well as porosity (Fig. 13b) decreased
as curing time increased. This phenomenon is well known and is
(13) attributed to autogenous self-healing phenomenon.
• The incorporation of nutrients and bacteria + nutrients as adjuvant
affected these both parameters especially at short time curing. The
porosity and water absorption were less important for concrete with
bacteria + nutrients than for control ones. This is explained by the
formation of CaCO3 inside the voids (microcracks, porosities, ….)
and is in agreement with [37]. It must be noted that the reported
results have been obtained for curing duration of concrete less than
28 days.
To separate the effect of bacterial from autogenous self-healing ef-
fects, a variation coefficient is introduced and is expressed as
(x )ti (x )tcontrol
rx (t ) = (x )tcontrol
. “x” denotes the porosity or water absorption
(WA48), the index “i” indicates the type of concrete (nutrient or bac-
teria + nutrient) while “t” is the curing time.
The evolution of rporosity and rWA48 (Fig. 14) revealed a more sig-
nificant decrease for concrete with microbial than nutrients adjuvant.
This decrease was stabilized and reached a threshold at 90 days for
nutrient-concrete while it carried on for bacteria-concrete even at 210
days. This reduction was due to the filling in pores and micro-cracks by
calcite produced through Bacillus Subtilis activity. This result is in
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T.H. Nguyen, et al. Cement and Concrete Composites 104 (2019) 103340

Fig. 11. EDS spectrum of self-healing products at the uncracked (a) and cracked area (b).

Fig. 12. Raman spectrums in the cracked area.

agreement with those of the literature related to the effects of in- this model was checked and it's predictive ability was confirmed
corporating other types of bacteria in concrete up to 60 days (Fig. 15).
[9,20,21,64]. In addition, the effects of bacteria type on microbial
products are illustrated in Fig. 14: an ureolytic bacterium isolated from n (%) = 2.26 × WA 48 (%) with R2 = 0.98 (14)
marble sludge [9,21], Sporosarcina pasteurii [20] and an alkaliphilic/
alkalitolerant bacteria obtained from rhizospheric soil and from marble
sludge [64]. The concentration cells was constant and equal to
105 cells/ml for the mentioned researches and urea was added to the 3.3. Development of the mechanical properties of concrete with age
culture medium. The observed discrepancies between results from Refs.
[5,21] can not be explained. The dependency of concrete properties on time was investigated and
Omary et al. [65] have proposed an expression relating water ab- the evolution was determined according to Eurocode 2 standard related
sorption coefficient, WA 48 , and porosity, n , (Eq. (14)). The validity of to “Design of concrete structures” [40], as described by Eq. (15):

Fig. 13. Water absorption (a) porosity (b) versus time.

8
T.H. Nguyen, et al. Cement and Concrete Composites 104 (2019) 103340

Fig. 14. The variation of water absorption coefficient (a) and porosity coefficient (b).

can be established that the development of fcm against age is well

described by EC2 given by Eq. (15) for all mixtures even those in-
corporating bacteria (Fig. 16a).
• The effects of bacteria on the compressive strength is maximum at
the short curing age, here 28 days (Fig. 16b). At 90 days the var-
iation coefficient is less important than for 28 days. The increase is
mainly due to the pores filling inside the concrete specimens and the
modification of pore size due to microbiologically induced calcium
carbonate precipitation. This improvement led to more dense ma-
trix. These results are consistent with those obtained for porosity
and water absorption (Fig. 13).
• The compressive strength enhancement and the general trend of
variation coefficient are in agreement with the literature results and
strongly depend on the type of bacteria as well as on the con-
centration of cells (a concentration of 105 cells/ml for [9,20,21,64]
instead of 2.8 × 108 cells/ml for [26]).

3.3.2. Splitting tensile strength

Fig. 15. The evolution of water absorption as a function of the porosity of
The evolution of splitting tensile strength, fctm, sp , with concrete age,
concrete.
for each mixture, was assessed according to EC2 as described by Eq.
fctm, sp (t )
(15) where fctm, sp (28days )
= cc (t ) with (α = 1 for t < 28 and α = 2/3 for
1
(t ) 28 2 t > 28).
= cc (t ) with cc (t ) = exp s 1
(28days ) t
(15) Fig. 17a shows that obtained experimental values of fctm, sp are well
fitted by EC2 model.
with (t )
the normalized mechanical property which may be the The evolution of r fctm, sp is illustrated in Fig. 17b. It appears that the
(28days )
mean compressive strength (fcm), splitting tensile strength (fctm,sp) or incorporation of nutrients and Bacillus Subtilis led to an increase in the
dynamic modulus of elasticity (Ed), t the age of concrete in days, and s splitting tensile strength. The maximum value was achieved for 90 days
the coefficient depending on cement type. In this study, s = 0.2 for CEM then it decreased. This trend was observed for bacteria as well as nu-
II. trient concrete. As expected, this increase is less important for Nutrient
To separate the effect of bacterial from autogenous self-healing for a samples comparing to Bacteria ones. The results are consistent with the
(x )ti (x )tcontrol
given concrete age, a coefficient expressed as rx (t ) = was microscopic observations and microstructural analysis (see paragraph
(x )tcontrol
calculated where x is the considered property and i denotes that con- 3.1). The presence of Bacillus Subtilis resulted in a decrease in both
crete is formulated by adding either nutrient or bacteria. porosity and pore size, leading to an improvement of fctm, sp . In addition,
this phenomenon is more significant for curing ages less than 90 days,
after the Bacillus Subtilis activity decreased.
3.3.1. Mean compressive strength
The effect of bacterium Bacillus Subtilis on the compressive strength
at different ages and the verification of EC2 model is illustrated in 3.3.3. Elastic modulus
Fig. 16. The development of fcm against age of all concrete mixes The experimental results of dynamic modulus of elasticity (Ed) are
(Control, Nutrient and Bacteria) was studied on the basis of experi- given in Fig. 18. It can be outlined that, Ed increased when nutrients
mental results and those of the literature [9,20,21,26,64]. According to and bacteria were added. This growth was slightly more significant
EC2, the normalized mean compressive strength is expressed by Eq. with the presence of bacteria. This increase is mainly due to the con-
f (t ) solidation of pores inside the concrete specimens with micro-
(15) with f cm = cc (t ) and = 1.
(28days )
cm biologically induced calcium carbonate precipitation and attributed to a
The evolution of fcm is reported on Fig. 16 where it appears that:
reduction in the porosity as established previously (Fig. 13 in paragraph
3.2). These results show that the addition of Bacillus Subtilis improved
• An increase of f cm is observed for all concrete mixes. Moreover, it
Ed which is necessary for the design of concrete structures.

9
T.H. Nguyen, et al.
Fig. 16. The verification of EC2 model (a) and variation of compressive strength (b) with age.
The assessment of Ed was conducted at different ages in Fig. 18a. It
E (t )
can be observed that Eq. (15) expressed as E (28d days) = cc (t ) and = 0.3
d
well described the evolution of Ed made with nutrients and bacteria.
This figure shows that EC2 model can be applied for concrete with and
without bacteria.
Omary et al. [65] established a relationship between porosity and
elastic modulus of concrete (Eq. (16)). The validity of this expression is
confirmed in this work (Fig. 19).
Ed (GPa) = 65 × exp[ b × n (%)] with and b = 0.03 (16)
3.4. Capillary water absorption results
The transport of aggressive fluids and gas is affected by the porosity,
pore size distribution, connectivity and pores tortuosity as well as the
exposure conditions and properties of the diffusing substances (surface
tension, viscosity, density, hydrophilicity) [66]. Concrete specimens
with bacteria showed a significantly less water absorption compared to
control samples (Fig. 20) due to both mechanisms of the continuous
hydration and the bacteria-induced precipitation (with the bacterial-
treated specimen). The results were also observed by other authors for
less exposure time, until 28 days with Bacillus mucilaginous and Brewers
yeast [23] and 91 days with Sporosarcina Pasteurii [29]. SEM
Fig. 17. The verification of EC2 model (a) and variation of splitting tensile strength (b) with age.
10
Cement and Concrete Composites 104 (2019) 103340
observations (paragraph 3.1) showed that these products have filled
cracks and porosities leading to the decrease of pore size. Water ab-
sorption tests are related to capillary pores without given indications on
the pore size and tortuosity. However, capillary water absorption tests
provide indications on portal microstructure (pore size, tortuosity and
interconnectivity). For example, water capillary lift is slower in smaller
pores than in larger ones while the level of water elevation in the ca-
pillaries is inversely proportional to the pore size.
Hence, the sorptivity coefficient, kO, was calculated at 24 h ac-
cording to ASTM C1585-04 [44] for the different concrete formulations
and at different curing age. The obtained values summarized in Table 2
show clearly that k0 decreased as the duration increased. Moreover, the
results highlighted that the reduction of sorptivity was more significant
when microbial adjuvant was added to the concrete formulation.
These results obtained through capillary tests corroborated those
obtained through SEM analysis. Indeed, the reduction of sorptivity
coefficient indicates that pore size and the interconnectivity have di-
minished for nutrient and bacteria concrete mixes. A relative decrease
in the sorptivity of about 60% was noticed at 91 days concrete age that
was maintained up to 210 days. This is in agreement with the findings
of R. Siddique et al. [64], who noticed a reduction in the range of 50%
and 70% for the sorptivity coefficient at 28 and 56 days respectively. S.
Bhaskar et al. [34], V. Achal et al. [38] indicated that the addition of
bacteria resulted in a dense microstructure and a decrease in capillary
T.H. Nguyen, et al.
Fig. 18. The verification of EC2 model (a) and variation of Young's moduli (b) with age.
Fig. 19. The evolution of Young's moduli as a function of the porosity of con-
crete.
water uptake.
3.5. Air permeability
The effects of adding Bacillus Subtilis on the gas permeability of
concrete is presented in Fig. 21. It can be concluded that:
• The permeability decreased as the curing time increased (Fig. 21a)
for all tested concrete. The similar evolution trend as for the porosity
was observed.
•
The incorporation of nutrients and nutrients + bacteria as adjuvant
improved the resistance to gas penetration. The permeability was
less important for concrete with bacteria + nutrients than control
ones. This is explained by the formation of CaCO3 inside the voids
(microcracks, porosities, ….). The observed results are in agreement
with those related in the literature using Bacillus sphaericus bacteria
concrete tested at 28 age days [36,37].
To separate the effect of bacterial self-healing from autogenous one,
(Kint )tnutrient / bacteria
the variation coefficient, rpermeability (t ) = (Kint )tcontrol
was calculated and the results were reported in Fig. 21b. It appears
clearly that the addition of Bacillus Subtilis induced a decrease in Kint up
to 210 days while the effect of nutrients was significant until 90 days,
duration after which a steady state was reached. The same trend was
observed for the porosity evolution (paragraph 3.2). It is evident that
Cement and Concrete Composites 104 (2019) 103340
the presence of calcium carbonate products, by microbial-induced ac-
tion, improved the resistance to gas permeability.
Moreover, relationships in the form of y = e x were established
with good correlation between the intrinsic permeability and the por-
osity on one hand and sorptivity coefficient on the other hand was il-
lustrated by Eq. (17) and Eq. (18) (Fig. 22).
Kint (10 17m2 ) = 0.01e 0.4× n (%) with R2 = 0.99 (17)
Kint (10 17m2 ) = 0.22e103777× ko (m / s) with R2 = 0.91 (18)
3.6. Rapid chloride permeability test (RCPT)
Corrosion of reinforcing steel due to chloride ions diffusion is one of
the most frequent environmental attacks that lead to the deterioration
of structures. The pore structure is among the important parameters
controlling the concrete durability against chloride ions penetration. As
established previously, incorporating Bacillus Subtilis as microbial ad-
juvant conducted to a decrease in the total porosity accessible to water,
the sorptivity and the concrete permeability. Therefore, it can be ex-
pected that ions diffusion will significantly decrease. This has been
confirmed experimentally by measuring the chloride ions diffusion
depth in Fig. 23 and by calculating the non-steady-stade migration
coefficient Dnssm (m2 / s ) (Eq. (4)).
Fig. 24 Shows a significant improvement of the concrete durability
when nutrients and/or Bacillus Subtilis were added to the formulation.
On the other hand, the effect of Bacillus Subtilis was more relevant than
nutrient one. Moreover, it appeared that the improvement associated
with bacterial healing was efficient up to 28 days. This result is in ac-
cordance with those of the literature [9,20,21,64]. It should be outlined
that incorporating Bacillus Subtilis as microbial adjuvant without urea
was more efficient than microbial adjuvants including urea used in the
literature [20,21,64].
The pH variation of NaCl solution before and after tests is given in
Fig. 25. It can be outlined that the variation of pH was less important
for Bacteria specimens. This smaller change of pH means that the
bacterial specimen has not absorbed too much Cl− ions due to the
lower porosity and sorptivity coefficient of bacteria concrete by com-
parison to the control and nutrient ones. Nutrient concrete has lower
(Kint )tcontrol porosity (Fig. 13b) and sorptivity coefficient (Fig. 20) than control one
100 , because urease activity occurs when nutrient medium was added to the
concrete mix as bacteria can be found in aggregates, mixing water, ….
Correlations between the non-steady chloride migration coefficient,
Dnssm, and permeability (Fig. 26) as well as between Dnssm and sorp-
tivity coefficient were established. Obvious that the sorptivity coeffi-
cient impacted more the chloride migration coefficient. Relationships
11
T.H. Nguyen, et al. Cement and Concrete Composites 104 (2019) 103340

Fig. 20. The rate of water absorption versus time.

Table 2 and yeast extract) and Bacillus Subtilis without urea as a microbial
Values of k0 (cm/s1/2) at 24 h calculated according to ASTM C1585-04 stan- adjuvant could be an effective way to promote microbial CaCO3 pre-
dard. cipitation in sufficient quantities to enhance sustainability and to pro-
Concrete formulations Age before capillary tests mote self-healing of concrete. This microbial adjuvant was added to the
mix design of C35/40 strength class concrete. Three formulations were
28 days 90 days 210 days studied: the control concrete, concrete containing nutrient and concrete
elaborated with microbial adjuvant. Different experiments were, then,
Control 2.3 ± 0.058E-03 2.0 ± 0.032E-03 1.5 ± 0.037E-03
Nutrient 1.9 ± 0.056E-03 1.4 ± 0.12E-03 1.3 ± 0.072E-03 conducted on these three formulations up to 210 age days. The ob-
Bacteria 1.7 ± 0.027E-03 8.0 ± 0.054E-04 6.0 ± 0.046E-04 tained results highlight the following conclusions:

are described by Eq. (19) and Eq. (20):

• Ureolysis of Bacillus Subtilis in yeast extract and peptone medium
provides CaCO3 precipitation and therefore microbial self-healing of
17 2 based cement concrete (CEM II/A). SEM observations showed that
Dnssm (10 12m2 / s ) = 9e 0.4 × Kint (10 m ) and R2 = 0.88 (19)
the products are in the form of bouquets-like, rhombohedral-shaped
and needles-like. The 400 μm cracks width of bacterial concrete
Dnssm (10 12m2 / s ) = 7e 49456ko (m / s) and R2 = 0.93 (20)
were completely closed after 44 days of water immersion. On the
other hand, only few quantities of calcites were observed on the
4. Conclusions crack of specimens with nutrient and negligible precipitations for
control one due to the autogenous self-healing. The percentage of
This work was intended to show that the use of nutrients (peptone calcium carbonate and the kinetics of self-healing process in the

Fig. 21. The gas permeability coefficients (a) and variation of Kint (b) at different ages.

12
T.H. Nguyen, et al. Cement and Concrete Composites 104 (2019) 103340

Fig. 22. Coefficient of air permeability versus the porosity (a) and the sorptivity coefficient (b), ko, of concrete.

Fig. 23. The chloride depths (a) and variation of chloride depths (b) at different ages.

Fig. 24. The non-steady chloride migration coefficient (a) and variation of Dnssm (b) at different ages.

bacterial specimens were significantly higher than those without
bacteria. These observations proved that peptone and yeast extract
without urea act as source of nutrients for Bacillus Subtilis and
promote its urease activity.
• Bacteria concrete exhibited better resistance to capillary suction,
water absorption and total porosity accessible to water than nutrient
concrete and control concrete. The reduction in sorptivity coeffi-
cient was important when microbial adjuvant was added to the
concrete suggesting that pore size and interconnectivity have
diminished. This reduction is significant after 210 days (60%)
especially because no urea was added to concrete formulation
contrary to literature results obtained up to 56 days.
• A substantial decrease in gas permeability was noticed when mi-
crobial adjuvant was incorporated to concrete with a relative re-
duction of about 70% at 210 days. These results are encouraging
compared to previous researches [36,37] which obtained a gas
permeability decrease by adding Bacillus sphaericus, urea and nu-
trients of 32% at 28 days while in this study a 50% relative decrease

13
T.H. Nguyen, et al.
Fig. 25. The change of pH in NaCl solution (a) and variation of pH (b) at different ages.
Fig. 26. Coefficient of Dnssm versus Kint (a) and the sorptivity coefficient (b), ko, of concrete.
was achieved.
• Chloride ions diffusion have significantly decreased by in-
corporating in concrete bacillus subtilis in yeast extract and peptone
medium. The results showed that the efficiency of microbial ad-
juvant has been effective up to the age of 28 days. After, no sig-
nificant changes were observed up to 210 days. The pH change
confirmed that bacterial concrete samples have not absorbed too
much Cl-ions due to their lower porosity, sorptivity and gas per-
meability coefficients by comparison to control ones. Elastic mod-
ulus, mean compressive strength and splitting tensile strength have
strongly decreased by adding microbial adjuvant. The evolution of
these properties was studied up to the age of 210 days. It was es-
tablished that Eurocode 2 design code can be successfully applied to
predict the properties and therefore the mechanical behavior of
bacteria-concrete.
• Relationships established previously for concrete without microbial
adjuvant can be extended to bacteria concrete (water absorption
coefficient and Young's modulus as a function of porosity).
• Relationships have been established to correlate permeability to
porosity and sorptivity and the non-steady chloride coefficient to the
intrinsic permeability and sorptivity.
Acknowledgments
The authors would like to acknowledge Pr. Pascal GONNON; OMYA
SAS for providing limestone filler and Dr. Damien SEYER; University of
Cergy Pontoise for providing bacteria.
14
Cement and Concrete Composites 104 (2019) 103340
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