Construction and Building Materials 226 (2019) 492–506

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Construction and Building Materials

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Synthesis and characterization of bio-immobilized nano/micro inert and

reactive additives for feasibility investigation in self-healing concrete
Nafeesa Shaheen a, Rao Arsalan Khushnood a,b,⇑, Wasim Khaliq a, Habib Murtaza a, Rafay Iqbal a,
Muhammad Humza Khan a
a
NUST Institute of Civil Engineering (NICE), School of Civil and Environmental Engineering (SCEE), National University of Sciences and Technology (NUST), Sector H-12,
Islamabad 44000, Pakistan
b
Department of Structural, Geotechnical and Building Engineering, Politecnico di Torino, Corso Ducadegli Abruzzi 24, Turin 10129, Italy

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Direct induction and immobilization

were used for formulating self-
healing concrete.

Iron oxide and bentonite particles
were used for immobilization of
microbes.

Microbial instigated precipitation at
cracks was analyzed by SEM, EDX,
XRD and TGA.

Bacillus subtilis immobilized via iron
oxide particles healed cracks
successfully.

Bentonite was least-effective in
preserving microbes due to its
reactive nature.

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

Article history: Bacteria-based self-healing concrete can be made more efficient by using immobilization techniques,
Received 15 October 2018 hence shielding the bacteria from crushing during mixing to remain dormant till the development of con-
Received in revised form 2 June 2019 crete cracks for the uniform healing of developed cracks. In this study, two intrusion approaches, direct
Accepted 19 July 2019
induction and immobilization, are deliberated by using ‘Bacillus subtilis’ bacteria for self-healing concrete.
Furthermore, for immobilization’s evaluation, two media namely iron oxide nano/micro particles (INMPs)
and bentonite nano/micro particles (BNMPs) were selected. Compressive and tensile strengths were com-
Keywords:
puted at 3, 7 and 28 days. Moreover, field emission scanning electron microscopy (FESEM), X-ray diffrac-
Bio-inspired self-healing
Bacillus subtilis
tion (XRD), X-ray fluorescence (XRF) and thermo-gravimetric analysis (TGA) were employed for
Nano/micro iron oxide particles monitoring self-healing efficiency. Results revealed that immobilization through INMPs media was best
Nano/micro bentonite particles among all intrusions ensuing successive healing of cracks up to 1.2 mm width having 85% recovery in
Compressive strength compressive strength after pre-cracking. Whereas, BNMPs immobilization and direct induction exhibited
Split tensile strength crack healings up to only 0.15 mm and 0.45 mm crack healing widths with 45% and 65% strength recov-
ery, respectively.
Ó 2019 Elsevier Ltd. All rights reserved.

Abbreviations: INMPs, Iron oxide Nano/Micro particles; BNMPs, Bentonite Nano/Micro particles; GNPs, Graphite Nano Platelets; LWA, Light Weight Aggregate; ACI,
American Concrete Institute; ASTM, American Society for Testing and Materials; SP, Superplasticizer; Ca(OH)2, Calcium Hydroxide; .CaCO3, Calcium carbonate; OPC, Ordinary
Portland cement; SEM, Scanning electron microscopy; TG, Thermogravimetry; EDX, Energy dispersive X-ray; XRF, X-ray fluorescence.
⇑ Corresponding author at: Department of Structural, Geotechnical and Building Engineering (DISEG), Politecnico di Torino, Corso Ducadegli Abruzzi 24, Turin 10129, Italy.
E-mail addresses: rao.khushnood@polito.it, arsalan.khushnood@nice.nust.edu.pk (R.A. Khushnood).

https://doi.org/10.1016/j.conbuildmat.2019.07.202
0950-0618/Ó 2019 Elsevier Ltd. All rights reserved.
 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506
1. Introduction
The Self-healing concrete has the potential to mitigate the prob-
lems associated with cracking without external intervention and
an emulating way of sustainability by eluding the substantial envi-
ronmental impacts [1]. It can be formulated using numerous tech-
niques, broadly categorized as autogenous and autonomous
healings [2]. Autogenous healing includes the formation of hydra-
tion products to fill the cracks subsequent to secondary hydration
of anhydrous particles while autonomous healing is achieved by
the addition of micro-organisms, mineral admixtures, polymers
or expansive agents [3–6]. Among them, bio-inspired self-healing
is under prime consideration in the recent era in order to accrue
incentives subsequent to amplified strength and consumed CO2
as a result of microbial activity [7,8].
The idea of crack healing via microorganism’s additions into
concrete originated in 1998 by Ramakrishnan and coworkers [9].
Bacillus pasteurii and Pseudomonas aeruginosa microbes were
investigated for remediation of artificially cracked cementitious
mortar specimens. Later on, Ghosh, Mandal, Chattopadhyay and
Pal [10] used Escherichia coli and shewanella specie for strength
improvement in cementitious mortar specimens in 2005. Then, in
2007, Jonker [11] incorporated microbes into the concrete matrix
during mixing for making a homogenized autonomous healing sys-
tem. Furthermore, self-healing cementitious specimens were for-
mulated and designated as a two-component healing system,
using Bacillus pseudofirmus and Bacillus cohnii which were cul-
tured from high alkaline lakes for catalyzing the conversion of an
organic compound into calcite to attain the copious amount of
healing precipitations [12,13].
Profoundly, bio-influenced healing of cementitious systems
depends upon the production of calcium carbonate which is
directly related to the high alkalinity of concrete, dissolved inor-
ganic compounds, nucleation sites, and presence of calcium ions
throughout the mixture [14]. Moreover, it also depends upon the
microbial type and its concentration, curing procedures and
material used for immobilization of intruded microbes [15,16].
Immobilization techniques of preserving the microbes in the
protective carriers secured an imperative position in formulating
superlative self-healing cementitious systems owing to their con-
tribution in extending microbial survival by preventing them
crushing during mixing and inducing dormancy [17,18]. Initially,
expanded clay (EC) particles were employed for immobilization
of microbial spores and calcium lactate which were used as a
replacement of conventional course aggregates. These healing
agents amplified crack healing aptitude by attaining a maximum
of 0.46 mm crack healing widths with improved durability of
concrete, too [3,19]. Afterward, the immobilization of microbes
through different media initiated to ensure their persistence into
highly alkaline cementitious environments to achieve relatively
higher crack healing rates complimented with strength regains
[20]. Wang et al., investigated the potential of using polyur-
ethane sheet, silica gel, diatomaceous earth (DE) and hydrogels
for immobilization of Bacillus sphaericus in fabricating self-
healing cementitious mortar. Among them, hydrogels achieved
a maximum crack healing width of 0.5 mm followed by DE hav-
ing a maximum of 0.17 mm crack repairing widths [6,21,22]. The
immobilization of Bacillus sphaericus spores using micro-
encapsulation technique was also studied in which melamine
capsules having a size of 5 lm were used. Although, the use of
a micro-encapsulation technique, improved crack healing rate
up to 40–80% with a maximum of 0.97 mm healed crack width
however the compressive strength was degraded dramatically
up to 15–34% due to the formation of weak ITZs [23]. For
improving interfacial bonding of cementitious grains and
493
preventing the microbes from leaching, microbes of genus Bacil-
lus were coated by geo-polymer (metakaolin) [24]. The ceram-
site, as a carrier was used in another study for immobilization
of bacteria and nutrients separately, which exhibited successful
enhancement of area repair rate up to 87.5% having a maximum
crack width of 0.5 mm, complemented by the decrease in water
permeability and increase in flexural strength [25]. The potential
of lightweight aggregate (LWA) for microbial intrusion into the
cementitious environment was studied by different researchers.
The shortcoming of such self-healing cementitious system was
the reduction in intended mechanical properties as compared
to conventional concrete systems [26–28]. Furthermore, Khaliq
and Ehsan [29] investigated the effect of intrusion methods on
the efficiency of Bacillus subtilis in self-healing concrete using
three different mechanisms. These were a direct intrusion, intru-
sion through carrier medium namely lightweight aggregate
(LWA) and graphite nanoplatelets (GNP). Intrusion through
GNP immobilization was found effective at an early age while
LWA immobilization exhibited greater crack healing at later ages
[29]. Bio-immobilized zeolite was incorporated in conventional
and fiber reinforced mortar for comparing the microstructural
and crack healing properties. Results confirmed the improved
compressive strength with reduced chloride ion penetration
resulting from plugged pores of 0.07 mm widths [30]. In another
study immobilization through expanded perlite (EP) was com-
pared with direct induction and bio-immobilized expanded clay
(EC) [31]. Findings of experimentation evidenced that EP immo-
bilized bacteria exhibited efficient crack healing maximum of
0.79 mm and higher strength recovery relative to EC-
immobilized self-healing concrete exhibited the maximum of
0.45 mm crack healing width.
The state of the art highlights the viability of various materi-
als as immobilization media and their potential feasibility in
self-healing cementitious systems. Despite all, there is still room
for further investigation of immobilization media for formulating
long-lasting, resilient and cost-effective concrete having the abil-
ity to ensure persistent microbial survival for relatively longer
durations and attaining more crack-healing widths without dis-
tressing mechanical characteristics. To the best of the author’s
knowledge, a maximum of 0.97 mm crack healing width has
been reported while the mechanical strength of that system
was declined up to 34% [23]. For attaining higher crack widths
homogenized system of uniform dispersion of bio-influenced
nano-sized particles must be devised. Merely, GNPs were inves-
tigated as bio-influenced nano-media, but their carbonaceous
nature having anti-bacterial characteristics badly affect the sur-
vival of microbes [29]. Hence it is necessary to investigate new
media, impudent to anti-bacterial properties for formulating
homogenized dispersion of microbes throughout the matrix for
microbial activity on the discrete level of crack initiation. More-
over, one of the major hindrances in the global application of
self-healing concrete is its manufacturing cost and requisition
of complex technology. In the present study, for making long-
lasting, resilient and cost-effective concrete, indigenous raw
materials; namely iron oxide and bentonite were used as innova-
tive microbial carriers and synthesized in the laboratory into
nano/micro sized particles to ensure uniform dispersal of micro-
bial spores for maintaining identical crack healing rates. The
inspected immobilizing media, iron oxide is an inert media and
retains its structure in the host matrix while bentonite is poz-
zolanic in nature and capable to contribute to autogenous heal-
ing as well. Moreover, these media have never been previously
investigated in the context of microbial survival in self-healing
concrete.
494 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506

2. Experimental program 8

7
2.1. Materials Exponential phase Death phase

No. of viable cells (log)

6
2.1.1. Concrete constituent materials Stationary phase
Ordinary Portland cement CEM-I (grade 53) conforming to 5
ASTM C-150 having a specific gravity of 3.05 g/cm3 and an average 4
particle size of 20 lm was used in the study. Siliceous sand form
local source was used as fine aggregates having a fineness modulus 3
of 2.6 and density of 2.83 g/cm3 along with Margalla crush, a local
2
source name, as coarse aggregate with a fineness modulus of 6.77
and density of 1.37 g/cm3. The physical and chemical properties of 1 Lag phase
CEM-I are summarized in Table 1. The chemical composition in
0
oxide form was determined using X-ray fluorescence technique 0 20 40 60 80 100
(XRF).
Time (hours)

2.1.2. Germination of microorganism Fig. 1. Different phases of Bacillus subtilis life cycle curve.
B. subtilis is grampositive (having peptidoglycan layer) bacteria
commonly found in soil [32]. It has a rod-shaped structure, can
form protective endospores in the alkaline environment and This was the longest phase spanning over 48 h as evident from
endure extreme pH and temperature [33]. Before preparing a sam- the life cycle curve shown in Fig. 1. The third one is the stationary
ple of culture from a fresh batch of B. subtilis was sent to South phase that indicates the growth limitation of bacteria caused by
Korea for the identification and verification of their type. B. subtilis depletion of necessary nutrients, accumulation of waste materials,
used in the study has American type culture collection (ATCC) toxic metabolisms and formation of an inhibitory product which
number 11,774 which is usually manufactured under ISO changes the pH and temperature of the solution. In the stationary
9001:2008 certification. phase, the growth rate becomes almost equivalent to the death
The life cycle of B. subtilis microbes in a new growing media is phase. This particular phase spanned over to 16 h as depicted by
quite pertinent in the selection of incubation periods to have a a horizontal straight line of the life-cycle curve (Fig. 1). In the death
maximum strength of inducted microbial species in the selected phase, the microbes completely lose their ability to reproduce. It
media. The growth curve was prepared in controlled microbiology begins to die due to the unfavorable conditions in the form of
lab using the standard protocol given by Prescott et al., 1999 [34] unavailability of nutrients, unfavorable environment and the sub-
after rigorous efforts of 72 h as given in Fig. 1. The curve has four sequent accumulation of metabolic waste products. Only those
distinct phases corresponding to changes in microbial population organisms could resist in such conditions that are able to produce
with time. endospores. The growth curve reveals that B. subtilis strain incu-
When a microorganism is intruded into the fresh medium, it bated at 48 h will result in achieving maximum concentration to
requires some time to adapt the pristine environment. This phase be immobilized inside the cementitious matrix.
is known as the lag phase, in which microbes are maturing but The B. subtilis culture was incubated in the controlled microbi-
do not have the ability to divide and reproduce. During the lag ology laboratory (25 °C temperature; 95% relative humidity). For
phase, microbes evolve for the synthesis of ribonucleic acid the germination of mentioned microbes, the blank medium was
(RNA), enzymes and other molecules formation. The lag phase of selected. The process of microbial solution preparation is illus-
B. subtilis ranges up to 2 h as determined through the bacterial life trated in Fg.2 stepwise. For retrieving B. subtilis from glycol stock
cycle curve (Fig. 1). The second phase is characterized by cell dou- nutrient ager was poured in the plate (Fig. 2(a and b)) after that
bling and known as the exponential phase. The micro-organisms microbial stock was swabbed on a plate and incubated in the static
begin to replicate the DNA by binary fission at a constant rate incubator (Fig. 2(d)). Later on, grownup microbial colonies as
and thereby increase the metabolic process. The broth solution shown in Fig. 2(e) were inoculated in nutrient broth. Nutrient
was exploited at the maximal rate and the number of bacteria broth used for this purpose comprising of lab-lemco powder
increased logarithmically with a single cell split into two, which (1.0 g/l), yeast extracts (2.0 g/l), peptone (6 g/l) and sodium chlo-
further subdivided into four, eight, sixteen, thirty-two and so on. ride (5.0 g/l) as shown in Fig. 2(f). After inoculum, NB broth was
incubated in shaker for 48 h. For sporulation, difco sporulation
medium (DSM) was used by mixing KCl (0.2 g/l), 1 M MgSO4
Table 1
(0.5 ml) and MnSO4 (.5g/l). After autoclaving, 1 M CaCl2 (0.5/g)
Chemical and physical properties of materials used.
and 0.5 ml of 1 mM FeSO4 (0.5 ml) was added and incubated for
Parameters CEM-I BNMPs INMPs 4 days at 37 ͦC with shaking at 200 rpm. Cells were pelleted at
Aluminum oxide (Al2O3) 9.86 20.18 0.39 9000 rpm for 20 min at 4 ͦC. Spore inside the LB are shown in
Silicon dioxide (SiO2) 18.9 54.54 0.31 Fig. 4(a) and marked via red circles. B. Subtilis spores usually
Magnesium oxide (MgO) 1.62 4.21 0.4
appear as white beads (rhombo-hexagonal) having a size range
Ferric oxide (Fe2O3) 3.43 8.59 86.5
Calcium oxide (CaO) 60.1 7.22 0.13 of 0.87–1.41 mm [35].
Phosphorus pentoxide (P2O5) 0.065 1.1 The blank solution was used as a reference to monitor the
Potassium oxide (K2O) 1.18 3.91 0.06 microbial concentration based on the measured absorbance value
Sodium oxide (Na2O) 0.83 1.23 0.03 of microbial solution using HACH DR 2400 Portable spectropho-
Sulfur trioxide (SO3) 2.62 – –
tometer shown in Fig. 2(h). The 0.5 ml quantity of blank solution,
Zinc oxide (ZnO) – 0.16 –
Titanium oxide (TiO2) – 0.92 4.12 was placed in a spectrophotometer with a selected optical density
Specific gravity 3.04 2.81 4.8–5.26 of 600 nm. After reading the blank solution by the machine, 0.5 ml
Blain fineness (cm2/gm) 1720 4800 – of microbial solution was used in a replacement at the same
Average particle size 20 mm 6.129 mm 6.63 mm
optical density of 600 nm. Finally, the concentration of microbes
Loss on ignition 1.03 5.428 0.1–0.3
in the solution was measured using the expression derived by
 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506 495

(c) B. subtilis spores (e) Microbial colonies (f) Inoculum in NB (g) Incubation in Shaker

(a) NB ager (b) Ager plating for growth of

microbial colonies (d) Incubation (h) OD measurements

Fig. 2. Microbial solution preparation.

D50 =2.57mm
Fabrication of nano particles D50 =6.63µm

(a) Milisized Iron oxide (b) Planetary ball (c) Soil compactor (d) INM Pused in the (e) immobilized
particles mill PM 400 HM-530 study INMPs
Fig. 3. Phases involved in synthesis of INMPs.

Spore

INMPs

(a) SEM image of bacterial spores in (b) Bacteria spores immobilized through
nutrient broth INMPs
Fig.4. FESEM micrographs.

Ramachandran et al., Y = 8.59  107X1.3627 [36]. Where Y is the
microbial concentration per ml and X is the reading at an optical
density (OD) 600, using the above-mentioned approach spore con-
centration in the investigated specimens was maintained equiva-
lent to 2.3  108 cells/cm3. Calcium lactate solution was used as
a precursor.
Field emission scanning electron microscopy (FESEM) of B. sub-
tilis spores in nutrition broth also evidence their homogenized dis-
tribution in the selected immobilizer is shown in Fig. 4(a) clearly
indicating bacterial spores [37].
For effective immobilization, protocol given by Seifan et al.,
2018 [38] was used with slight modification. Immobilization
media was sonicated for 1 min in distilled water then spores solu-
tion of B. subtilis was mixed 4.5% (v/v) and incubated at 30 °C
@145 rpm for 30 min for ensuring adequate immobilization.
2.1.3. Immobilization media
2.1.3.1. Iron oxide nano/micro particles (INMPs). INMPs were used as
a protective media for the intrusion of B. subtilis inside the con-
crete owing to their biocompatibility, large surface to area ratio,
496 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506
(a) Natural extract (b) Crusher
Fig. 5. Processing of BNMPs.
strong adsorption capacity, chemical inertness and superparamag-
netism [39,40]. INMPs as bacterial immobilizer have been
successfully employed in the fields of medicines, bioprocesses
and bio-fertilizer industries [41–43]. INMPs used in the study were
extracted from local natural iron ore named as ‘‘hematite”.
Availability of hematite a-F e2 O3 is more common in Pakistan and
it mainly comprises more than 60% of iron [44]. INMPs have been
previously investigated for improving pull out strength of concrete
[45], inducing piezo-resistivity into cementitious matrix [46] and
as a self-healing agent for bituminous materials [47]. In the recent
study, an effort has been made to explore its feasibility as an
immobilizer of B. subtilis in self-healing concrete.
The INMPs were fabricated using raw iron obtained from heavy
mechanical complex, Taxila [33.7463° N, 72.8397° E], Pakistan as
shown in Fig. 3(a). The milling operation was performed using
planetary Ball mill as shown in Fig. 3 (b) to have the particle refine-
ment in macro/micro ranges and soil compactor (HM530) for ultra-
refinement into nano/micro phase. The milled material was sieved
using #200 sieve to further segregate the remaining coarser grains.
The particle size of INMPs ranged between 0.259 and 17.37 mm as
determined through particle distribution given in Fig. 6(a). The
chemical composition of INMPs is summarized in Table 1 using
XRF. The incubated B. subtilis strain on INMPs appears to be a
blackish solution shown Fig. 3(e). The FESEM micrographs given
in Fig. 4(b) affirms the homogenous distribution of B. subtilis
spores in nutrients broth and then on the selected immobilizer of
INMPs. INMPs adhered to bacteria in electrostatic and hydrophobic
mode which is prone to release due to change in pH and upon
ingress of water and oxygen [31,48,49]. After inducing these spores
into concrete, their survival was continuously evaluated via bio-
mineralization process assessed through healed crack measure-
ments, strength recovery index, scanning electron microscopy,
energy dispersive X-ray spectroscopy and thermo-gravimetry.
Fig. 6. Particle size distribution of (a) INMPs (b) BNMPs using Laser Granulometry.
D50=6.129 µm
(c) Ball milling (d) Bentonite powder
Hematite powder obtained from a local source, employed in
research predominately consisted of ferrite (Fe) as verified from
energy dispersive X-ray (EDX) spectrum shown in Fig. 7(a), Fe con-
tents are 67.55% of total weight of the sample.
2.1.3.2. Bentonite nano/micro particles (BNMPs). Bentonite is cost-
effective, locally available and environment-friendly material.
Technically bentonite has a lot of potentials to be used as pozzolan
in concrete [50,51]. Previously, bentonite has been used to improve
bacterial survival inside the soil and emanate as an efficient carrier
for enhancing bacterial survival [52]. The density and water
absorption of concrete decreases with the insertion of bentonite
as a cement replacement [53]. An effort has been made to use ben-
tonite as an immobilization media to B. subtilis in self-healing con-
crete. Bentonite was locally obtained from Jehangira, Pakistan, site
[33.9642° N, 72.2186° E] having natural bentonite reserves of
approximately 26 km2 with an average bed depth of 3.5 m [54].
The average particle size 2.5 mm of raw bentonite was successfully
reduced to 6.129 mm using the milling scheme as elaborated in
Fig. 5. Laser granulometry was performed to determine the
detailed particle size distribution as given in Fig. 6(b). Chemical
composition was analyzed through XRF and EDX as summarized
in Table 1 and Fig. 7(b), respectively. The summative presence of
silicon dioxide (SiO2), aluminum oxide (Al2O3) and iron oxide
(Fe2O3) by more than 75% of the total weight affirms the pozzolanic
nature of BNMPs as standardized in ASTM: C 618-08a [55].
2.2. Mix proportions
Four diverse types of mixes were investigated in this study hav-
ing a designed fc’ of 28 MPa. A constant water-cement ratio (w/c)
of 0.4 was used for all the formulations with 1% superplasticizer.
Calcium lactate (precursor) was added 1% of cement weight, into
 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506 497

Fig. 7. EDX spectrum of (a) INMPs and (b) BNMPs.

Table 2
Designed mix proportions.

Mixes Cement (kg/m3) Sand (kg/m3) Coarse aggregate (kg/m3) (W/C) % Carrier medium(kg/m3) Bacterial culture (l/m3)
CM 480 920 1050 0.4 – –
Mix A 480 920 1050 0.4 – 5.9
Mix B 480 920 1050 0.4 16.4 INMP 5.9
Mix C 480 920 1050 0.4 16.4 BNMP 5.9

the solution containing bacterial spore, nutrient broth and a carrier
medium. Mixing proportions of the investigated formulations are
given in Table 2.
CM designated the reference formulation with no bacterium
intrusions while Mix ‘‘A” carried B. subtilis spores transmitted
through mixing water. Mix ‘‘B” and Mix ‘‘C” also contained B. sub-
tilis microbes immobilized on INMPs and BNMPs, respectively. For
immobilization of microbes, incorporating media INMPs and
BNMPs mixed with nutrient broth in the separate bottles and were
placed in an autoclave at elevated temperature and pressure for
15–20 min duration. Then a single colony of B. subtilis was inocu-
lated into the solution under the sterilized condition. After the
inoculation process, the solution was placed in an incubator at
37 °C for 48 h.
2.3. Casting of specimens and testing regimes
In total 108 concrete cylindrical specimens of (150  300 mm)
were casted having 32 specimens against each among the four
investigated formulations. The prepared specimens were kept
under controlled conditions for the initial 48 h at 90% relative
humidity and 25 °C temperature. The demolded specimens then
moist cured till their targeted age of testing.
The testing regime was sub-divided in three phases. Firstly
mechanical properties were tested in terms of compressive and
tensile strengths. Compressive and tensile strengths were evalu-
ated at regular intervals of 3, 7 and 28 days after curing of speci-
mens. In the second phase, crack healing mechanisms were
monitored through visual, mechanical and micro-graphical means.
Real cracking conditions were simulated to induce multiple realis-
tic structural and non-structural cracks. In the literature, research-
ers have reported different arrangements for crack creation
comprising of split tensile test, compression test, 3-point bending
test and use of steel fibers for controlled cracking [26,56,57]. Dong
et al. [58] loaded specimens up to 60% of compressive strength for
crack formations. In the present study, specimens were pre-
cracked up to 85% of their compressive strength for visible cracks
having relatively wider crack widths for simulating real cracking
conditions to analyze the self-healing performance. The induced
cracks of different aperture were measured on the specimens by
using a crack width measuring microscope HC-2950 and were
marked for further monitoring of self-healing. The pre-cracked
samples were again subjected to immersed curing until the healing
498 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506
evaluation periods of 28 days. Moreover, for internal healing eval-
uation, regain in compressive strength was measured. For this pur-
pose, specimens were pre-cracked up to 0.8 of fc’ at the age of 3, 7
and 28 days then they were moist cured up to 28 days. After that
compressive strength test was performed for estimating the heal-
ing rate. Meanwhile, FESEM analysis was also employed for inspec-
tion of healed specimens. In the third phase, the healing precipitate
was characterized and its morphology was examined through XRD,
TGA, XRF and EDX spectrographs after 28 days of moist curing of
cracked specimens for endorsement of calcite formation and inves-
tigation of its morphological characteristics.
3. Results and discussions
Results obtained from the systemized testing regime are pre-
sented and discussed in this section. Mechanical properties of
bio-influenced self-healing concrete containing immobilized B.
subtilis were evaluated by compressive and tensile strength tests.
Crack healing efficiency was examined through visual inspection,
mechanical and micro-graphical means in addition to morpholog-
ical observation and characterization of healed precipitate through
XRF, EDX, XRD and TGA.
3.1. Mechanical properties of bio-influenced self-healing concrete via
immobilized B. Subtilis
3.1.1. Compressive strength analysis
The compressive strength of the investigated four concrete
mixes were measured using MCC8 compression test machine con-
forming to ASTM test standards C-39 [59] and results are summa-
rized in Fig. 8. It can be seen that the addition of B. subtilis
improved the compressive strength of all types of concrete mixes.
Specimens of CM endured 18 MPa compressive strength at
3 days testing. The compressive strength of these specimens kept
on increasing with the passage of time due to the ongoing hydra-
tion of CM specimens. At 7 and 28 days, compressive strengths
were observed 26 MPa and 32 MPa, respectively. In Mix A speci-
mens having water as carrier media depicted slightly higher
strength than CM specimens. So, it is inferred that the inclusion
of B. subtilis directly through mixing water improved compressive
strength by 8% compared with CM. This strength increase may be
attributed to refinement in the microstructure of the resultant
matrix due to bio-synthesized calcite. The results are in harmony
with the study performed by the Ramachandran, et al. [36] that
B. subtilis are a better choice as compared to B. pasteurii which
presented insignificant strength increase.
CM
Coompressive strength (MPa)
40
Mix A
Mix B
30
Mix C
20
10
0
3 7
Curing time (days)
Fig. 8. Compressive strength development in bio-influenced self-healing concrete
using different immobilizers.
Samples with INMPs as conducting media showed the maxi-
mum compressive strength of 37 MPa after 28 days of curing
which is almost 21% more than the control samples. The increased
compressive strength is in agreement with the results of the study
carried out by Sikora, et al. [60]. Compared to other techniques,
INMPs offered as the best conducting media for B. subtilis with
the improved microstructure of the concrete supplemented by
enhanced cracks healing rate having amended durability. Also,
due to the ultrafine size of INMPs, it acted as a filler material,
resulting in a more compacted microstructure [61].
Specimen incorporated with BNMPs as carrier media depicted a
slight change in compressive strength as compared to control sam-
ple similar results are reported in the literature [62] and is attrib-
uted to the pozzolanic nature of bentonite and weaker interaction
between BNMPs and coarser aggregate. Further strength reduction
can be attributed to dilution of cementitious content [63]. The poz-
zolanic bentonite starts reactivity after about 28 days inside the
cementitious matrix [64] and such reactivity affects the healing
efficiency in BNMPs.
3.1.2. Splitting tensile strength
An indirect measure to evaluate the bond strength between
deposited media and the adjacent matrix through splitting tensile
strength tests is considered. A split tensile strength test was per-
formed according to procedure conforming to ASTM-C496/
C496M standards [65]. Splitting tensile strength values were mea-
sured for bio-influenced self-healed concretes and compared to CM
as shown in Fig. 9. The figure demonstrated the split tensile
strength of the investigated formulations at varying intervals of
3, 7 and 28 days. The result endorsed the relating strong interfacial
bond between the bio-synthesized calcite and the adjacent cemen-
titious matrix.
It is evident from results that the addition of microbes
improved the tensile strength of all formulations. Specimens of
CM attained 2.37 MPa split tensile strength at 3 days which kept
on increasing up to 3.16 MPa at 28 days of testing. A similar trend
of tensile strength increment was observed in Mix A and Mix B. In
Mix A, the addition of B. subtilis enhanced split tensile strength up
to 7% compares to CM at 3 days of testing. Likewise, split tensile
strength of Mix A specimens, increased up to 3.5% and 2% at 7
and 28 days of testing, respectively. Mix B, containing
bio-immobilized INMPs attained higher splitting tensile strength
values among all mixes at 3, 7 and 28 days of testing with a
percentage increase of 13%, 12% and 7.5% compared to CM, respec-
tively. These results were in accordance to a study conducted by
Kishar, et al. [66]. Mix C having BNMPs secured 8% and 4% more
split tensile strength values after 3 and 7 days as compared to
CM
Split tensile strength (MPa)
Mix A
4
Mix B
Mix C
3
2
1
28 0
3 7 28
Curing time (days)
Fig. 9. Split tensile strength of bio-influenced self-healing Concrete.
 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506
CM while the reduction was observed after 28 days in comparison
to CM. Split tensile strength values of BNMPs were lagging at 7 and
28 days for all four formulations and a similar trend was reported
in the literature [50,62].
3.2. Crack healing investigation
3.2.1. Visual evidence
Visual inspection is one of the most effective nondestructive
testing criteria to quantitatively gauge healing efficiency of con-
crete. The traces of any visible calcium carbonate precipitation
were monitored in the pre-cracked specimens after the specified
intervals of 3, 7 and 28 days. The widths of healed cracks were
gauged via a crack width measuring microscope. Crack sealing of
all four mixes at the healing age of 28 days is demonstrated in
Fig. 10. The healing was measured in millimeters (mm) by moni-
toring the difference of initial crack and healed crack widths at
specified intervals of time.
In control samples, there were no traces of calcium carbonate as
it did not contain B. subtilis to provoke microbial action for effec-
tive healing of cracks. A little healing was attained in some micro
cracks (Fig. 10) that may be associated with autogenous healing
of referenced formulations. Autogenous healing is a natural pro-
cess of crack healing that triggers in presence of moisture when
calcium hydroxide and carbon are available in water instigating
precipitation of calcium carbonate crystals. Additionally, the con-
tinuous hydration process of cement particles and the physical
clogging of cracks by detached loose particles also contribute to
autogenous healing. Mix A, using water as conducting media
depicted limited crack healing that was partial and non-uniform.
Since the mixing water is partially consumed in the hydration pro-
cess and partially evaporates, therefore the absence of media com-
promises the survival of B. subtilis furthermore. The small content
of water molecules confined inside the gel pores of the matrix pre-
served the bacteria and contributed in partial healing of micro-
cracks [31]. The healing efficiency also increased with the passage
of time as 0.08, 0.15 and 0.44 mm healing was observed after 3,7
and 28 days of pre-cracking, respectively. Water presented in the
10 mm 10 mm
Negligible healing in control sample (CM)
10 mm
1.2 mm completely healed
10 mm
10 mm
Uniform healing in samples with iron oxide (Mix B)
Fig. 10. Healing efficiency of all four mixes.
499
micro and macropores of the concrete had evaporated and bacteria
in these pores couldn’t survive for the healing of cracks. Fig. 10
reveals that specimens of Mix B incorporating INMPs as the poten-
tial carrier showed significant crack healing and gave promising
results. INMPs were uniformly distributed in the cementitious
matrix and their presence at every nano-site is responsible for
the efficient and uniform healing of cracks. The healing rate also
increased with time as 0.11, 0.31 and 1.2 mm crack widths were
healed effectively after 3,7 and 28 days of pre-cracking respec-
tively. In Mix C, using BNMPs particles as the conducting media,
non-uniform and partial healing was observed. At early age, heal-
ing efficiency was a bit higher which was gradually reduced with
the passage of time that may be associated with pozzolanic nature
of bentonite in cementitious environments.
Crack healing efficacy for all formulations is compared in
Fig. 11. It is evident from that, INMPs are highly compatible with
Bacillus species resulting in better crack-healing among all formu-
lations both at initial and later stages. INMPs served as the promis-
ing media to preserve B. subtilis in the dormant stage till the
development of cracks and ensured effective sealing of cracks up
1.2
CM
1 Mix A
Healing width (mm) Mix B
0.8
Mix C
0.6
0.4
0.2
0
3 7 28
Healing time (days)
Fig. 11. Crack healing of specimens of all four formulations.
Partially healed
10 mm 10 mm
Partial healing in water inducted samples (Mix A)
Partially healed
10 mm
Partial healing in samples with bentonite (Mix C)
500
to 1.2 mm width, which is maximum in comparison to previously
reported crack healed widths in bio-influenced self-healing con-
crete. The maximum reported healed crack width in literature is
0.97 mm that was attained using microencapsulation of spores
by Wang et al., [23] followed by 0.81 mm which was achieved
via successful bio-immobilization of GNPs by Khaliq et al., [29].
Besides, expanded perlite contributed in 0.79 mm healing of cracks
while expanded clay attained a 0.45 mm crack healing width [31].
Ceramsite(sand) as microbial carrier sealed 0.4–0.5 mm crack
widths effectively [25] whereas, hydrogel healed maximum up to
0.5 mm crack healings [22]. Moreover, Wang et al., [6] employed
diatomite earth as an immobilizer to attain a maximum crack heal-
ing width of 0.15–0.17 mm.
3.2.2. Regain in compressive strength
For evaluating self-healing efficiency, specimens of formulated
concrete mixes were pre-cracked at 80% of their compressive
strength and then re-immersed in curing tank to gauge strength
regain in compression for 28 days. The objective of investigating
compressive strength regain after pre-cracking was to assess the
effectiveness of healing precipitate and monitoring its contribution
toward the strength developments and durability enhancement of
concrete.
The specimens were pre-cracked at 3, 7 and 28 days up to 80%
of their ultimate compressive strength (0.8Xfc0 = 0.8 * fc) and
tested in compression to evaluate the self-healing efficiency in
terms of percentage regained in compressive strength (RCS) after
respective healing periods using the following expression given
as Equation (1).
 
Cu  Cr
RCSð%Þ ¼ 1   100
Cu
where,
Cu = Referenced ultimate compressive strength as reported in
Fig. 8
Cr = Regained compressive strength after curing of pre-cracked
specimens
Regained compressive strength values of all formulations were
shown in Fig. 12 while percentages of compressive strength regain
were compared in Fig. 13. Specimens of CM pre-cracked after
3 days of curing attained 14 MPa compressive strength which
was 44% of maximum compressive strength value (fc’). The
strength recovery values of specimens pre-cracked after 7 and
35
CM
30 Mix A
Compressive Strength (MPa)
Mix B
25
Mix C
20
15
10
5
0
3 days 7 days
Pre-cracked age (days)
Fig. 12. Regained compressive strength development in bio-influenced self-healing
concrete using different immobilizers.
N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506
100 CM
90 Mix A
80
Strength Regain (%)
Mix B
70 Mix C
60
50
40
30
20
10
0
3 days 7 days 28 days
Pre-cracked age (days)
Fig. 13. Self-healing efficiency in Bio-influenced self-healing concrete.
28 days of curing were 11 and 10 MPa which were 34% and 31%
of ultimate strength value, respectively. Higher strength regains
rate of CM specimens pre-cracked at the curing age of 3 days is
attributed to the hydration kinetics which is higher at earlier ages
owing to the presence of anhydrous particle and decreased with
the passage of time on limiting the hydration reaction. In Mix A,
regained compressive strength of specimens pre-cracked after
3 days of curing was 21.6 MPa, about 65% of referenced ultimate
compressive strength while specimens pre-cracked at 7 days of
curing secured 48% strength regains. Moreover, 28 days strength
regained was about 32% which was comparable with CM. The addi-
tional compressive strength regains confirmed the evidence of
microbial activity in Mix A. Mix B dominated the other investigated
formulations at the analyzed pre-cracking ages of 3, 7 and 28 days.
ð1Þ Maximum strength regain 31.5 MPa (85% of ultimate compressive
strength) was attained in ‘‘Mix B” carrying immobilized Bacillus
species on INMPs for specimens pre-cracked at 3 days of curing
which was reduced to 60% and 46% for pre-cracked specimens at
7 and 28 days, respectively. Whereas, Mix C was capable of regain-
ing up to 45% of its initial compressive strength after 28 days of
curing for specimen’s pre-cracked at 3 days of curing while 37%
and 31% recovery in strength was observed for specimens pre-
cracked for 7 and 28 days. The marginal difference in the regained
compressive strength of the Mix C with CM evident the BNMPs
least potential to be used as immobilizer for B. subtilis in cement
environment. This belongs to one among the interesting findings
of this research. The reactive nature of bentonite material towards
slaked lime generated as hydration product, makes it ineffective to
preserve bacterial life.
It was observed that strength regained reduced with increasing
pre-cracking ages. All four formulation showed maximum strength
regained for specimens pre-cracked at 3 days of curing while least
was for specimens pre-cracked at 28 days of curing. This gradual
decrease can be an advocate with the two possible phenomena.
The first one relates to the decline in natural healing at later ages
due to decrease in the hydration of cementitious grains. The sec-
ond phenomenon links with the establishment of dense and rela-
tively strong microstructure at later ages exceeding 7 days, the
viability of microbes were compromised due to pressure applied
by denser microstructure. It is endorsed from the results, the addi-
tion of B. subtilis contributed to higher strength regains which is
attributed to calcite production subsequent to microbial activity.
The tests confirmed the bio-compatibility of INMPs with Bacillus
species leading to improved healing efficiency via bio-
28 days mineralization.
3.2.3. Scanning electron microscopy
In addition to visual examination and mechanical assessment of
healed concrete specimens, all four formulations were subjected to
 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506
microstructure image analysis using field emissions scanning elec-
tron microscopy (FESEM). The deposited layer inside the cracks
was carefully scratched and observed on higher magnification
using FESEM after 28 days of pre-cracking.
As calcium carbonate formation is considered responsible for
the crack healing in bio-self-healing concrete [67], therefore, the
production of calcium carbonate crystals is a prime consideration.
Calcium carbonate crystals are usually established in three differ-
ent polymorphs which are named calcite (trigonal), aragonite
(orthorhombic) and vaterite (hexagonal) [68]. Calcite is the most
stable form of calcium carbonate which is produced by microbial
action [69]. The mechanism involves is the reaction of calcium lac-
tate with oxygen in the presence of Bacillus species [70]. The
chemical reaction is given in Eq. (2), further illustrates the calcium
carbonate crystallization with a byproduct of water and carbon
dioxide gas formation.
CaC6 H10 O6 þ 6O2 ! CaCO3 þ 5CO2 þ 5H2 O
The produced CO2 gas as a result of metabolic action discussed
in Eq. (2), further adds in the precipitation of CaCO3 by interacting
Fig. 14. Field emissions scanning electron microscopy (FESEM) analysis of 28 days healed samples.
501
with Ca(OH)2 as given in Eq. (3). This interaction results in the
transformation of unstable Ca(OH)2 into stable CaCO3 crystals that
strengthen the healing mechanism of bio-influenced self-healing
concrete [71,72].
CaðOHÞ2 þ CO2 ! CaCO3 þ H2 O ð3Þ
FESEM images of all four formulations are shown in Fig. 14.
There is no indication of crack recovery in the FESEM image of
the CM mix at 28 days. As, CM does not contain microorganisms,
neither any traces of calcium carbonate are found here.
Whereas, in other three formulations, rhombohedra calcium
carbonate crystals are detected by FESEM imaginary. Calcium Car-
bonate crystals were identified chemically via spot EDS analysis
technique and further visual endorsements were made while com-
paring it with the previous literature based on their crystallo-
graphic morphology [27–29,73–76].Comparatively, higher
content of calcium carbonate crystals is presented in Mix B con-
ð2Þ
taining INMPs as a conducting media. Whereas, FESEM imaginary
for Mix A and Mix C shows a lesser number of calcium carbonate
crystals. It can be concluded that the healing efficiency of Mix B
CaCO3 Crystals
Rod-shaped Microbe
502 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506

is more as compared to other formulations which is attributed to a
higher quantity of calcium carbonate crystals [77].
3.3. Morphological characterization of healing precipitate
3.3.1. Energy dispersive X-ray analysis
Energy dispersive X-ray (EDX) is an analytical technique used to
quantify the elemental compositions and chemical characteristics
of materials. Preliminarily, EDX analysis of INMPs and BNMPs
was carried out for authentication of constituent elements. Like-
wise, EDX tests of four formulations were performed to character-
ize the emitted healing materials and investigate its possible
formation kinetics as shown in Fig. 15. The primary goal was the
confirmation of the total content of calcium carbonate presented
in healing precipitations.
Elemental EDX spectrums of healing precipitation in CM reveals
that 6.61% calcium is present in the mixed powder as shown in
Fig. 15(a) and its amount is normal as presented in the ordinary
concrete [78]. EDX of the other three formulations confirmed the
higher content of calcium element in the deposited layer. The
enhanced deposition of calcium oxide inferred that the bacteria
in concrete had consumed calcium lactate and produced calcium
carbonate. Bacterial survival is also confirmed by results (Fig. 15)
as the larger content of calcium in these conducting media’s is
responsible for production of more calcium carbonate. EDX spec-
trum of healing precipitation extracted from Mix A specimens
offered 31.41% of calcium element quantity which is identical to
Mix C with a value of calcium as 31.18%. EDX of Mix B portrayed
maximum contents of calcium element with a number of 41.37%
in the healing precipitate. Percentage weight analysis of samples
with iron oxide indicated that bacteria survived effectively and
produced more calcium carbonate as compared to other formula-
tions. EDS on bacterial cell wall is shown in Fig. 15(e). Calcite pre-
cipitation on bacterial cell wall is clearly evident from the results
as B. subtilis provides nucleation sites [79].
3.3.2. X-ray diffraction analysis
For further investigation of self-healing kinetics to examine the
morphology of the induced CaCO3 crystal and phase purity of the
samples, the healing precipitate was subjected to X-ray diffraction

Weight (%)
0 20 40 60

C 19.05
O 44.38
Element

Si 28.37
K 0.26
Ca 6.61
Fe 1.33

(a) CM

Weight (%)
0 20 40 60
C 7.16
O
Al 2.82 49.07
Element

Si 5.25
S 1.59
K 0.84
Ca 31.41
Fe 1.86

(b) Mix A

Weight (%)
0 20 40 60

C 10.4
O 46.23
Mg 0.47
Element

Al 0.39
Si 0.83
K 0.35
Ca 41.37

(c) Mix B
Fig. 15. EDX spectrums of healing-powder of all formulations.
 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506 503

Weight (%)
0 20 40 60
C 4.78
O
Mg 0.47 49.99

Element
Al 3.42
Si 5.66
S 2.91
Ca 31.18
Fe 1.59

(d) Mix C

Weight %
0 20 40 60

C 35.73
O

Elements
Mg 0.02 57.08
Si 0.03
Ca 7.15

(e) Spot EDS ofbacteria

Fig. 15 (continued)

analysis (XRD). The specimen was carefully scratched out of the
induced cracks and examined through X-ray diffraction technique.
Copper (Cu) was selected as an X-ray target because it can be cool
easily, due to its high thermal conductivity, and capable to produce
strong Ka and Kb lines [80,81]. The diffractograms were recorded
at a wavelength of 1.54 Å with distinctive peaks attained at differ-
ent 2-theta (2h) orientations as shown in Fig. 16. The only poly-
morph of calcium carbonate detected by XRD was calcite. It can
be seen from Fig. 13 that the dominant peaks obtained at 2-theta
(2h) orientation of 29.93° which belongs to the pure calcite as evi-
denced by Harrington [82]. The presence of calcite is again an indi-
cation of successful microbial activity. This also confirms that the
induced material in the cracks is calcium carbonate and is in har-
mony with the results obtained from previous studies [25,29,30].
investigated through X-ray fluorescence (XRF). XRF is an experi-
mental technique used for complete chemical analysis of speci-
mens and used widely for cement and concrete to obtain reliable
elemental characterization [83]. An XRF analyzer determines the
elemental composition of a sample by measuring the fluorescent/
secondary X-rays emitted from a sample when excited by a pri-
mary X-ray source. Each of the elements in a specimen produced
a unique set of characteristic fluorescent X-ray that is unique for
the concerned element’s qualitative and quantitative composi-
tions. The detailed XRF analysis of healing material is given in
Fig. 17.
The scratched powder is comprised of three main oxides named
as SiO2, Fe2O3 and CaO. The presence of a considerable amount of
CaO in induced precipitate evidenced the biosynthesis of CaCO3
as a result of microbial metabolic action.

3.3.3. X-ray fluorescence

The elemental composition of induced precipitation via immo-
bilized microbial cells on INMPs extracted out of cracks was further
3.3.4. Thermogravimetric analysis
Thermogravimetric analysis (TGA) is a method of thermal anal-
ysis which is used to examine the changes in physical and chemical
properties of materials as a function of increasing temperature
(with constant heating rate), or as a function of time with constant
temperature and constant weight loss [84]. Likewise, TGA can pro-
vide information about chemical phenomena including chemisorp-
tion, decomposition and solid-gas reactions [29].
Thermogravimetric analysis of powder scratched from the
healed crack of specimens carrying Bacillus species immobilized
via INMPs is given in Fig. 18. The thermal decomposition of pure
calcite ranges in between 600 and 800 °C [85] with the individual
chemical mechanism outlined in Eq. (4).

CaCO3 ðsÞ ! CaOðsÞ þ CO2 ðgÞ ð4Þ

Fig. 18 clearly evidences that the major weight loss by 6.5%

Fig. 16. XRD spectra of induced precipitation via immobilized bacterial cells in occurred in the referenced temperature range of 600–750 °C that
INMPs. may be associated with the decomposition of CaCO3. This further
504 N. Shaheen et al. / Construction and Building Materials 226 (2019) 492–506

Element Mass Mol Intensity

(%) (%) (cps/mA)

SiO2 27.03 35.78 15634

K2O 1.13 0.95 1664
CaO 29.03 41.17 48171
TiO2 0.93 0.92 1283
Cr2O3 1.53 0.80 3744
Fe2O3 39.33 19.59 103658
SrO 0.99 0.76 3719

Fig. 17. XRF spectra of induced precipitates via immobilized bacterial cells on INMPs.

102 5. Split tensile strength of bacterial concrete demonstrated higher

values as compared to control concrete at 3, 7 and 28 days,
100 Δm = 3% excluding BNMPs which attained lowest 28 days split tensile
strength.
98
6. Selection of immobilization material for self-healing agents is a
Weight (%)

96 key parameter as some reactive materials like BNMPs reduced

the crack healing efficiency, whereas, inert materials like INMPs
94 improved crack-healing efficiency.
92 It is recommended to have a detailed investigation on the quan-
Δm = 6.5%
tum of microbial cells wrapped and released via immobilizer dur-
90
ing the bio-healing process in future studies.
88
Declaration of Competing Interest
86
0 200 400 600 800 1000 The authors declare that they have no known competing finan-
Temprature (°C) cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Fig. 18. Thermo-gravimetric analysis of induced precipitate via immobilized
bacterial cells.
Acknowledgments

The research presented in this paper was sponsored by National

affirms the bio-mineralization of CaCO3 in consequence of micro-
bial metabolic action.
4. Conclusions
In the study, two types of nano/micro sized particles as innova-
tive carriers have been synthesized for investigation of their poten-
tial feasibility as bacterial immobilization media for self-healing
cementitious materials and compared with direct induction of bac-
teria. Based on cracks efficiency and microstructural properties
achieved through presented techniques, the following conclusions
are drawn.
1. SEM, EDX, XRD and TGA studies on concrete specimens proved
visually and chemically that incorporated bacteria through all
media’s can produce ample amount of carbonaceous minerals
which have the potential to seal the cracks.
2. INMPs gave promising outcomes by sealing cracks up to 1.2 mm
and regained compressive strength up to 85% after crack forma-
tion resulting from uniform dispersion of bacteria.
3. Direct induction of bacteria in concrete healed the cracks up to
0.44 mm with 65% strength recovery and gave a better result
than BNMPs.
4. BNMPs showed the least crack healing efficiency only up to
0.2 mm along 45% strength regain. Pozzolanic nature and its
reaction with cement hydrates could be regarded as the reason
for the least crack healing.
University of Sciences and Technology (NUST), Islamabad, Pakistan.
For a part of tests, the laboratory and faculty support provided by
IESE and SCME at NUST are appreciated. This research did not
receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors. Any opinions, findings, and
conclusions in this paper are those of authors and do not necessar-
ily reflect the views of sponsors/material providers.
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