Structures 28 (2020) 2646–2660

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Structures
journal homepage: www.elsevier.com/locate/structures

Effect of direct addition of two different bacteria in concrete as

self-healing agent
M. Pourfallahi a, A. Nohegoo-Shahvari a, *, M. Salimizadeh b
a
Department of Civil Engineering, Bandar Abbas Branch, Islamic Azad University, Bandar Abbas, Iran
b
Hormozgan Environmental Department, Organization of Environment, Bandar Abbas, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: In this paper, a suitable and practical method to make bacterial concrete on a large and bulky scale is presented.
Self-healing concrete In this method, first, bacteria compatible with concrete are extracted and identified. The bacteria are then added
Bacteria to the concrete in solution in a simple way. To find bacteria compatible with concrete, first, the soil near the roots
Compressive strength
of plants (rhizosphere) and two types of cement called Portland pozzolanic cement (PPC) for Darab city and
Water absorption
Portland cement type 2 (PC2) for Hormozgan-Bandar Khamir were used. After extracting the cultured samples, it
was observed that bacteria grew only in the soil sample and did not grow significantly in the cement samples.
Therefore, the cultivation and reproduction of bacteria from concrete fragments were investigated. At this stage,
concrete samples were prepared with two types of cement (PPC and PC2). Then, some concrete powder con­
taining all the constituent materials was used to extract bacteria. The resulting bacteria were identified by the
Deoxyribonucleic Acid (DNA) testing. Bacterial samples obtained from the concrete made of PPC with certainty
exhibited the genus and species of Bacillus paralicheneniformis, but in the case of bacterial samples of concrete
prepared with PC2 can only be bacterial Cited by Bacillus sp. Afterward, 4 mixing designs were designed to make
concrete from the two abovementioned types of cement, with the addition of bacteria and without the addition of
bacteria. In this mixing design, bacterial sediments were added to the solution and directly to the concrete
mixing design. Finally, the compressive strength, water absorption and crack recovery in concrete samples were
investigated. The results indicated that the presence of bacteria had slight effect on the compressive strength of
concrete. The water absorption percentages of bacterial concrete samples with PPC and PC2 were 0.07 and
0.19% higher than those of non-bacterial samples, respectively. Both types of bacteria had the ability to repair
cracks. Uncontrolled cracks in concrete samples were repaired to a width of at least 39.82 μm.

1. Introduction this regard, a number of consecutive studies have been conducted in

research centers around the world whose main purpose was to propose
Nowadays, with the growing population in recent decades, the need some corrective suggestions to overcome these shortcomings of con­
for housing constructions as well as the sustainable and inexpensive crete. Accordingly, there are two aspects to prevent and heal the cracks
infrastructures has become inevitable. Furthermore, many scientists and if they occur. It should be mentioned that the cracks might be occurred
engineers seek a practical way to design perfect, ideal, and durable by overloading, volumetric changes due to its high temperature, creep,
materials [1–5]. Here, it is worthwhile to mention that concrete is one of shrinkage, improper construction methods, incorrect design or Alkali-
the building materials containing good public acceptance [6]. This is Silica Reaction, and periods of freezing and thawing [7].
because concrete is known as the most important and common building In this regard, five samples of the self-healing methods that have
materials owing to its invaluable properties such as plastic form before been proposed up to now can be described as follows [6–13]:
setting time, good fire resistance, easy access, and good compressive
strength. Nevertheless, the existence of some considerable limitations, • Chemical encapsulation
such as poor tensile strength and low resistance to cracking, has • Mineral admixtures
attracted many researchers’ attention to overcome these challenges. In • Chemical in tubing

* Corresponding author.
E-mail address: [email protected] (A. Nohegoo-Shahvari).

https://doi.org/10.1016/j.istruc.2020.10.070
Received 26 March 2020; Received in revised form 30 September 2020; Accepted 27 October 2020
Available online 10 November 2020
2352-0124/© 2020 Institution of Structural Engineers. Published by Elsevier Ltd. All rights reserved.
M. Pourfallahi et al. Structures 28 (2020) 2646–2660

• Intrinsic healing with self-controlled tight crack width Bacillus Megateium Bsknau, Bacillus Licheniformis Bsknau, and Bacillus
• Bacterial encapsulation Flexus Bsknau, survived. Then, they compared the results obtained from
Bacillus Megateium Bsknau with the gene bank. Their results indicated
Certainly, the aforementioned methods, despite their advantages, that the suitability of these bacterial strains for the use in the concrete
have a number of limitations. For example, in the chemical encapsula­ increased both strength and healing of the cracks, which could be
tion method, difficulty of making the capsules, placement of the capsules effective due to the reduction in the concrete cracks by calcite, which
in the mold, and difficulty of releasing the healing agent can be was examined by scanning electron microscopy. Bundur et al. [28]
mentioned. Meanwhile, in the expanding mineral admixtures method, explored the effect of adding Bacillus Pasteurii plant cells to cement-
the adverse effect of expansion due to non-healing can be expressed. based concrete materials. De koster et al. [29] employed Geopolymers
Regarding the bacterial concrete, the need for many prerequisites, particles to protect bacteria as a coating on the surface. Their results
bacteria preservation in the concrete, and concern about affecting the confirmed that, by determining the range of the cracks as well as the
mechanical properties of concrete can be expressed. Thus, this paper saturation mechanism of particle coating, they were better protected
intends to develop an optimal method for the concrete healing. than the uncoated particles were. Kim and Lee [30] tested the surfaces of
Here, among the above methods, the bacterial concrete method has concrete samples by macro and micrography to analyze, form, and
been considered owing to its non-pollution. The self-healing method by distribute the calcium carbonate crystals generated by the bacteria. It
adding bacteria is one of the most interesting methods to heal cracks in should be noted that the size of the calcium carbonate crystals precipi­
concrete structures [13–16]. This method utilizes special bacteria such tated by the bacterial medium was larger than that by the non-bacterial
as Bacillus Pasteurii, Bacillus Sphaericus, Striya kelly and Bacillus medium. They also examined the capillary adsorption rate of the con­
Subtilus, which have high strength to Alkali environments that are crete samples. They concluded that regarding the amount of water ab­
usually found in nature. These bacteria remain in a dormant state until sorption from the surface of the concrete samples, the efficiency of
the cracks occur in the concrete, and then they will be activated after bacterial methods was still lower than that of conventional permanent
cracking [17–22]. When they begin to work, they greedily swallow the waterproofing materials such as epoxy resin and silicone.
water, and then produce large amounts of calcium carbonate precipi­ Kadapure [31] utilized ash as an alternative to reduce the effect of
tation. After that, they precipitate inside the cracks, so that the cracks cement on the bacterial concrete. He showed that the use of the Bacillus
are filled using integrated and solid precipitation [20–25]. Sphaericus in the concrete could improve the mechanical properties and
A substantial point is that oxygen that is an important factor in durability of concrete (with or without ash) at all ages. If 30% of the ash
reinforcement corrosion is consumed during this chemical reaction. was replaced by the cement, the mechanical properties of the bacterial
Hence, reinforcement corrosion is also prevented, resulting in increasing concrete after healing were similar to the ordinary 28-day concrete.
the service life of reinforced concrete structures [23]. This self-healing Sahoo et al. [20]employed the Ureolytic bacteria to improve the various
process does not take more than a few days [24]. The self-healing pro­ properties of cement and concrete mortars. Their results indicated that
cess can also occur either inside or outside the microbial cell, or even at a the resistance tests could depend on pH and temperature. Luo and Qian
distance from it inside the concrete environment. In the last two de­ [13] explored the effects of bacterial additives on the cement mortar.
cades, the scope of research on self-healing concrete has increased owing Their experimental results confirmed that the bacterial-based additives
to the importance of increasing the life of structures. In these conducted could be used to design a self-healing system; however, the compressive
researches, some factors affecting the durability of the concrete, such as strength of the samples was slightly lower than that of the samples
water absorption, chlorine permeability, and mechanical properties of without bacterial additives at the early age of concrete.
the bacterial concrete like increasing compressive strength, have been Siddique et al. [21,32] investigated the effect of the Cement
prioritized. In the following, some of these researches are briefly Baghouse Filter Dust (CBFD) on the mechanical properties of the con­
presented. crete with partial replacement of the conventional Portland cement (0%,
Muynck et al. [4] conducted one study conducted on both water 10%, 20%, 30%) in which the bacteria were encapsulated. This study
absorption and chlorine permeability, and investigated the bacterial found that the bacterial concrete with 0% CBFD had the best results for
carbonate on the durability of mortar samples under different porosity. compressive strength, but by increasing the CBFD percentage, the
They showed that the calcium carbonate precipitates could reduce the compressive strength was decreased. Williams et al. [33] and Zhang
water absorption up to 65 to 90%, depending on the porosity of the et al. [6], employed the rainfall Biogenic calcium carbonate and the
samples. Fedko [25] investigated the effect of hot and humid climatic Calcium Precipitation Activity of Minerasion bacterial as a way to
conditions on the coastal region of the United States on samples of 51 × enhance the durability and crack, respectively. Arthi and Dhaarani [14]
51 × 51 mm3 via a standard crack width of 0.4 mm. The bacteria used in evaluated the results of an experimental study on the effect of Bacillus
this study were Bacillus Subtilus and Bacillus Pasteurii. Their results Subtilus and Bacillus Licheniformis on the compressive strength, split
confirmed that the compressive strength of the samples exposed to high tensile strength, flexural strength, water absorption, and self-healing
humidity and temperature was higher than that at room temperature. properties. In this study, bacteria with a concentration of 105 cells per
Tittelboom et al. [26] exploited Ureolytic bacteria, such as Bacillus milliliter were considered. Their results revealed that the compressive
Sphaericus, which are capable of precipitating CaCO3. Their results strength of the samples made by Bacillus Subtilus and Bacillus Lichen­
revealed that culturing pure bacteria were unable to kill the occurred iformis was increased to 8% and 15%, respectively. Moreover, the ten­
cracks. However, when the bacteria were protected in silica gel, they sile strength of the concrete cubes was increased by 6.66% and 12.69%
filled the cracks completely. Kannan et al. [27] showed that healing using Bacillus Subtilus and Bacillus Licheniformis, respectively.
using the Cement Kiln Dust (CKD), along with Bacillus Halodrans, had a Furthermore, the flexural strength of the concrete cubes was increased
positive effect on the properties of the CKD concrete. Accordingly, by by 6.87 and 25.9% using Bacillus Subtilus and Bacillus Licheniformis,
adding 10% bacteria to the concrete, the compressive strength was respectively, whereas the amount of the water absorption of the con­
increased, and water absorption was decreased by 20%. Moreover, by crete was decreased by 46.93% and 52.04%, respectively.
adding more than 10% bacteria to concrete, the resistance reduction was Maresca and Moser [35] and Li [36] screened to identify the
achieved by decreasing the hydration reaction as well as the amount of concrete-compatible bacteria, and then investigated bacteria such as
cement. Bacillus, Bacillus Sphaericus and calcium lactate as compatible agents
Krishnapriya et al. [17] utilized the bacteria isolated from Alkali soil for the self-healing concrete. Accordingly, the bacteria were encapsu­
samples from a cement factory, and then tested them to prepare the lated for use in the concrete in which the metabolic activity of active
calcium carbonate. For this purpose, they identified three samples of the bacterial spores, oxygen consumption, and recovery of material function
isolated bacteria using gene sequencing. In this paper, three bacteria of were measured in the form of water intensity recovery. Their results

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M. Pourfallahi et al. Structures 28 (2020) 2646–2660

indicated that an effective healing method of bacteria could be consid­ the bulk concreting due to some limitations and high costs. Thus,
ered for some specific applications of the marine concrete. Tziviloglou choosing an appropriate and practical method of employing bacteria in
[37] developed a novel method of the self-healing concrete. In this the concrete can have positive effects on the self-healing, strength and
paper, the bacteria were admixed with the granules and then used to durability of structures in the long term. In this regard, in the next part of
make a fresh mortar. This method increased self-healing in concrete, so this research, the steps of extracting bacteria compatible with the con­
that the samples were able to healing themselves in water after being crete are described. Hence, the bacterial concrete samples are prepared
broken. after determining the type of bacteria. To make the concretes, the bac­
Alonso et al. [18] considered different bacterial species isolated from teria are added to the concrete mix as additives and solution, so that this
the soil of the Lango region of Mexico for further study. Their results process allows the preparation of mass bacterial concretes. In the final
indicated that addition of the bacteria increased the compressive section, strength tests, water absorption and bacterial characterization
strength, durability and resistivity, whereas it decreased the perme­ are carried out on the concrete samples, and then the results are
ability of mortar samples. Patil et al. [1], using experimental research discussed.
results, investigated the effect of Bacillus Subtilus on the compressive
strength of concrete, healing of the concrete cracks, and cost analysis of 2. Materials and methods
the bacterial concrete. Their results confirmed that the bacterial con­
crete helped to increase the strength of concrete by the calcium pre­ 2.1. Isolating bacteria
cipitates, resulting in increasing the compressive strength by 20%. Vijay
et al. [12] assessed the bacteria used in the concrete and then studied Initially, to separate the bacteria from two types of cements, PPC,
how to utilize it as a self-healing agent. At the end, they provided a brief PC2, and soil near the roots of plants (Rhizosphere) were utilized. It
description of the various properties of the concrete added with the should be noted that the soil was prepared from the rhizosphere medium
addition of bacteria. The results of this study revealed that the bacteria (a micro-ecological region in the immediate vicinity of tree roots),
had a positive effect on the compressive strength of both Portland located in Mellat Park in Bandar Abbas. It is worthwhile to mention that
cement mortar and concrete mixtures. In addition, the use of bacteria most of the soils in Iran in these regions contain several calcareous
reduced the permeability of water and the chlorine permeability. Zhang properties and Alkali reactions in which approximately 40% of the soil
et al. [34] investigated the feasibility of expanded perlite as a novel weight is calcium carbonate. In the case of PPC due to the use of very
bacteria carrier on quantifying cracks-healing in concrete via immobi­ good quality Clinker Type II in the production of this type of cement, not
lization of Bacillus cohniiwas. Mostofinejad et al. [2,5,16,38–44], in only provides its compressive strength, but also acts like anti-sulfate
several studies, evaluated the effect of S. pasteurii and B. Subtilus bacteria cement in terms of the chemical properties, which will have a special
on the water absorption, permeability, and compressive strength. and effective application in corrosive sulfate environments. After all, the
Additionally, they examined the effect of chloride permeability on the hydration heat of this cement, along with its calorific rate, is lower than
concrete mortar and the Lightweight Aggregate Concrete. Their results that of other cements, so that in the mass concreting, it prevents a sharp
showed that the water uptake was decreased to an acceptable extent, rise in the temperature of the concrete core as well as the formation of
while the compressive strength was increased. Furthermore, the chlo­ thermal cracks .PC2 is employed when there is a possibility of slow
rine infiltration in the experimental samples was decreased as compared sulfate infiltration, and as the sulfate concentration in groundwater is
to the control samples. slightly higher than usual, it acts as a drainage structure. Furthermore,
Tan et al. [11] described that the bacterial concrete was a concrete the type 2 cement usually produces lower heat than the Type 1 cement
whose properties had been modified using some bacteria selected from does. Concerning this moderate hydration heat, such cement can be
both Bacillus Sphaericus and Bacillus Pasteurii. The results of this study utilized in large volumes. In this regard, Figs. 1 and 2 illustrate the
showed that when 10% of the cement was replaced with Bacillus isolating bacteria process from a liquid and solid culture medium.
sphericus, in the concrete compressive strength test, it showed 10.8% Note that at the beginning of the bacterial culture, both lactose broth
higher resistance than the sample made with Bacillus sphericus. More­ medium and agar (solid) culture medium are prepared according to the
over, in tensile test, 29.37% was higher, and the flexibility was 5.1% instructions for preparation of the bacterial medium. Afterward, the
stronger than the control concrete. It should be noted that the concrete agar (solid) culture medium exposed at 4 ◦ C is got out from the refrig­
created by Bacillus Pasteurii had lower strength than that of the Bacillus erator and then placed at room temperature to be adapted to the envi­
Sphaericus. By investigating the bacterial concrete, Mondal et al. [45] ronment. Then, all plates are placed in a Laminar Flow Hood with a UV
stated that the increase in the maximum compressive strength occurred lamp for 20 min until they will be sterilized. At the same time, the needle
at a specific bacterial concentration, which was not necessarily the (fildoplanite) is heated on a flame to be sterilized, which after cooling, a
highest level of the bacterial concentration. However, high concentra­ drop of bacteria is taken from the culture lactose broth medium using a
tions of bacteria could be more effective in healing the cracks. Never­ needle, and then it is cultured linearly on the agar culture medium (as
theless, in cases where increasing the compressive strength was more can be seen in Fig. 3). This process is separately implemented for both
important, the optimal bacterial concentration should be determined. cement and soil samples. From each of them, three samples are then
Here, a noteworthy point that appears to be the conclusion of pre­ taken for the culture, and then the bacteria are cultured as a suspension
vious studies was the role of bacteria in the strength and durability of the containing bacteria.
self-healing concrete. Although the use of certain bacteria may be useful After getting out the cultured samples, in the nutrient agar medium,
in most cases and have a good function, it cannot be extended to all it can be observed that only in the soil sample taken from the rhizo­
cases, since aggregates, atmospheric sediments, and other concrete sphere medium, the bacteria are grown. In addition, no significant
constituent materials affect the dominant bacteria in the concrete. growth is observed in the samples of PPC and PC2 (see Fig. 4).
Meanwhile, some other researchers have offered similar views. Accordingly, owing to the small percentage of soil in concrete, the
Another important factor affecting the dominant bacteria is the bacteria in the soil are excluded, and then the culture and propagation of
implemented methods of the bacterial extraction, which still contain bacteria are investigated from concrete supports. Initially, two cubic
some challenges and require more research that is extensive. Accord­ samples of 15 × 15 × 15 cm concrete are made of two types of PPC and
ingly, finding a bacterium compatible with the concrete environment PC2. The constituent materials of this concrete have been prepared from
can play a critical role in achieving the desired aim. Furthermore, the mines of Hormozgan Province, and Table 1 presents the concrete mix
providing a suitable method that can employ bacteria as a healing agent design.
for the cracks and improve the strength parameters of concrete, is one of After making the concrete and taking the samples out from the mold,
the existing challenges; in this respect, it is impossible to employ them in they are processed in ordinary water within 7 days. Afterward, using a

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Fig. 1. Cultivation of two samples of cement and soil in broth lactose medium.

Fig. 2. Autoclave agar and sterile plates by laminar hood.

200-ton hydraulic jack, the samples are broken, and some concrete growth of bacteria in the concrete powder. Figs. 6 and 7 display an
powder containing all constituent materials is consumed to isolate the incubator and the process of making agar medium for the concrete
bacteria (Fig. 5). pieces, respectively. After the growth of bacteria in the nutrient agar
According to the bacterial culture instructions, first the nutrient medium, four purification steps are applied to identify DNA. Thus, in
broth culture medium and then the agar nutrient are prepared for the each step, using a linear method, the bacteria obtained from both

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M. Pourfallahi et al. Structures 28 (2020) 2646–2660

Fig. 3. Sterilize agar medium by laminar and culture bacteria.

13,000 g for 5 min. Furthermore, 500 µL of the supernatant was trans­

ferred to a new eppendorf with the 500-µL phenol- chlorophorm solu­
tion. This stage was repeated for several times. Finally, double the
volume of the extracted top solution was added pure ethanol, centri­
fuged again and collected white precipitate, rinsed by 70% ethanol and
dry at 37 ◦ C.

2.2.1. Assessment of DNA quality extracted by electrophoresis

For standard agarose gel electrophoresis, 5 µL of DNA samples was
separated in 0.7% (w/v) gel stained with ethidium bromide. Electro­
phoresis conditions were 100 V for 45 min in 0.5x TBE buffer (89 mM
Tris, 89 mM boric acid and 2 mM EDTA). (See Fig. 9).

2.2.2. Performing polymerase chain reaction (PCR) steps

Fig. 4. Growth of soil bacteria and lack of growth of bacteria of two cement The 16S rRNA genes of the isolates were PCR- amplified using
samples in agar medium. primers 8F and 1492R and sequenced. Sequences were compared to the
non-redundant nucleotide database at national center for biotechnology
information (NCBI) using Basic Local Alignment Search Tool (BLAST N)
Table 1 and were assigned to a species based on percent similarity.
Concrete mixing scheme.
Weight ratio of Concrete Components (1 m3) Concrete 2.2.3. Bacteria propagation
The nutrient broth culture medium was considered to propagate the
W/c Water Gravel Sand Cement
bacteria. According to the instructions (13 g per 1 L of distilled water)
0.52 190 600 1200 375 Fc = 25 MPa
for each sample separately, 13 g of Nutrient broth powder was poured
into 1 L of distilled water, and the solution was thoroughly mixed and
concrete samples made of PPC and PC2 are placed in an incubator at clarified by heating. The medium was then sterilized in an autoclave at
35 ◦ C for 72 min. This process is repeated 4 times until the bacteria are 121 ◦ C for 15 min. After getting out the culture medium from the
pure and completely uniform in appearance and color (see Fig. 8). autoclave device, they were cooled to ambient temperature. A needle
adds the culture nutrient broth added separately to some of the samples
purified in the fourth step. Afterward, these samples were placed in an
2.2. DNA extraction method incubator at 35 ◦ C for 72 h to grow the bacterial (Fig. 10).

DNA extraction was carried out after isolation and purification by the
phenol-chloroform extraction method (Sambrook et al. [46]). Organic 2.3. Manufacture of bacterial concrete
(phenol–chloroform) extraction used sodium dodecylsulfate (SDS)
and proteinase K for the enzymatic digestion of proteins and nonnucleic To make the concrete, PPC and PC2 were employed. Other concrete
acid cellular components. A mixture of phenol:chloroform:isoamyl components such as washed sand and 1.5 cm crushed sand were then
alcohol (25:24:1) was then added to promote partitioning of lipids and prepared from Minab City (Table 2). To remove dust, the sands were
cellular debris into the organic phase, leaving isolated DNA in the washed with water and then dried completely at room temperature. The
aqueous phase. Then, 20 mg of cultured media was transferred to a 1.5- samples were divided into four general designs for construction. It
mL eppendorf tube and 500 µL of extraction solution (50 mM Tris-HCL, should be noted that the amount of materials required was the same in
pH8.0; 25 mM EDTA and 400 mM NaCl), 20-µL 20% SDS, and 10 µL all designs. The difference between the two designs was in the existence
Proteinase K (10 mg/mL) were then added. Afterward, the extract was of the bacterial precipitates and the type of cement, and the other two
homogenized and incubated at 50 ◦ C for 3 h. After incubation, 500-µL designs were considered controls. In the designs containing bacterial,
phenol was added and shaken for 15 min. Centrifugation was done at the water-substituting bacteria were utilized for 5% by the weight of

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Fig. 5. Steps of making, shaping and breaking concrete.

components.
After weighing the materials and preparing the molds, the concrete
constituent materials are poured into the mixer according to the mixing
design. Accordingly, the sand was first mixed for 2 min, after which the
cement was added to the mixture, and cement was added until the color
of the mixture became uniform. At this point, for the concrete via bac­
teria, first ordinary water was added, and then the precipitates of the
propagated bacteria from the same type of cement were added. Ordinary
water was used for the control samples. After the complete mixing of the
materials, fresh concrete was poured into 150 mm molds, and then the
molds were opened after 24 h, and then 150 × 150 × 150 mm samples
were processed in ordinary water for 28 days (see Fig. 11).
To generate different levels of damage on the concrete samples with/
without bacteria, on the 28th day, 50 tons of force was applied to create
micro-cracks in the sample. To prove the self-healing after cracking,
some photos were taken from all samples, and then the cracked samples
were wetted and dried. In this method, in order to be as close as possible
to the real conditions, in this cycle, ordinary water was used for rainy
Fig. 6. Nutrient broth culture medium containing pieces of both types of days, and the free environment was considered for sunny days. In this
concrete, placed on a shaker inside the incubator. method, the bacterial concrete was placed in ordinary underwater for
24 h in a laboratory environment. It was then dried in the open air for 24
cement. Concrete mixing design was designed according to Topic 9 of h, during which no effect of temperature and humidity changes was
the National Building Regulations of Iran [47], and then, the concrete considered. After 6 weeks of wet and dry cycle, all cracked samples were
construction and processing were condcuted in the climatic conditions photographed. The MIP4Student Material software was utilized to
of Hormozgan Province. determine the width of the cracks.
Therefore, in the concrete mixing design, some special rules to
implement concrete in the coastal areas of the Persian Gulf and the Sea 3. Tests performed on bacteria and concrete
of Oman were considered in accordance with Topic 9 of the National
Building Regulations of Iran [47]. It is worth mentioning that according To investigate the growth and survival of bacteria in the environ­
to the special criteria of the region, the maximum ratio of water to mental conditions of cement paste and hardened concrete, catalase test,
cement should be considered 0.4, which requires additives to reach this pH test, and urease test were performed on the bacteria of both concrete
amount of water to cement ratio. For this reason, the water ratio of samples. Compressive strength and water absorption of the samples
cement was considered 0.52. Table 3 lists the values of concrete were also investigated. Then the obtained results were analyzed.

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Fig. 7. The process of making agar medium, cultivating two concrete samples on it and pacing it inside the incubator.

Fig. 8. Purification of bacteria.

3.1. Catalase test they all have the ability to use oxygen as the final electron acceptor,
catalase-negative bacteria include anaerobic and facultative anaerobic
Catalase is an enzyme found in almost all living organisms. The bacteria. These bacteria are only fermenters and do not use oxygen for
enzyme catalase is made up of free rhizomes within the cytosol. This cellular respiration. Therefore, in this test, bacteria extracted from
enzyme breaks down hydrogen peroxide into oxygen and water. More­ concrete were examined for aerobic or anaerobic.
over, it is one of the most important enzymes to protect cells against
oxidative contamination by hydrogen peroxide. In addition, this enzyme 3.2. pH TEST
is one of the enzymes that have high decomposition power, so that one
molecule of catalase is able to decompose 6 million molecules of Any microbe can grow in a certain range of pH, which can be either
hydrogen peroxide into water (H2O) and oxygen in one minute. Here, it wide or limited, but the best growth is possible in a range called the pH
should be noted that the Catalase-positive bacteria include absolute optimum. This particular pH requires the mutual adaptation of organ­
aerobic bacteria as well as the facultative aerobic bacteria. Although isms to their natural environment. The specific pH range for the

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Fig. 9. Extraction of DNA from bacteria in both concrete samples.

Fig. 10. Bacteria amplified from both concrete samples to make bacterial concrete.

bacterial growth is between 4 and 9 with an optimum of approximately concrete samples were examined.
6.5 to 7.5. According to Section 9-10-4-3 of the Topic 9 of the National
Building Regulations of Iran [47], the pH of water used in the concrete 3.4. Compressive strength test
should not be less than 5 or more than 8.5. In this regard, the samples of
bacteria prepared from both types of concrete were tested at pH 5, 9 and The compressive strength tests of samples on 7 and 28 days of con­
12. struction were conducted according to the Standard ASTM C 39 [48].
The average compressive strength of three samples was considered for
3.3. Urease test each group.

The basis of the urease enzyme test is the study of the activity of the 3.5. Water absorption test
enzyme urease, which some organisms could produce and hydrolyze
urease to carbon dioxide, water and ammonia. Ammonia is converted to To perform the water absorption tests according to the standard
ammonium carbonate in the solution, causing the environment to ASTM C 642-97 [49], at first the bacterial and control concrete samples
become Alkali and to raise the pH. According to the implemented test, were dried in the incubator for 24 h at 105 ◦ C, and they were immersed
the values of the urease enzyme for the bacteria isolated from the in water for half an hour after cooling. The weighing of the samples was

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carried out for the first time after cooling (w saturated), and the second
time after removing them from water and drying the surfaces with a

Specifications

Coarse Sand
napkin (w oven dried). The water absorption of the samples was calculated

Sand fine
by the following formula:

Sample

Sand
wsaturated − woven dried
water absorption(%) = × 100 (1)
woven dried

(ASTM C 136)
Sand Softness
Coefficient

3.6. Scanning electron microscope (SEM)

3.65
–
–
On the 28th day of construction, the 150mmx150mmx150mm con­
crete cubes were sent to the laboratory for SEM analysis. The SEM is a
equivalent

type of scanning electron microscope producing sample images by

D2419)
(ASTM

scanning the sample with a focused beam. When the secondary electrons
Sand

(%)

86
hit the sample, an image is created by visualizing the surface topography
–
–
and the property of the material is observed. Owing to the limited size of
the concrete cubes, a piece with 10mmx10mmx10mm dimensions was
Granulation
Los Angeles Weight Loss Against Abrasion

cut from them. According to the different types of cements in this study,
for each type of cement, two concrete cubic samples with and without
Type

bacteria were considered.

–
Number of Machine

4. Results
Revolutions Per
(ASTM C131, C535)

4.1. Identified bacteria

Minute

500

Based on the database using BLAST algorithm the bacteria used in

–

this study which originally was Bacillus paralicheneniformis from PPC

Wear

and Bacillus sp from PC2. Application of Bacillus paralicheneniformis in

(%)

24

microbial concrete has not been done before. Maresca et al. [35] re­
ported that different environment conditions can affect dominant bac­
Material front on
the sieve score 4

teria and similar results were obtained in present research because

(ASTM D5821)
Fracture in a

dominant bacteria in two concretes (from two different cements) were

different.
(%)

87
84
–

4.2. Catalase test results, pH and urease

Extension

The results of the catalase test on the bacteria purified from the
(BS 812 part105)

(%)

16
15

fourth return, it is determined that the PPC catalase is positive, and PC2
–
Extension and

is catalase negative. On the other hand, pH test showed that the bacteria
Thinning
thinning

had the highest growth at pH = 5 (as described in Table 4). Therefore,

(%)

the most suitable pH is suggested between 5 and 8.5 to make bacterial

10
14
–

concrete; however according to the results, these bacteria have also

grown at pH = 12. According to the urease test, the value of urease
Appearance
Specific gravity and water absorption

enzyme for the bacteria isolated from concrete sample with PPC is 58.
2.716
2.721
2.787

Furthermore, this value is 48 for bacteria isolated from the concrete

sample prepared with PC2. The results of this test indicate that the
bacteria in both types of cement contain the enzyme urease and are also
2.665
2.647
2.658
True
(ASTM – C127, 128)

able to produce carbon dioxide (Fig. 12).

absorption

4.3. Analysis of 7 and 28-day compressive strength test results

Water

(%)

0.9

1.7

The compressive strength of concrete is considered one of the most

1

important properties of concrete. The compressive strength of concrete

Compact

shows the general picture of concrete quality. The compressive strength

g/cm3

1.51
1.54

of concrete depends on the ratio of water to cement, fine-grained ag­

–

gregates, water, and additives. Here, it is worthwhile to mention that the

ratio of water to cement is the most significant factor in the compressive
Specifications of aggregates.

condensing

strength of concrete.
g/cm3
Non-

1.47
1.39
1.66
Unit volume weight

4.3.1. 7-Day compressive strength

The average 7-day compressive strength of control and bacterial
(ASTM-C29)

samples is shown in Fig. 13. As shown, the compressive strength of

bacterial concrete with PPC was 1.94% higher than its control sample.
mixture
grained
coarse-
Table 2

g/cm3
Dense

Moreover, bacterial concrete with cement PC2 was 5.65% lower than its
1.53

control sample.
–

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M. Pourfallahi et al. Structures 28 (2020) 2646–2660

Table 3
Bacterial Concrete mixing scheme.
Weight ratio of Concrete Components (1 m3) Concrete

W/c Bacteria Water Gravel Sand Cement

0.52 18.75 190 600 1200 Fc = 25 MPa 375

Fig. 11. Construction, forming, Open molds and process concrete with and without bacteria.

Table 4
Results of pH reading with spectrophotometer by OD 660 method.
PH 5 PH 9 PH12

Control 0 0 0
Cement ppc 0.160a1 0.018a 0.025a
Cement pc2 0.124a 0.098a 0.041a
1
absorption.

Fig. 13. 7-day compressive strength of samples with and without bacteria with
both types of cement (ppc) and (pc2).

precipitation; thus, it seems Calcium carbonate sediments could improve

the strength parameters of concrete and repair cracks. Choosing the
concentration of bacterium is important to improve these parameters.
Mendel and Ghosh [50] reported that not only concentration of bacteria
but also precipitation pattern is dependent on different causes such as
Fig. 12. Urease test results on bacteria of both cements. bacteria type. They also reported that 103 density cells in mL is the
highest compressive strength and 107 cells in mL is the best choice to
4.3.2. 28-day compressive strength repair the cracks. But Chahal et al. [51] reported 105 for optimized
The 28-day average compressive strength of control and bacterial density in compressive strength and water absorption. The ratio of water
samples is shown in Fig. 14. As shown, the compressive strength of and cement and ratio of cement and sand are different and this differ­
bacterial concrete with PPC is 2.48% lower than that its control sample. ence is dependent on bacteria precipitation. If density of bacteria were
In addition, for bacterial concrete with PC2, it is 1.96% less than con­ too low, the precipitation would not block the pores.
trolling sample. Adding bacteria to the concrete causes CaCO3

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M. Pourfallahi et al. Structures 28 (2020) 2646–2660

of gram-positive Bacillus bacteria is the appearance of the bar, which are

often obligate aerobe or facultative anaerobic. Fig. 16 depicts the PPC
bacterial sample. The bar shape of the bacterial and the formation of
(white) calcium carbonate precipitates can be seen. Additionally, in this
figure is a sample of PC2 bacterial concrete, which shows all bar shapes
of bacterial and the formation of (white) calcium carbonate precipitates.
According to the DNA identification, it is determined that the genus of
each bacterium is Bacillus, but their species are different. A closer look at
the pictures reveals that the appearance of the bacteria is slightly
different from each other, but the formation of calcium carbonate pre­
cipitates in both concrete samples is similar in appearance, and the only
difference is that these precipitates grow better in the PPC sample. In the
control samples (PPC and PC2) (Fig. 16), as the figure illustrate, the
existence of bacteria or specific calcium carbonate precipitates is not
Fig. 14. 28-day compressive strength of samples with and without bacteria visible. However, it appears that there is a small amount of bacteria in
with both types of cement (ppc) and (pc2). the control PPC sample. This is because these bacteria are derived from
concrete, and should normally be present even in the control samples.
4.4. Half-hour water aborption analysis The only difference between samples with/without bacteria is the
addition of bacteria to the sample of bacterial concretes. This has led to
The strength and durability of concrete are directly affected by the formation of calcium carbonate precipitates and healing of cracks in
number, type, size and percentage distribution of pores in the cement bacterial samples.
paste, aggregates and the joint surface between the cement paste and the
aggregate. In this paper, the concrete constituents material and its 4.6. Analysis of cracks creased on samples
storage environment were in the coastal areas of the Persian Gulf, while
this area contained water, soil and atmosphere pollution via harmful To prove the self-healing on 4 concrete samples, some uncontrolled
chemical elements for concrete, high humidity, temperature and solar cracks were created, of which one samples with bacteria and one control
radiation to the extent that it contributed to the process of concrete sample for both types of concrete were prepared with PPC and PC2
demolition. Inevitably, the permissible values of concrete permeability separately. Fig. 17 is related to the PPC bacterial and control samples. As
were set at a maximum of 3% in accordance with Topic 9 of the National can be observed, the cracks up to 39.82 μm were healed in PPC bacterial
Building Regulations of Iran [47]. concrete. It should be noted that the formation of these cracks was un­
According to Fig. 15, the percentage of the water absorption of all controlled, and the bacteria in the concrete of cracks with different
samples with/without bacteria in both types of cement was lower than widths up to this width provided the maximum healing. Therefore, it is
the allowable range, revealing the proper bond of the cement paste with possible that the bacteria will be able to heal larger cracks. Fig. 18 is
its components. Nevertheless, the existence of bacteria had slight effect showed the PC2 bacterial and control samples. As can be observed, the
on the water absorption of the samples. According to Fig. 15, the control cracks up to 1.17 μm were healed in PC2 bacterial concrete. The healing
concrete sample with PPC cement had 0.07% lower water absorption of cracks up to this width was much lower than that of the concrete
than its bacterial sample. In addition, the PC2 control concrete sample sample prepared with PPC. As mentioned earlier, the formation of these
had 0.19, being lower water absorption percentage than its bacterial cracks is uncontrolled, and the bacteria in the concrete provide the
sample. maximum healing of the cracks with different widths up to this width.
Accordingly, it is possible that bacteria can heal larger cracks. On the
other hand, as shown in Figs. 17 and 18, no effect of calcium carbonate
4.5. The SEM analysis formation and self-healing precipitates is observed in the PPC and PC2
control samples. Although, All conditions in creating cracks and being
To compare the results, four concrete samples are scanned by elec­ under wet and dry cycles were the same for all samples.
tron microscopy. Two samples of concrete include bacteria, while two
samples of concrete are regarded as control. According to the DNA 5. Discussions
identification in Section 4.1, it is determined that both bacteria in the
concrete samples are of the Bacillus genus. The most important feature In previous research, researchers, who paid attention to reinforce the
strength, considered bacterial concentrations in the range of 103-107
cells per ml [50]. However, researchers, who focused more on crack
healing, usually used higher bacterial cell centrifuges of approximately
107-109 cells per ml [50]. Several studies have also shown the optimal
concentration of bacteria to maximize the compressive strength of the
bacterial concrete. Although it is obvious that higher concentrations of
bacteria could lead to more calcite precipitation, it is observed that the
optimal concentration to reinforce the compressive strength must be
considered. Overall, some researchers have suggested that the most
appropriate amount of bacteria added to concrete is 2%. In this paper,
the added amount of bacteria in relation to cement is 5% and more than
the optimal concentration is considered.
The existence of this amount of bacteria did not improve the strength
parameters of the concrete, as compared to the control samples, so that
the 7-day compressive strength of PPC bacterial concrete was 1.82
higher than its control sample, and the PC2 bacterial concrete was 4.85
Fig. 15. Water absorption percentage of samples with and without bacteria lower than its control samples. At the 28-day compressive strength for
with both types of cement (ppc) and (pc2I). each concrete, they are 2.48 and 1.96 percent lower than their control

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M. Pourfallahi et al. Structures 28 (2020) 2646–2660

Fig. 16. Properties of different bacterial concrete compositions and control with both types of cement through SEM.

samples, respectively. Based on the results of the water absorption while it was up to 1.17 μm for the PC2 cement. Certainly, the formation
percentage, the samples for PPC and PC2 cements are 0.07 and 0.19 of these cracks is uncontrolled and there is a possibility of healing larger
percent of the samples, respectively. Thus, the control samples absorbed cracks for both concretes. Regarding the equality of all conditions for
less water. Although, the water absorption percent is very small, indi­ samples with/without bacteria in the formation of cracks and their
cating the equality of samples with/without bacteria. Nevertheless, the placement under wet and dry cycles, no healing has been observed in the
bacteria healed all the cracks created for the PPC cement up to 39.82 μm, cracks of the control samples for both types of cement.

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M. Pourfallahi et al. Structures 28 (2020) 2646–2660

Fig. 17. Control (D4) and bacterial (D3) concrete with cement ppc. Left after creating cracks, Right side after 6 weeks of wet and dry cycle.

6. Conclusions 3. According to the DNA identification and comparison with the NCBI
World Gene Bank, the genus and species of bacteria were determined
In this paper, an attempt has been made to analyze and study the with the concrete with PPC Bacillus paralicheneniformis and con­
feasibility of self-repairing bacterial concrete in a special way. This crete with PC2 Bacillus sp. This revealed that, by changing one of the
method is based on two principles: 1) Extraction of bacteria compatible concrete components, the bacteria that could grow and survive were
with the concrete from concrete components 2) Adding bacteria in the different in those conditions.
solution directly to the concrete mixing plan. 4. According to the implemented catalase test, it was determined that
To extract the bacteria, first the soil sample of the rhizosphere the concrete bacteria with PPC and PC2 cement were catalase posi­
environment and two types of cements (PPC and PC2) were used. Owing tive and negative, respectively. This difference would vary according
to the lack of proper growth of bacteria in both types of cement, the to the bacteria behavior.
extraction of bacteria from the concrete itself was considered. In these 5. The existence of bacteria had not much effect on the compressive
conditions, two concrete samples were made with the above-mentioned strength of the concrete. Accordingly, in the 7-day compressive
cements, and the bacteria were then extracted from the powdered strength of the samples, the bacterial concrete with PPC was 1.82
concrete. After purifying the bacteria, determination of the species of higher than its control samples. Furthermore, the bacterial concrete
bacteria, catalase, pH and urease tests was performed on them, and then with PC2 was 4.85 lower than its control samples. At the 28-day
the bacteria were added to the concrete as an additive in the amount of compressive strength, the samples for each concrete, they were
5% by the weight of cement as a solution. The result of adding bacteria 2.48 and 1.96 percent lower than their control samples, respectively.
did not have a positive effect on the resistance parameters, but the The reason for this decrease is that the percentage of bacteria is
cracks formed repaired on the samples of both types of cement. higher than the optimal concentration.
Overall, the results of this paper could be described as follows: 6. The water absorption percent of the bacterial concrete samples with
PPC and PC2 cements was 0.07 and 0.19% higher than that of their
1. The growth of bacteria isolated from the soil sample of the Rhizo­ control samples, respectively. Although this reduction is very small,
sphere medium was suitable, but the growth of bacteria of PPC and it can be attributed to the behavior of new bacteria used in concrete.
PC2 was unsuitable. Therefore, due to the small amount of soil in 7. Reading bacterial and control concrete samples by scanning electron
concrete, the use of soil bacteria alone was unsuitable for the self- microscopy and X-ray diffraction proved the existence of bacteria as
healing concrete. well as the formation of calcium carbonate precipitates in the con­
2. Isolation of the bacteria from concrete sample was a logical method, crete samples with both types of cement.
since its bacteria were better adapted to environmental conditions, 8. The bacterial concrete with the PPC cement could heal the cracks up
continuous concrete interactions, and pH of concrete. to 39.82 μm width, whereas the concrete with the PC2 cement could
heal the cracks up to 1.17 μm width. The formation of these cracks

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M. Pourfallahi et al. Structures 28 (2020) 2646–2660

Fig. 18. Control (H4) and bacterial (H3) concrete with cement pc2. Left after creating cracks, Right side after 6 weeks of wet and dry cycle.

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