Crack filling in concrete by addition of Bacillus subtilis spores –

Preliminary study •
Nicole Schwantes-Cezario a, Maria Vânia Nogueira do Nascimento Peres b, Thomas Kehrwald Fruet b,
Geovana Souza Ferreira Nogueira a, Berenice Martins Toralles a & Davi De Souza Cezario c
a
State University of Londrina, Londrina, Brazil. nicoleschwantes@hotmail.com; geovanasouzanogueira@gmail.com, betoralles@gmail.com
b
FAG University, Cascavel, Brazil. vaniaperes@gmail.com; thomas@fag.edu.br
c
State University of São Paulo, Ilha Solteira, Brazil. davi.scezario@gmail.com

Received: October 30th, 2017. Received in revised form: March 16th, 2018. Accepted: April 4rd, 2018.

Abstract
This study aimed to analyze the performance of two concrete mixtures with the addition of B. subtilis spores regarding their capacity of
filling cracks during the curing time, evaluating whether the addition can influence the compressive strength of the concrete. For so, six
concrete mixtures were studied divided in two mix compositions: 1:1:2 and 1:2:3 (cement: sand: gravel) with w/c of 0.33 and 0.45,
respectively. For each composition were added concentrations of 0, 0.3x108 or 1.2x108 of B. subtilis spores/mL. The results showed that
all the samples with spores addition presented crystals precipitation, possibly calcium carbonate, that visually filled the cracks. However,
more studies of microstructural analysis should be conducted to prove that the cracks were closed. The compressive strength presented
satisfactory results, because the addition of 1.2x108 spores/mL did not present significant differences in compressive strength at probability
level of 5%, when compared to the reference concrete.

Keywords: bioconcrete; concrete with addition of spores; Bacillus subtilis; crack filling; cracks.

Relleno de fisuras en concreto por la adición de esporas de Bacilos

subtilis - Estudio preliminar
Resumen
Este estudio tiene como objetivo analizar el desempeño de dos composiciones de hormigón con adición de esporas de B. sutilis con relación
a su capacidad de relleno de fisuras durante el periodo de cura, evaluando la influencia de la adición en la resistencia a compresión. Para
ello han sido estudiadas seis composiciones de hormigón, divididas en dos dosificaciones: 1:1:2 y 1:2:3 (cemento: árido fino: grava) en
peso, con relación a/c de 0,33 y 0,45. Para cada una de las mezclas se utilizaran concentraciones de 0, 0.3x108 or 1.2x108 de esporas de B.
sutilis/mL. Los resultados obtenidos han demostrado que todas las muestras con adición de esporas presentaron precipitación de cristales,
posiblemente carbonato de calcio, que fue visualizado en las fisuras. Más estudios microestructurales deberán ser realizados para comprobar
que de hecho ocurrió el relleno de las fisuras. La resistencia a compresión presentó resultados satisfactorios, pues no hubo diferencias
significativas en relación al hormigón de referencia, a nivel de 5% de probabilidad.

Palabras clave: biohormigón; hormigón con adición de esporas; Bacilos subtilis; relleno de fisuras; fisuras.

1. Introduction manifestations, which compromise its integrity and durability

[2]. Among these manifestations, cracking is a frequent
Concrete is the main material used in construction, as other problem, which in addition to an aesthetic damage, can cause
materials are limited compared to it, by either cost, ease of leakage and may compromise the entire structure [3].
execution, durability or flexibility to adapt to various geometric Moreover, in environmental terms, the production of
shapes [1]. However, concrete is subject to pathological concrete is unsustainable [4], for the manufacture of the main

How to cite: Schwantes-Cezario, N., Peres, M.V.N.doN., Fruet, T.K., Nogueira, G.S.F., Toralles, B.M: and Cezario, D.deS., Crack filling in concrete by addition of Bacillus
subtilis spores – Preliminary study. DYNA, 85(205), pp. 132-139, June, 2018.
© The author; licensee Universidad Nacional de Colombia.
Revista DYNA, 85(205), pp. 132-139, June, 2018, ISSN 0012-7353
DOI: http://doi.org/10.15446/dyna.v85n205.68591
 Schwantes-Cezario et al / Revista DYNA, 85(205), pp. 132-139, June, 2018.

component, Portland cement, is a major global carbon tending to restrict bacterial growth [19].
dioxide emitter [5]. The emission of CO2 from the calcination Thus, this research aims at evaluating the crack filling and
process of limestone and from the combustion in the furnace the compressive strength of two concrete mixtures with
of the cement industry accounts for about 7% of global CO2 addition of B. subtilis spores isolated in Brazil [23] at
emissions, making it an important sector for mitigation different concentrations.
strategies concerning this pollutant gas [6].
In view of these problems and the need to make concrete 2. Microrganisms and growth conditions
a more durable and sustainable material, researches are being
conducted on the addition of different materials that provide 2.1. Materials
healing of concrete cracks [7].
In this context, there arises a technological innovation This study was conducted with B. subtilis AP91, isolated
that seeks to minimize the problem of cracks in concrete in Brazil from leaves of long grain rice. This bacterium has
structures, the bioconcrete. The incorporation of bacteria to the potential to bioprecipitate CaCO3 according previous
the concrete matrix has been used from the bio-precipitation researches [23]. Initially the bacteria was reproduced from
of minerals, with the goal of closing pores and cracks. Thus, two culture medium: Mueller Hinton (MH) (Sigma-Aldrich,
the bacteria are considered a self-healing agent of concrete USA) and Trypton Soy Broth (TS) (Sigma-Aldrich, USA).
cracks, because they have the ability to precipitate minerals These two culture medium were tested to verify in which one
that close the cracks autonomously [8]. the bacteria have the more effectively reproduction ability.
However, obtaining a greater understanding of the basic From the analysis of this tests, it was select the best culture
mechanisms and principles of materials that are able to close medium, which provided the highest bacterial growth,
concrete cracks is still a challenging goal. If this goal is enabling to obtain the experiment.
achieved, it will undoubtedly lead to the development of a The culture media (MH and TS) were formulated to 500
new generation of self-healing materials [7]. mL and autoclaved for 15 minutes at 121°C and 1 atm. After
The mineral precipitated by the bacteria in cement sterilization, the bacteria were inoculated in the culture
materials is from calcium carbonate (CaCO3), which can be medium and samples were placed in shaker TE 400 (Tecnal)
precipitated by microorganisms from several mechanisms. for 66 hours at an average temperature of 29.1ºC to induce
The precipitation of CaCO3 promoted by bacteria is classified bacterial growth.
as a Biologically Induced Mineralization (BIM) [9]. This After the stir period, the solution with the bacteria was
process will result in minerals formed from three possible washed in order to remove the culture medium and to obtain
ways: enzymatic hydrolysis of urea, dissimilation of nitrates a more concentrated solution of bacteria. For this, the culture
and aerobic metabolic conversion of calcium salts [10]. This media was centrifuged in Falcon tubes for 20 minutes at 3600
last one results in minerals that are formed from an rpm spin, the supernatant was removed and replaced with
involuntary metabolic activity [11]. 0.85% saline solution [1177]. The experiment was repeated
After investigation about equations that could summarize twice (n = 2).
possible ways for CaCO3 bioprecipitations, researchers found Then, the solution containing bacteria was stored for two
three main results (Eqs. 1-3) for these biochemical reactions days at the temperature of 8ºC to induce spore formation. It
in which the cell surface works as a nucleation site [12]. is important to mention that spores formation are
fundamental for the survival of the bacteria in the
Ca2+ + CO32- ↔ CaCO3 (1) cementitious materials.

HCO3- ↔ CO32- + H+ (2) 2.2. Quantification of bacteria

HCO3- + OH- ↔ CO32- + H2O (3) Quantification of the bacteria grown in MH and TS were
performed with the spectrophotometer UV340GX (Gehaka),
This bioprecipitations happen as the bacteria finds a with reading at 600 nm. For this test, three samples were
calcium source such as limestone rocks or cements. This used: the control with 0.85% of saline solution, the bacteria
source, due its negativity, tends to approach the cell wall of grown in MH dissolved in 0.85% saline solution and the
the bacteria forming a complex. This complex precipitates bacteria grown in ST dissolved in 0.85% saline solution. To
calcium carbonate due to the reaction with ion CO3 (Eq. 1). calculate the concentration of bacteria in the medium through
The carbonate and biocarbonate ions dissolved in solutions the absorbance, Equation 4 was applied [17].
are formed mainly from the CO2 produced during the
microbial respiration (Eq. 2 and 3) [12]. 𝑌𝑌 = 8.59 ∗ 107 ∗ 𝑋𝑋1.3627 (4)
Some bacteria that produce minerals by BIM have been
used to repair limestone monuments [13-16] and to fill pores Where:
and cracks in concrete and other cementitious materials [17- • X = absorbance (A);
22]. • Y = Spore concentration per mL.
The success of the use of bacteria to close pores in cement After complete this stage, the culture medium that
materials is directly related to their ability to form spores [8]. resulted in the highest cell growth was identified and then the
The pH of the concrete is highly alkaline, has no nutrients procedure was repeated with this medium to obtain the
and there is oxygen only in its open pores and on its surface, required concentrations in the experiment, which are 0.3x108

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and 1.2x108 spores/mL according to previous experiment

with the bacteria B. pasteurii and Pseudomonas aeruginosa
[17].

2.3. Concrete mixture proportions

For the production of concrete mix, the following

materials were used: Portland cement, high early strength
(CPV ARI, Brazilian denomination); fine aggregate: medium
quartz sand; coarse aggregate: gravel 19 mm of basalt, water
and B. subtilis spores dissolved in 0.85% of saline solution.
The aggregates were characterized according to Brazilian
standard [24].
The cement specifications and the requirements of NBR
5733 (Brazilian standard) [25] are presented in Table 1.
Mixture proportions are presented in Table 2. The mix
proportions used were 1:1:2 and 1:2:3 (cement: sand: gravel),
in mass, with water to cement ratio of 0.33 and 0.45, Figure 1. Cracking simulation.
respectively. For each composition used, two treatments were Source: The authors.
made with the addition of B. subtilis spores to the mixing
water in the concentration of 0.3x108 and 1.2x108 spores/mL.
Besides, in the mixing water it was added 0.85% saline greater than 95% until the test ages, which was 7 days for
solution to maintain the medium isotonic for the bacteria. cracking, 8, 15, 22, 29, 36 and 43 days for the monitoring
The concrete mixture was performed in a vertical shaft visual of crack filling, and 28 days for the compressive
mixer and bacteria concentrations were added to the mixing strength test.
water.
For each treatment, six cylindrical specimens (100x200 2.4. Simulation and analysis of cracks
mm) were made, being three for visual analysis of crack
filling (n=3) over the curing time and the other three for the In order to evaluate the cracks filling by spores addition
compressive strength test at 28 days (n=3). It should be noted compared to reference, a 2 centimeters slice was extracted
that, for each mix compositions, reference specimens were from each concrete (n=3). These slices were sent to the
made without bacterial spores, with 0.85% sodium chloride hydraulic press on the seventh day of healing to apply a
to ensure the same conditions as the specimens with spores. strength to simulate a crack, as shown in Fig. 1.
Specimens were molded in 100x200 mm cylindrical The visual monitoring of cracks during the curing period,
samples according to NBR 5738/2015 (Brazilian standard) at 8, 15, 22, 29, 36 and 43 days, was conducted by images
[26]. The specimens were demolded after 24 hours and stored taken from an optical microscope with an attached camera
in a dripping wet chamber at 20ºC ± 2ºC and relative humidity (Olympus), with magnification of 100x. To ensure that the
analysis was always held in the same position of the crack, it
Table 1. was demarcated. It is worth mentioning that from this method
Characteristics of cement CPV ARI (Brazilian denomination) and the of cracking simulation it was not possible control it’s
requirements of NBR 5733 (1991). thickness.
Compressive strength (MPa) Cement setting
Ages 24 Blaine(cm²/g) Start End 2.5. Compressive strength
3 days 7 days 28 days
hours (min) (min)
NBR The compressive strength test was performed with non
≥ 14 ≥ 24 ≥ 34 - ≥ 3000 ≥ 60 ≥600
5733* cracked specimens of 100x200mm at the age of 28 days (n =
CPV ARI 27 37 42 48 5330 160 265 3) according to a NBR 5739/2007 (Brazilian denomination)
Source: Brazilian standard [25]. [27].

Table 2.
2.6. Statistical design
Mixture proportions.
Mix Cement Sand Gravel Water NaCl Treatment A statistical analysis was performed only for the
w/c
proportions (g) (g) (g) (g) (g) (spores/mL) evaluation of compressive strength, from a completely
0 randomized design experiment (CRD), in a 2x3 factorial
1:1:2 0.33 5.750 5.750 11.500 1.900 16.15 0.3x108 scheme, consisting of two concrete mixtures (1:1:2 and 1:2:3)
1.2x108
and three bacteria content (0, 0.3x108 and 1.2x108). In this
0
1:2:3 0.45 3.860 7.710 11.560 1.740 14.79 0.3x108
statistical scheme, the two concrete mixtures were evaluated
1.2x108 with all bacterial contents, with three replications.
Source: The authors. For the statistical analysis, the software Sisvar was used
to perform an analysis of variance [28].

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Figure 2. Mean absorbance and concentration of bacteria in each culture

medium.
Source: The authors.

3. Results and discussion

Figure 3. Particle size analysis of sand.
3.1. Quantification of spores Source: The authors.

From the two culture medium used in the experiment, the

spores quantification of each medium was performed in
accordance with Eq. 1., Fig. 2 shows the results of absorbance
and concentration of spores/mL of culture media MH and TS.
Through the analysis of Fig. 3, it was observed a higher
absorbance values and concentration of spores in the medium
MH, of 1.28 and 1.21x108 spores/mL, respectively, while under
the same conditions, the medium TS presented, respectively, 0.29
and 1.61x107 spores/mL. From this result, only the culture
medium MH was selected for this experiment.
The reproduction procedure was repeated only with the
medium MH, which presented the highest concentration of
spores/mL, until it reached the concentrations of 1.2x108 and
0.3x108 spores/mL, which was quantified by
spectrophotometer.

3.2. Characterization of aggregates

Sand was performed by Particle size analysis [24]. The

results of these test were presented in Fig. 3.
The fine aggregate used was a medium sand. The particle
size analysis is shown in Fig. 3. It was found that the grading
curve is inside the limits established by NBR 7211/2009
(Brazilian standard) [29]. Analyzing the grading curve it
should be noted that it has a continuous particle size, with
desirable characteristic for use in cementitious materials due
to the better packaging of the grains in the structure of the Figure 4. Visual monitoring of crack filling in the specimens of composition
material. For the grading curve, it were calculated the 1:1:2.
maximum size, specific mass and unit mass. The values Source: The authors.
found were 2.36 mm, 2.62 g/cm³ and 1.57 g/cm³,
respectively. Besides that, the gravel has maximum size of 19
mm. It can be seen that in all the compositions in which bacteria
were added, there was a crystal precipitation from the 15th
3.3. Crack filling visual analysis day on, possibly calcium carbonate (CaCO3) according to
literature [20,22,30-35]. Besides that, the same strain was
The visual analysis of crack filling was performed with used in another study [23] that performed an analysis in vitro
one crack per specimen until 43 days of curing, being that of the bioprecipitation of calcium carbonate by pH change
three specimens of each concrete were analyzed (n=3). The during biofilm formation. The authors found that the B.
images obtained from an optical microscope with attached subtilis AP91 were able to precipitate crystals of CaCO3 with
camera are shown in Figs. 3 and 4. the same morphology as other researches [11,36-40].

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Figure 6. Stereomicroscopic images of crack-healing process in control

mortar specimen before (a) and after 100 days healing (c), in bio-chemical
agent-based specimen before (b) and after 100 days healing (d).
Source: [3].

Figure 5. Monitoring the crack filling in the specimens of composition 1:2:3.

Source: The authors.

Other investigators conducted the studies in the same way,

first studying whether the bacterium has the ability to
precipitate CaCO3 and in the sequence analyzing how that
addition would behave in the cement matrix [9,41].
The filling of cracks up to 0.19 mm were visible in
concretes with addition of spores of B. subtilis. However, in
the samples without spores addition, precipitated material Figure 7. Compressive strength of concretes.
was not visible and the crack remained with the same feature Source: The authors.
throughout the curing period analyzed.
Although the cracks have different thickness sizes, it was
visually found that in the concretes with addition of B. 3.4. Compressive strength
subtilis spores there was filling of the cracks by precipitation
of crystals, even if the crack were of greater thickness The compressive strength test was performed on the 28th
compared to the reference concrete, without spores. Besides, day of curing and the results are shown in Fig. 7. There was
in the reference concretes this filling was not visualized in a difference in resistance between the compositions due to
this analysis. It is noteworthy that this analysis was just the proportions of materials used and the water/cement ratio
visual. To a better detailing a microstructural analysis would (w/c), with higher resistance for the mix proportion 1:1:2
be necessary. compared to w/c of 0.33 and without addition of bacteria, and
Fig. 6 shows the crystals formed by calcium carbonate the lowest resistance for the mix proportion 1:2:3 with w/c of
precipitation promoted by the bacterium B. alkalinitrilicus 0.45.
added with a nutrient source and curing totally submerged Through the analysis of the results, it was noted that there
under water for the healing regime [3]. It was observed that was a decrease in the mean resistance of samples with added
there was closure of the cracks from 0.13 to 0.40 mm thickness spores when compared to the reference. This decrease in the
after 100 days of curing. Similar results were found in this mix proportion 1:1:2 was of 20.06 and 16.58% for the
research. Besides the above referenced authors, similar results treatments 0.3x108 and 1.2x108 spores/mL, respectively. For
using the bacterium B. sphaericus have been presented [18]. the mix proportion 1:2:3, the difference was greater,
Thus, it was observed that the crystals precipitation corresponding to 63.63 and 23.28% for the treatments
promoted by the addition of B. subtilis spores under the 0.3x108 and 1.2x108 spores/mL.
conditions analyzed was visually satisfactory in crack filling. There was an increase in the resistance of the samples with
However, for more specific studies a microstructural analysis 1.2x108 spores/mL compared to the ones with 3.0 x107
would be required. spores/mL in both mix proportions, demonstrating that the

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amount of bacteria added to concrete influences the It was noted that there were significant differences in
compression strength. However, it is necessary to test a larger compressive strength by varying both the concentration of
number of bacteria concentrations for a better understanding. spores added and the mix proportion. A significant difference
According to the literature [31], calcium hydroxide (Ca(OH)2) between the compressive strengths of the two mix
is the most important source of calcium for concrete, but it does compositions were expected, since the w/c ratio is a factor
not alter mechanical resistance. On the other hand, calcium directly related to it [6].
carbonate (CaCO3) contributes to increase this resistance, Regarding the addition of bacterial spores, it was
which makes the bioprecipitation a positive contribution [31]. observed that the lowest concentration of spores resulted in a
It should be noted that the crystals precipitated by bacteria in minor compressive strength, significantly differing from the
cementitious materials, calcium carbonate stand out [12, 20, reference (without addition of spores). The concentration of
22-23, 30-35]. But, to confirm that the crystal precipitated by 1.2x108 spores/mL presented minor compressive strength
B. subtilis was calcium carbonate more studies need to be than the reference; however, there was no significant
conducted, since this research is a preliminary test. difference between them.
Based on this analysis (Tukey test), it’s possible that the
3.4.1. Statistical analysis addition of spores of B. subtilis could be used in concretes
with these specific mix proportions without significant
The statistical analysis was performed in Sisvar [28] and influences in compressive strength, nevertheless it is
the results of analysis of variance are shown in Table 3. The necessary more investigations about the use of this kind of
sources of variation were the mix proportions of concrete and material, since this study is a preliminary analysis.
concentration of added spores, as well as the interaction of
these two sources of variation. 4. Conclusion
There was a significant difference (P < 0.05) at 5%
probability, for the parameter compressive strength in the From the experimental results, it was verified that there
variation of Mix proportion (M) and Spores Concentration was a visual crack filling when the B. subtilis spores were
(C), indicating that there are variations to compressive added in two studied concentrations (0.3x108 and 1.2x108
strength according to the concentration of spores added and spores/mL). However the analysis by microscope with
to the mix proportion analyzed. attached camera is just a visual analysis. To confirm that
Table 4 presents the tests of means performed for the there was a closure of cracks a microstructural analysis would
parameter compressive strength with variation of Mix be necessary.
proportion (M) and Table 5 present the test of means for the The addition of 0.3x108 spores/mL resulted in a
parameter Spores Concentration (C). statistically significant decrease (at 5% probability) of the
compressive strength of the specimens, while the addition of
Table 3. 1.2x108 spores/mL did not present statistically significant
Mean squares and summary of the Analysis of Variance (ANOVA). differences when compared to the reference concrete. It
Sources of variation Mean squares
possible happened because the highest concentration of
Spores Concentration (C) 189.00 *
spores was capable to produce more crystals that could
Mix proportion (M) 768.00 *
increase the strength. Although further analysis need to be
Interation (C x M) 19.51 ns
performed to confirm this theory.
Residue 33.84
Thus, these preliminary study has shown that the addition
* Significant at 5% probability by the Fisher test; ns: non-significant
Source: The authors. of B. subtilis spores isolated in Brazil to unarmored concrete
could be beneficial and this practice should be developed in
order to obtain a material capable of self-healing and
Table 4. consequently increase its lifetime. For this, further studies on
Tukey test for compressive strength with variation in mix proportion. the concentration of addition of these spores are required, as
Mix proportion Mean Square well as on the adding procedure, curing process and
1:2:3 38.050 a microstructural analysis.
1:1:2 43.275 b
DMS 8.218 Acknowledgements
CV (%) 13.120
Source: The authors. The authors are grateful to Assis Gurgacz College, State
University of Londrina and CNPQ for all financial support.
Table 5.
Tukey test for compressive strength with the addition of B. subtilis spores
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N. Schwantes-Cezario, received the BSc. in Civil Engineering in 2015, the

MSc. degree in Building Engineering and Sanitation in State University of
Londrina, Brazil in 2016, and is a PhD student in Civil Engineering at State
University of Londrina, Brazil.
ORCID: 0000-0002-3254-6396

M.V.N.N. Peres, received the BSc. in Civil Engineering in 2004, the MSc.
degree in Structures in Federal University of Rio Grande do Sul, Brazil in
Área Curricular de Ingeniería Civil
2008, and is a PhD. student in Structures by Federal University at Rio
Grande do Sul, Brazil. Currently is a professor at Centro Universitário
Fundação Assis Gurgacz (FAG), Brazil. Oferta de Posgrados
ORCID: 0000-0002-3986-3503
Especialización en Vías y Transportes
T.K. Fruet, received the degree in Biological Sciences in 2010, the MSc.
degree in Conservation and management of natural resources in State
Especialización en Estructuras
University of Western Paraná in 2013 and is a PhD student in Biology at Maestría en Ingeniería - Infraestructura y Sistemas
State University of Maringá, Brazil. Currently is a professor at Centro
Universitário Fundação Assis Gurgacz (FAG), Brazil.
de Transporte
ORCID: 0000-0002-3521-600X Maestría en Ingeniería – Geotecnia
G.S.F. Nogueira, graduated in Civil Engineering in 2016 at the State
Doctorado en Ingeniería - Ingeniería Civil
University of Londrina, and is a MSc. degree student in Civil Engineering at
State University of Londrina, Brazil. Mayor información:
ORCID: 0000-0002-7059-257X
E-mail: asisacic_med@unal.edu.co
B.M. Toralles, received the BSc. in Civil Engineering in 1982, the MSc. Teléfono: (57-4) 425 5172
degree in Civil Engineering in Federal University of Rio Grande do Sul,
Brazil in 1986, and the PhD degree in Enginyeria de Camins Canals i Ports
- Universitat Politècnica de Catalunya - Barcelona Tech, Spain, in 1996 and
PhD. in Universitat Politècnica de Catalunya, Spain, in 2013. Currently is an
associate professor at the State University of Londrina, Brazil.
ORCID: 0000-0001-8828-7250

D.S. Cezario, received the BSc. in Mechanical Engineering in 2014 at the

State University of São Paulo and post graduated in Labor Safety at the State
University of Maringá, Brazil in 2017.
ORCID: 0000-0001-6137-731X

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