Case Studies in Construction Materials 11 (2019) e00250

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Case Studies in Construction Materials

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Impact of the self-healing agent composition on material

characteristics of bio-based self-healing concrete
Hana Schreiberová, Petr Bílý* , Josef Fládr, Karel Šeps, Roman Chylík,
Tomáš Trtík
Department of Concrete and Masonry Structures, Czech Technical University in Prague, Faculty of Civil Engineering, Thákurova 7, 166 29
Praha 6, Czech Republic

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

Article history: This paper investigates the composition of the biological self-healing agent based on its
Received 13 February 2019 impact on material characteristics of concrete. A direct addition of the agent – a mixture of
Received in revised form 10 May 2019 bacterial spores and nutrients – into concrete matrix has been investigated by many studies
Accepted 15 May 2019
in recent decades. Under certain conditions, the applied microorganisms proved to be able
to produce CaCO3, and researchers used this biocalcification process to autonomously seal
Keywords: microcracks in concrete. Thus, this bio-based material could potentially heal itself and lead
Bio-based concrete
to a more durable and economic structure. However, it has been shown that the self-
Self-healing concrete
Nutrients
healing agent, especially the indispensable nutrients, can positively or negatively influence
Mechanical properties the material characteristics. In this study, some of the most suitable and frequently
proposed nutrients (calcium lactate, calcium nitrate, calcium formate, urea, and yeast
extract) were directly added into cement mortar during the mixing process and their
impact on material characteristics – compressive strength, flexural strength, and rheology
– was evaluated and compared. Results show that calcium nitrate, calcium formate,
calcium lactate, and urea have generally a potential to increase the compressive strength,
especially in early ages. In contrast, the applied dose of yeast extract resulted in a drastic
drop of compressive strength when compared to the control series, thus further
optimization of the concentration is needed. The flexural strength was affected rather
negligibly by the proposed nutritional admixtures.
© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

In the field of cementitious building materials, microbiology has traditionally been included in the research from the
perspective of negative effects: biodeterioration [1], microbial stains [2], and human health issues. However, in some cases,
the interaction between the material and microorganisms can result in positive actions as well. One of them, the so-called
biocalcification process, has been extensively studied in the previous decades with the aim to increase the durability of both
existing and new structures.
The ability of certain microorganisms to produce CaCO3was firstly reported by Murray, Irvine, and Steinmann [3] in the
late 19thcentury. Since then, researchers have deepened the knowledge of the microbially induced calcite precipitation and

* Corresponding author.
E-mail address: petr.bily@fsv.cvut.cz (P. Bílý).

https://doi.org/10.1016/j.cscm.2019.e00250
2214-5095/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
2 H. Schreiberová et al. / Case Studies in Construction Materials 11 (2019) e00250

proposed numerous potential applications: protection and remediation of stone surfaces [4], biological repair systems of
cracked concrete surfaces [5], and bio-based self-healing concrete.
This paper focuses on the lastly mentioned: a type of concrete with enhanced natural autogenous healing capacity by the
incorporated self-healing agent (calcite-producing bacteria in the form of spores accompanied by nutrients). As it is widely
known, the durability of concrete structures is largely influenced by the formation of microcracks. These cracks do not
endanger the load-bearing capacity directly, however, if the crack widths exceed a certain level, the cracking might result in a
failure of the cover layer of the embedded steel reinforcement. Thus, it may lead to a total failure of the structure. In the
novelty biological concrete, the incorporated bacterial spores are activated due to water ingress caused by the cracking, and
they start to metabolize the supplied nutrients. This action results in a formation of CaCO3 by which the already existing
cracks are then gradually sealed.
As the concept of sustainable development has emerged in the popularity growth in the field of building materials, the
bio-based self-healing concrete has been extensively studied in the recent decade. Numerous investigations identified
suitable bacterial genotypes and described their limitations [6–9]; they proposed different types of bacteria protection [5,6,
10–13]; and they examined the self-healing efficiency through in-vitro and in-situ experiments [14,15].
This paper concentrates on the composition of the self-healing agent, specifically on the nutrients. The selection of the
nutritional compounds is primarily based on the mode of metabolic activity of the applied bacteria. Generally, the calcite
precipitation by microorganisms is based on the shift of carbonate ions (CO3
2), which is formed due to the presence of
carbon dioxide released by the metabolic activity of bacteria, under conditions of the high pH and high calcium ion (Ca+2)
concentration to calcium carbonate [16]. However, several metabolic pathways are known to result in the carbonate ions
formation: aerobic respiration [17], urea hydrolysis [18], and nitrate reduction [10].
All of the mentioned bio-calcification pathways need a suitable metabolic activator and calcium source. A wide range of
these compounds was proposed and analyzed from the perspective of the crack sealing efficiency [19], the impact on
mechanical properties of concrete [20,21], and economic factors [22]. However, the selection based on the presented factors
demand further clarification. Only a very limited number of studies compared the impacts of the nutrients to a larger extent
and the reported results differ quite significantly. At the same time, it is clear that the effect of the nutrients on mechanical
properties of concrete has a profound impact on the practical applicability of bio-based self-healing concrete. The fact that
the bacteria together with selected nutrients can heal the cracks in concrete has no practical relevance if the mechanical
properties of concrete are deteriorated by the nutrients at the same time.
In this study, a variety of nutrients (yeast extract, urea, calcium nitrate, calcium formate, and calcium lactate) was
proposed based on the reported impacts on the compressive strength, economic comparison, and compatibility with
concrete. Subsequently, the pre-selected nutrients were directly added into a cement mixture and a large number of small
prism specimens was prepared. This paper then evaluates the impacts of the applied nutrients and discusses the most
suitable self-healing agent composition. The results can serve as a guidance for other researchers dealing with optimization
of bio-based self-healing concrete mixtures.

2. Materials and methods

2.1. The nutrients selection

In order to achieve truly self-healing material, it is necessary to incorporate the healing agent directly into the mortar
matrix. Although numerous studies proposed and investigated encapsulation of the complete agent – bacteria and nutrients
– the possibility of an unintentional leakage from the protective carrier or encapsulation of the bacteria only must be
considered for ensuring functionality and safety of the novelty material. Consequently, it is inevitable to determine the
impact of these compounds to the materials properties.
With the intention of optimizing the self-healing agent, applied nutrients and their dosages were pre-selected based on
the already performed experiments presented in available literature, economic factors, and widely known compatibility
with concrete.
The economic factor, hence the commercial applicability of the bio-based self-healing concrete, was approximately
determined on the basis of the product prices on the web Alibaba.com to date 09/2018. This mode of evaluation was inspired
by Palin et al. [22]. The price comparison was carried out only between the calcium sources, as the addition of yeast extract
and possibly urea is unavoidable depending on the mode of the metabolic pathway of the selected bacteria. The detailed
description of the pre-selection can be seen in Table 1.
Based on the research, following nutrients were selected: calcium lactate (C6H10CaO6), calcium nitrate (Ca(NO3)2), and
calcium formate (Ca(HCOO)2) at a concentration of 3% to cement weight as calcium sources, yeast extract at 0.85% to cement
weight as nutritional carbon and nitrogen source, and urea at a concentration of 2.5% to cement weight as addition in case of
a self-healing agent with ureolytic bacteria.
In our study, the impact of incorporation of bacteria itself to cement mortar was not determined. Firstly, previous studies
did not report any significant change in mechanical properties caused by bacterial spores, thus only negligible differences
were expected in general. Secondly, the selection of the most suitable genus of bacteria is primarily a matter of evaluation of
the calcification potential and its behavior in various conditions such as low and high temperatures, different humidity and
 H. Schreiberová et al. / Case Studies in Construction Materials 11 (2019) e00250 3

Table 1
An overview of the most frequently proposed nutrients; their impact on the compressive strength in various dosages reported in previous studies; approx.
prices from Alibaba.com; and summarizing comments.

Type of Dose [% by Approx. shift in the Ref. Price Comments

nutrient cement compressive strength [$/1000 kg]
weight] relative to ref. sample
at 28 days
calcium 9.60% 58% [20] 1400 Positive effect on the compressive strength reported; frequently applied in the
lactate 4.80% 118% [20] bio-based self-healing concrete research; proved efficiency for the
3.00% 122% [21] biocalcification process.
2.50% 130% [20]
2.00% 116% [21]
1.00% 103% [21]
1.00% 118% [17]
1.00% 108% [19]
calcium 3.00% 103% [21] 900 Positive effect on the compressive strength reported; commercially available;
formate 2.00% 105% [21] used as a hydration accelerator.
1.00% 119% [21]
calcium 9.60% 118% [20] 200 Positive effect on the compressive strength reported; commercially available;
nitrate 4.80% 119% [20] used as a hydration accelerator; the key chemical compound for nitrate
3.00% 79% [21] reducing bacteria.
2.50% 109% [20]
2.00% 87% [21]
1.00% 108% [21]
calcium 1.00% 103% [19] 7200 Commercially unavailable; expensive.
glutamate
calcium 1.00% 73% [17] 1200 Negative effect on the compressive strength reported.
acetate
calcium Presence of chloride can lead to degradation and reinforcement corrosion; not applicable in reinforced concrete in large doses.
chloride
urea 0.60% 85% [20] 250 The key chemical compound for ureolytic bacteria.
0.30% 67% [20]
0.12% 101% [20]
yeast 1.00% 65% [17] 7000 The key metabolic activator; a negative effect on the compressive strength;
extract 0.60% 76% [20] maximum dosage of 0.85% proposed by Wang et al. [11]
0.25% 80% [20]
0.12% 77% [20]

nutritional supply. Thus, a comparison of different bacteria from the perspective of the mechanical properties changes
seemed to be not needed at this point of our research.

2.2. Mortar specimen preparation

In order to determine the impacts of the pre-selected nutrients mentioned above, series of mortar specimens were
fabricated. The aim of the study was to compare the effect of different nutrients on mechanical properties, not to determine
the absolute values of mechanical properties of a concrete mixture. The component of concrete that is affected by nutrients is
the cement paste, not the aggregates, and therefore one can be sure that particular nutrients will have similar effect on
concretes as they have on mortars.
The base for all the series was ordinary Portland cement CEM I 42.5 N (obtained from Závod Mokrá, Czech Republic) and
sand with grains 0.1–1 mm and 1–2 mm (obtained from Provodínské písky a.s., Czech Republic). The water-to-cement ratio
was 0.5 and the cement-to-sand ratio was 3 (Table 2).
The nutrient series were prepared by an addition of the calcium lactate (C6H10CaO6.5H2O, purity  98%, obtained from
Carl Roth GmbH + Co. KG, Germany) at a concentration 3% to cement weight (LAC), calcium nitrate (Ca(NO3)2.4H2O, purity 
99%, obtained from Lach-Ner, s.r.o., Czech Republic) at a concentration 3% to cement weight (NIT), calcium formate (Ca
(HCOO)2, purity  99%, obtained from Sigma-Aldrich spol. s.r.o., Czech Republic) at a concentration 3% to cement weight

Table 2
Mixing proportion of mortar used for all of the series.

Material Amount
[kg/m3]
CEM I 42.5 N 586.00
Water 293.00
Sand (1-2 mm) 439.50
Sand (0.1-1 mm) 1318.50
4 H. Schreiberová et al. / Case Studies in Construction Materials 11 (2019) e00250

Table 3
Applied nutritional admixtures to the basic cement mortar.

Series Admixture Amount Amount

[% by c. w.] [kg/m3]
CTRL – – –
LAC Calcium lactate 3.00 17.58
NIT Calcium nitrate 3.00 17.58
FORM Calcium formate 3.00 17.58
YE Yeast extract 0.85 4.98
UR Urea 2.50 14.65

(FORM), yeast extract (obtained from Carl Roth GmbH + Co. KG, Germany) at a concentration 0.85% to cement weight (YE),
and urea (NHCONH, purity  99.5%, obtained from Ing. Petr Švec - PENTA s.r.o., Czech Republic) at a concentration 2.5% to
cement weight (UR). The group without any further addition served as a control sample (CTRL). The overview of the applied
additions to the cement mortar can be seen in Table 3.
Mortar prisms 40x40x160 mm3 were prepared in triplicate sets for each group for the flexural and compressive test in 3, 7
and 28 days. Specimens were demolded after one day and placed in a room with temperature (20  2 C) into plastic water-
filled containers where they were cured until 12 h before testing.

2.3. The rheology of fresh cement paste

Cement flow test was performed in an attempt to determine the impact of the selected nutrients on the rheology of fresh
cement paste. Immediately after the mixing process, a part of the cement paste of all the series was removed and submitted
to the cement flow test using a cement flow table.
A cone placed on a 280 mm diameter table was filled with the currently tested mortar group in three layers and tamped
with 9 drops of the table (3 drops per layer). The cone was thereafter removed and spreading of the cement paste was
measured. Subsequently, the sample was dropped 15 times freely and the final spreading was measured. This procedure
largely corresponds with a standard cement flow table test [23]. As the aim of the rheology test was primarily to compare the
nutrient impacts, not a fully standardized procedure was needed in this paper.

2.4. The flexural and compressive strength

After the end of the respective curing period (i.e. after 3, 7 and 28 days from casting), all of the series were submitted to
mechanical testing. Firstly, a three-point bending test according to [24] was performed on 40x40x160 mm3 mortar
specimens using a calibrated electric loading machine. The bending tests were run in a deflection-controlled mode with a
loading rate of 0.3 mm/min, and the maximal load values were recorded and analyzed using a controlling software (SMAPS).
The final flexural strength of the tested mix designs was determined according to the following equation:
3F max l
f cf ¼ 2
ð1Þ
2bh
Where fcf is the flexural strength; l is the distance between support rollers; b and h are the width and height of the tested
specimen, respectively; and Fmax is the maximum applied load.
Secondly, the compressive strength was determined. The test was performed on the specimen halves that were created
after the three-point bending test according to [24]. The specimens were evenly loaded with force at the rate of 2.5 kN/s
using a calibrated hydraulic loading machine, and the mode of failure was observed to exclude incorrectly damaged
specimens. The maximum applied load was recorded and analyzed using a controlling software (SMAPS).
The final compressive strength of the tested mix designs was determined according to the following equation:
F max
f cc ¼ 2
ð2Þ
b
Where fcc is the compressive strength; b is the dimension of the loading area; and Fmax is the maximum applied load.
The triplicate sets for each investigated mix design group led to 3 independent values of the flexural strength and 6 values
of the compressive strength at each of the tested time (i.e. 3, 7 and 28 days). For the purpose of comparing the impact of the
applied nutrients, the obtained values were averaged by the arithmetic mean and their standard deviation (SD) was
determined according to the following formula:
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
uP
uN
u ðxi 
xÞ2
t
SD ¼  i¼1 ð3Þ
ð N
1Þ

Where xi is the obtained value; x̄ is their arithmetic mean value and N is the number of values.
 H. Schreiberová et al. / Case Studies in Construction Materials 11 (2019) e00250 5

Table 4
Results of cement flow table test.

Series Diameter before drops Diameter after drops

[mm] [mm]
CTRL 100 115
LAC 100 150
NIT 100 130
FORM 100 130
YE 100 160
UR 100 145

3. Results

3.1. The rheology of the fresh cement paste

In this work, the impact of the investigated nutritional admixtures on the rheology was determined via a cement flow
table test. In general, the obtained results have an overall strong concordance with our expectations. In all of the cases, the
addition of nutritional admixtures resulted in an increased viscosity of the fresh cement paste as it can be seen in Table 4.
The addition of calcium nitrate and calcium formate resulted in the increase of 13% in the workability. As both of the
chemical compounds are commercially used as hydration accelerators, it is known that they influence the consistency of
fresh cement mortar quite dramatically, especially in higher dosages [25–27].
Calcium lactate and urea addition caused a noticeable increase of approximately 30% in the fluidity; this tendency was
also reported in previous research [21,28]. The highest fluidity value was obtained in the cement mortar with yeast extract;
the applied additive resulted in an immense increase of 40%.

3.2. The flexural strength

The performed three-point bending tests provide information about the impact of the applied nutrients on the flexural
strength of prepared cement mortar. On the whole, the shift due to the nutritional addition usually did not exceed 5% when
compared to the control series at 28 days.
The lowest values of the tensile strength in all ages were obtained in the cement mortar with yeast extract as a nutritional
source, which is in line with our expectations. In Table 5, a detailed overview of the obtained data (the mean values and their
standard deviations) is presented.
The data given in Table 6 and Fig. 1 display slightly better results of the flexural strength (an increase of around 10–15%) of
mortar with urea, calcium lactate, and calcium formate in early ages (3 days) when compared to control mortar. However, the
final flexural strength in 28 days did not seem to be affected extensively by those admixtures. Furthermore, although calcium
nitrate is commercially used as a hydration accelerator, the flexural strength was not improved by the chemical admixture in
any hardening stage.

Table 5
The flexural strength (the mean value and its standard deviation) measured by three-point bending test.

Testing age Series

CTRL LAC NIT FORM YE UR

3 days [MPa] 4.51  0.16 4.85  0.25 4.22  0.10 5.12  0.14 2.65  0.08 4.94  0.48
7 days [MPa] 5.23  0.52 5.03  0.34 5.23  0.36 5.40  0.37 4.14  0.13 5.86  0.17
28 days [MPa] 6.48  0.25 6.55  0.67 6.30  0.03 6.33  0.17 5.46  0.16 6.71  1.17

Table 6
The impact of nutritional admixtures on the flexural strength mean values compared to control.

Series 3 days 7 days 28 days

[%] [%] [%]
CTRL 100 100 100
LAC 107 96 101
NIT 94 100 97
FORM 114 103 98
YE 59 79 84
UR 109 112 104
6 H. Schreiberová et al. / Case Studies in Construction Materials 11 (2019) e00250

Fig. 1. Dependence of the mean values of the flexural strength on the measuring time.

3.3. The compressive strength

As outlined in the introduction, the impact of the self-healing agent on the compressive strength of concrete is one of the
key factors that noticeably affect the applicability of the novelty material. In order to compare different compositions of the
self-healing agent which were applied in previous studies, a number of cement mortar specimens with and without the pre-
selected nutritional admixtures were submitted to compressive strength tests. Detailed list of obtained results is provided in
Table 7.
For the sake of simplicity, in Table 8 and Fig. 2 the mean values of the compressive strength results are presented. The
results show that the pre-selected calcium sources (calcium formate, calcium nitrate, and calcium lactate) in the
concentration of 3% to cement weight do not noticeably endanger the compressive values at any age. In this study, the
addition of calcium lactate and calcium formate had a distinct tendency to even increase the values throughout the whole
curing period.
As calcium nitrate and calcium formate are used as hydration accelerators, a steeper growth of the compressive strength
was expected, and the results are in agreement with our assumption. Additionally, unlike calcium nitrate, the mortar with
calcium formate reached an even higher final value of the compressive strength when compared to the control series. The
strength measured at 28 days was improved by 30% in the case of calcium lactate, whereas the addition of calcium nitrate
resulted in a slight drop of 9%.
In this study, a higher dose of urea (2.5% to cement weight) had predominantly a positive impact on the compressive
strength in the investigated ages. The final value of mortar with urea did differ only minimally from the control series.

Table 7
The compressive strength (the mean value and its standard deviation).

Testing age Series

CTRL LAC NIT FORM YE UR

3 days [MPa] 22.88  2.04 27.70  3.50 26.44  1.60 37.96  2.97 10.65  1.17 30.80  2.75
7 days [MPa] 28.93  3.01 36.02  2.16 33.47  1.45 44.23  3.71 19.61  1.33 34.77  1.18
28 days [MPa] 45.31  4.50 58.76  2.18 41.13  3.42 57.63  2.10 35.12  2.98 44.39  4.23

Table 8
The impact of nutritional admixtures on the compressive strength mean values compared to control mortar.

Series 3 days 7 days 28 days

[%] [%] [%]
CTRL 100 100 100
LAC 121 125 130
NIT 116 116 91
FORM 166 153 127
YE 47 68 77
UR 135 120 98
 H. Schreiberová et al. / Case Studies in Construction Materials 11 (2019) e00250 7

Fig. 2. Dependence of the mean values of the compressive strength on the measuring time.

The addition of yeast extract in the dose of 0.85% to cement weight caused a drastic drop of the compressive strength.
Especially at the early age (3 days), the strength of the YE series reached as little as 47% of the strength of the control series.
Furthermore, two of the samples prepared for the testing at 3 days and 28 days have been spontaneously damaged and
excluded from mechanical tests; a large crack appeared on the samples when they were immersed in tap water in the storage
containers.

4. Discussion and conclusions

The aim of this study was to provide a comprehensive overview of the most promising nutritional compounds used as a
part of the self-healing agent in bio-based self-healing concrete. The criteria which determined the pre-selection were
compatibility with concrete and previously reported effects on its mechanical properties.
In this section, the obtained values of the compressive strength at 28 days, the major usability factor, will be briefly
compared to the previous results available in the literature, which are presented in Table 1. In this study, the 3% addition of
calcium lactate to the cement matrix caused a considerable improvement of about 30%, which is in agreement with the result
reported by Luo et al. [21]. The dose 3% of calcium nitrate resulted in a slight decrease of about 10%, which is about 2 times
less than in the experiment carried by Luo et al. [21]. The impact of 3% addition of calcium formate was also investigated by
Luo et al. [21]. However, the positive effect of calcium formate in our study reached up to 30%, whereas Luo et al. detected
only a negligible difference from the control series. The impact of the urea and yeast extract addition in our concentrations
were not previously directly determined by mechanical tests, thus our obtained data provide a useful extension of the known
impacts.
On the basis of the conducted research and our own examinations, this paper concludes that all of the pre-selected
calcium sources (calcium formate, calcium lactate, and calcium nitrate) are, at the proposed concentrations, applicable
without any considerable negative impacts on mechanical properties of cement mortar. Furthermore, the addition of
calcium formate and calcium lactate led to a cementitious material with significantly higher compressive strength values at
all ages. In addition, the presence of urea, the nutritional compound for ureolytic bacteria, at a concentration of 2.5% to
cement weight did not negatively affect the materials properties as well.
Based on the results, the addition of yeast extract to cement mortar seems to be the most problematic admixture. Our
data suggest that the concentration of 0.85% to cement weight causes a dramatic fall of the material’s strength at all ages.
Potentially, this negative effect could be balanced by the presence of the other nutritional compounds. The investigation of
the interactions will continue in our future studies.
It is important to acknowledge that the bio-based self-healing concrete will be, in all probability, rather financially
demanding material. It implies that it will find its use in complex and highly important parts of structures. For that reason,
future studies should certainly further optimize the self-healing agent in terms of not only the efficiency of the crack sealing
process but also from the perspective of the impacts on the materials characteristics.

Data availability statement

The experimental data used to support the findings of this study are available from the corresponding author (Petr Bílý, e-
mail petr.bily@fsv.cvut.cz) upon request.
8 H. Schreiberová et al. / Case Studies in Construction Materials 11 (2019) e00250

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Declarations of interest

None

Acknowledgments

This paper was prepared thanks to the support of the Science Foundation of the Czech Republic (GACR), project “Self-
healing of cementitious composites by bacteria-induced calcification” (no. 18-15697S).

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