Self healing geopolymer using endogenous carbonatogenic bacteria isolated

from a Jaddih mountain in Bangkalan, East Java

Annisa Nur Fitriani1,4, Edo Danilyan1,4, Dika Bagas Permana2,4, Dimas Haqqi W3,4, Gulsheren
Singh3,4
1
Departemen Biologi, Fakultas Sains dan Analitika Data
2
Departemen Teknik Sipil, Fakultas Teknik Sipil, Perencanaan dan Kebumian
3
Departemen Teknik Mesin, Fakultas Teknologi Industri dan Rekayasa Sistem
4
Institut Teknologi Sepuluh Nopember Surabaya

Abstrak
Geopolymer is an environmentally friendly alternative to concrete. Bio-based self-healing in
cement is a significant development for the advancement of bioconcrete as a solution to
reduce the occurrence of cracks. The aims of this study were to isolate potential bacteria as
repair agents for geopolymer concrete, to characterize isolates and precipitation of calcium
carbonate, and to evaluate the effect of the main ingredient in geopolymer concrete on
compressive strength. The research method was carried out through several stages, namely
bacterial isolation, urease test, preparation of geopolymer and isolate viability, then the
compressive strength test of concrete was carried out, and data analysis. Research activities
were carried out for 5 months starting from site surveys, observations, sampling, cultivation,
observations, data analysis, seminars, monitoring and evaluation, preparation of final
reports, and publications.
because it can cause loss of structural integrity
I. Introduction of a building (Monfardini et al., 2016).
Geopolymers have become an Conventional crack repair methods are
environmentally friendly alternative to often complex, expensive, and time
concrete due to their low carbon footprint and consuming. To prevent further crack
excellent mechanical and chemical properties. propagation, they must be treated immediately
Geopolymers can be prepared through after crack formation occurs (Szeląg, 2020).
reactions involving aluminosilicate sources, As a result, self-healing bioconcrete has
which can be derived from waste by-products emerged as a promising solution to this
such as coal fly ash, and alkaline solutions, problem. Previous studies have often favored
which can initiate the geopolymerization the use of non-pathogenic soil microorganisms
process (Cong et al., 2021). Several studies as crack filling agents because they may be
have reported geopolymer characteristics such safer and more sustainable than chemical
as high compressive/flexural strength, low repair methods (Chen et al., 2019).
shrinkage, acid and fire resistance (Xie et al., Bio-based self-healing in cement has
2019), and high temperature and chemical made significant progress, namely the
stability in geopolymers due to the use of development of bioconcrete advancements.
different precursors and mixture ratios When cracks form, air and water can reach
(Hassan et al. ., 2019). However, because it is dormant microbes, stimulating them and
a cement-like material, geopolymers are still encouraging them to deposit minerals, which
prone to cracking. When geopolymer is used then close the cracks (Khaliq et al., 2016).
as a building material, this is undesirable Despite these advances, it is questionable
whether the same methods used to construct
bioconcrete can also be used to construct
geopolymers having different physicochemical
structures. However, these conditions can also
be more dangerous for microbial growth.
Currently very few studies have investigated
the use of microorganisms in geopolymers for
self-healing applications and enhancement of
physical properties. Given the huge gap that
needs to be bridged in improving
biogeopolymers that can actually repair cracks
naturally, current research seeks to investigate
what locally available bacterial species can be
used as crack filling agents and geopolymers
based on fly ash, metakaolin and ceramic
grog. . And how the viability of bacteria in
these substances can be increased.
II. Method
This research was carried out from
December 2021 to April 2022. Bacterial
isolation was carried out in Bukit Jaddih, East
Java. Characterization and growth of isolates
was carried out at the Microbiology and
Biotechnology Laboratory, Department of
Biology, Faculty of Science and Data
Analytics, Sepuluh Nopember Institute of
Technology (ITS), Surabaya. The
measurement of the compressive strength of
geopolymer concrete was carried out at the
Department of Civil Engineering, Sepuluh
Nopember Institute of Technology. The
characterization of calcium carbonate
precipitation was carried out using a Scanning
Electron Microscope (SEM) at the ITS Energy
and Environment Laboratory.
There is a few steps to running this
experiment, such as :
● Bactery Isolation :
Screening of isolates that can precipitate
calcium carbonate was carried out by
cultivating isolates on CCP (Calcium
carbonate precipitation) media containing
(per liter) 20 g urea, 2.12 g NaHCO3, 10
g NH4Cl, 3 g nutrient broth, 30 mM
CaCl2, 20 g agar then the pH of the
medium was adjusted to 8.5 (Chen et al.,
2019). Bacterial isolates were incubated
at room temperature for 7 days.
Carbonatogenic bacteria were evaluated
for their ability to manifest a zone of
CaCO3 deposition around the colony.
● Urease Test :
A qualitative urease test on Christensen-
urea agar was carried out to determine the
ability of isolates to produce urease
(Labud et al., 2007). The isolates were
incubated at room temperature for 2-5
days. The color shift of the medium from
yellow to pink indicates urease produced
by carbonatogenic bacteria. Urease
hydrolyzes urea and releases ammonium
which increases the pH in the medium
until the color of the medium turns pink.
Morphological and biochemical
characterizations such as cell shape,
Gram stain, endospore staining, catalase
test, oxidase test, motility test, and
oxygen demand test were carried out
following standard protocols.
● Geopolymer Preparation and Isolate
Viability Test :
There are 2 geopolymer precursors used,
the first is a mixture of GGBS with Fly
ash with NaOH activator at 23 degrees C.
The second is a mixture of metakaolin
with ceramic grog with potassium silicate
activator. Geopolymer concrete curing
was carried out for 28 days.
Bacterial viability test was used to
determine the viability of bacteria after
being mixed with geopolymer precursors.
Concrete specimens after curing for 28
days were taken to be pulverized. The
crushed concrete samples were
transferred aseptically into a
physiological solution (0.9% NaCl), then
inoculated on CCP-agar media, the
colonies that grew showed the viability of
the isolates in the concrete raw material.
● Concrete Compressive Strength Test :
The compressive strength of concrete is
the ability of concrete to accept
compressive forces per unit area.
 Although there is a small tensile stress in
the concrete, it is assumed that all
compressive stresses are supported by the
concrete. The compressive strength test
of concrete is carried out based on SNI
1974:2011. The calculation of the
compressive strength of the test object is
expressed by dividing the maximum load
received by the test object during the test
by the average cross-sectional area and
the result is rounded up to 1 decimal
place with units of 0.1 MPa.
● Data analysis :
Student's t-test was used to determine the
difference in compressive strength
between 2 types of geopolymer concrete
precursors. ANOVA was used to
determine the ability of calcium
carbonate precipitation between several
isolates. Data visualization and statistical
tests were carried out in the programming
language R ver.4.1.1.
III. Results and Discussion
Bacteria Isolation and Purification
Isolation and purification of
hydrocarbonoclastic bacteria yielded 15
isolates, with colonies able to produce CaCO 3
crystals surrounding the growth region when
grown on CCP agar (Fig.1). The CCP media,
which contains urea and CaCl 2 as precursors
of carbonate and calcium ion formation, is
used to detect the precipitation of CaCO 3
minerals (Kim et al., 2016). Because
developing isolates produce urease, a CaCO 3
precipitation zone forms surrounding the
bacterium colonies (Hammad et al.,2013).
Qualitative Urease Activity
Qualitative urease test was conducted
to differentiate urease producing bacteria
(Atlas, 2010). Based on the urease activity
test, among 15 isolates which produced
CaCO3, only 8 isolates were able to produce
urease which can be characterized by the color
changes on Christensen-Urea broth medium
from pale yellow into cherrish pink (Fig. 2).
Urea in the medium will be hydrolyzed by
isolates urease into ammonia and carbamate.
In the other stage it will be spontaneously
hydrolyzed and produce ammonia and
carbonic acid. Ammonia is alkaline
compound, so it will increasing the pH of the
medium. The color changes of phenol red
indicator into cherrish pink were caused by pH
variations in the medium becoming alkali
(Siddique and Chahal, 2011).
Isolate Identification and CaCO3
Production
According to Bergey's Manual
Determinative of Bacteriology, isolates SG3,
SG4, JB2, JB3, AT2, AT3 are Bacillus,
Sulfidobacillus isolates are Sulfidobacillus,
and Escherichia isolates are Escherichia.
Bacillus has the following features, according
to Holt et al. (1994): Gram-positive rod-
shaped cell that is motile, forms endospores, is
aerobic/ facultatively anaerobic, and has a
positive catalase reaction. Sulfido bacillus has
rod shaped cell, Gram-positive, non-motile,
endospores forming, aerobic/facultative
anaerobic, and positive catalase reaction. Rod-
shaped cells, Gram-negative, motile, non-
endospore-forming, aerobic/facultative
anaerobes, and positive catalase reaction are
all characteristics of Escherichia coli. Isolates
which grew in CCP-broth media formed
CaCO3 . Based on our observation Bacillus
JB2 has the highest value of CaCO 3 precipitate
compared to other isolates (Table 1).
Table 1. CaCO3 Production
CaCO3
Isolate Genus
(mg/ml)
SG 2 Sulfidobacillus 0.94
SG 3 Bacillus 1.12
SG 4 Bacillus 2.66
SG 5 Escherichia 2.64
JB 2 Bacillus 3.22
 JB 3 Bacillus 2.82
AT 2 Bacillus 3.02
AT 3 Bacillus 0.50
In a high urea and calcium
environment, bacteria's ability to produce
CaCO3 serves as a tolerance. It will produce
urease to hydrolyze urea and release carbonate
ions (CO32 ), then it will bind to calcium ions
(Ca2+) from CaCl2 to generates CaCO3 and
H2O (Chaparro-Acuna et al., 2017). The
formed CaCO3 crystals can be used as
additives in concrete raw materials which has
a role to strengthen materials in the raw
material (Krishnapriya et al., 2015). Besides of
concrete Furthermore, for concrete production
application this present study used Bacillus
JB2 since its able to produce the highest
CaCO3 amounts (3.22 mg/mL) compared to
other isolates
Concrete Preparation and Viability Test
The logarithmic phase and optimal
growth of Bacillus JB2 are after incubation for
10 hours, the cell density was calculated from
2.80 to 4.96 x 106 . The addition of bacteria
for concrete preparation was done using a
culture that had been cultured for 10 hours.
According to SNI 03-3976-1995, concrete
must be in moist for at least 7 days, the quality
could be better if the curing stage is carried
out for 28 days. The morphology of the
control concrete and concrete which added by
Bacillus JB2 isolates possess relatively the
same external morphology, so that the addition
of Bacillus JB2 as a hydrocarbonoclastic
bacteria did not affect the external
morphology of the concrete. The bacterial
viability test was carried out to determine the
viability of hydrocarbonoclastic bacteria after
being embedded in concrete. Bacterial
colonies that grew on CCP-agar media show
their ability to resist against harsh
environment inside the concrete.
Concrete Compressive Strength
Compressive strength is one of the
main properties of concrete, the results of
compressive strength tests on control concrete
and applicative concrete which have been
added by Bacillus JB2 are served in Table 2.
Based on Analysis of Variance (ANOVA),
there are differences in the concrete
compressive strength between control concrete
and concrete which is added by Bacillus JB2,
the LSD test (P-value 5%) shows a significant
difference. Based on Table 2, concrete with
the addition of Bacillus JB2 has a higher score
in maximum load and compressive strength
than control concrete. Increased strength of
concrete is mainly caused by CaCO3 crystals
which formed by bacteria that can fill the
micropores inside concrete, so that the size of
the pores will shrink and maximize the
compressive strength of concrete sample
(Chahal et al., 2012). The formation of CaCO3
by bacteria due to hydrolysis mechanisms of
urea which generates ammonia ions and
carbonate ions. Ammonia will increase the pH
of the medium, then the environment becomes
alkaline. That is favorable condition and
support the binding of carbonate ions and
calcium ions to generate CaCO3 crystals (Wei
et al., 2015)
Table 2. Compressive Strenght
Compressive
Treatment
Cell Density Maximum
(sel/mm3 ) Load (Kg)
Strength
(Mpa)
Control
30.44a
(without 0 24,273.03
isolate)
Bacillus JB 2
3.36 x 106 27,510.20 33.11b
(1)
Bacillus JB 2
2.85 x 106 26,862.48 33.30b
(2)
Bacillus JB 2
(3)
4.93 x 106 25,627.44 33.36b
IV. Conclusion
Eight isolates of hydrocarbonoclastic
bacteria are able to produce and form CaCO3
crystals. Concrete with the addition of bacteria
cells has a maximum load and compressive
strength of concrete higher than control
concrete. Therefore, Bacillus JB 2 bacteria can
be applied as additives to strengthen concrete
structures as a repair material for geopolymer
concrete, isolate and depositional
characteristics of calcium carbonate, as well as
evaluate the effect of the main ingredients for
making geopolymer concrete on the tensile
strength.
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