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A simple method to create hydrophobic mortar using bacteria

grown in liquid cultures
Citation for published version (WHAT):
Qu , Z. , Guo , S. , Zheng , Y. , Giakoumatos , EC , Yu , Q. , & Voets , IK (2021). A simple method to create
hydrophobic mortar using bacteria grown in liquid cultures. , [123744]. Construction and Building Materials297 ,

https://doi.org/10.1016/j.conbuildmat.2021.123744

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Machine Translated by Google

Construction and Building Materials 297 (2021) 123744

Contents lists available at ScienceDirect

Construction and Building Materials

journal homepage: www.elsevier.com/locate/conbuildmat

A simple method to create hydrophobic mortar using bacteria grown in

liquid cultures
a,1 a,1 a
Zhengyao Qu Ilja , Shuaiqi Guo b,c,d,ÿ,1 , Yifeng Zheng , Emma C. Giakoumatos b,c,d , Qingliang Yu ,

K. Voets b,c,d,ÿ
aDepartment of the Built Environment, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, the Netherlands
b
Laboratory of Self-Organizing Soft Matter, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, the Netherlands
c
Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, the Netherlands
d
Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, the Netherlands

article info abstract

Article history: The ingress of water-soluble aggressive species is a major cause to the reduced service life of concrete
Received 11 January 2021 structures. Bulk modification by adding hydrophobic agents in the cementitious materials is commonly
Received in revised form 14 May 2021 applied to reduce water penetration into the concrete matrix. However, conventional hydrophobic mod ifications
Accepted 23 May 2021
of concrete are associated with several unresolved major drawbacks such as high cost and tox icity. Here, we
investigate the impact of safe and easy-to-culture bacteria on the hydrophobic properties
of concrete. We demonstrate that hydrophobic concrete can be conveniently created by using the liquid
Keywords:
culture of bacteria without the need of growing biofilms as suggested by previous research, even with a
Hydrophobic materials
Water resistance strain that does not present innate hydrophobicity. These key findings greatly improve the feasibility for
Concrete the large-scale application of water resistance cementitious materials. Scanning electron microscopy and
Hydrophobic bacterium X-ray diffraction analyses indicated higher levels of micro-calcite formations with the increased addition
Hydrophilic bacterium of bacteria. These micro-calcite structures account for the dramatic increase in surface roughness which
leads to elevated hydrophobic properties of the hybrid concrete. This phenomenon is reminiscent of the
‘‘lotus-leaf effect”, with ultrahydrophobicity arising from the nano- and microscopic topologies of sur faces.
Bacteria-modified cement presents distinct heat-flow profiles during hydration consistent with
the increased calcite formation. Taken together, this research gives unprecedented mechanistic insight
into the design of microbial-modified concrete with hydrophobic properties particularly useful in floating
and offshore structures. Given the large repertoire of bacteria still to be used, our work sets the stage for
the future design of microbial-based hydrophobic concrete that can be tailored with additional functions
such as self-healing and odor removal.
2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

1. Introduction corrosion of the steel reinforcement of the concrete structures or

The durability of concrete is a major concern to design and
application of concrete structures in harsh environments [1–3].
The durability of concrete is normally defined as resist weathering
action, chemical attack, and abrasion while maintaining its desired
engineering properties [4,5]. Generally, concrete is a hydrophilic
material and water can be easily penetrated due to the capillary
forces when exposed to water [6]. The absorption of water and sol uble
aggressive ions pose a substantial safety risk as it may lead to
ÿ Corresponding authors at: Microbial Science Institute, Yale University, 840
West Campus Drive, West Haven, CT 06516, USA.
E-mail address: i.voets@tue.nl (I Feet).
1
These authors contributed equally to this work.
the degradation of the cement matrix [6,7]. When applied in brine
environments, such as in coastal areas and salt lakes, the penetra tion of
chloride could cause severe corrosion to steel bars in rein forced concrete
structures, resulting in a significant decrease in
mechanical strength and a sharp decline in service life [8]. To mit igate
these deterioration processes, concrete can be made water repellant by
endowing it with hydrophobic properties [9–12]. With
the advances of hydrophobicity and super-hydrophobicity tech nologies,
the design of concrete with highly hydrophobic proper ties offers a promising
route to improve its durability [12–15].
Conventionally, concrete is hydrophobized by surface or admix ture
treatments. Muhammad et al. [16] demonstrated that coating
the surface with nanomaterials is an effective method to render
cementitious materials water repellent. Indeed, the National

https://doi.org/10.1016/j.conbuildmat.2021.123744
0950-0618/ 2021 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Machine Translated by Google
Z. Qu, S. Guo, Y. Zheng et al.
Cooperative Highway Research Program (NCHRP) Report 244 indi
cated surface treatments can reduce up to 75% of water absorption in
concrete [17]. Epoxy coatings are especially effective in reducing the
water penetration of concrete [18]. It is also shown that silox ane
copolymers and hydrophobic silane admixtures can signifi cantly enhance
the water repellency of concrete [17,19]. Despite these progressions,
several key issues are yet to be resolved [16].
Most of the hydrophobic chemicals used in this way require rather high
temperatures for adequate dispersion, which increases the energy
consumption and complicates the manufacturing proce dures [6,16].
Silane-based chemicals can easily be mixed at an ambient temperature,
however, these are significantly more expensive than other coating
materials and are thus less appealing for large-scale applications [19]. In
addition, epoxy and silane are highly toxic raw materials that are harmful
to human health [20]. Finally, hydrophobic coatings can be damaged by
weathering and abrasion over time, which compromises their effectiveness
and warrants regular maintenance [21]. Bulk modification holds promise
for long term preservation of the hydrophobic properties of concrete, but
only if it is fairly inexpensive [6,22].
Recent advances in biotechnology highlight the potential of uti lizing
biological and living materials to accomplish functions hith erto achieved
by synthetic materials [23–26]. An inspiring example in the concrete field
is the development of a self-healing concrete through the addition of
alkaline-resistant spore-forming bacteria [27–30]. Microcracks which
develop on concrete surfaces expose the dormant bacteria within the
concrete to water and oxygen, reviving them to produce limestone and
‘‘heal” the cracks through biomineralization processes [31]. Bacteria can
also be utilized to engineer hydrophobic concrete. Grumbein et al [22]
discovered that addition of biofilms of a Gram-positive bacterium, Bacillus
sub tilis, significantly altered the wetting behavior of hybrid mortar making
it hydrophobic instead of hydrophilic. The underlying mechanisms and the
influence of bacterial agents on the cement hydration are yet to be
investigated. Moreover, for large-scale engi neering applications it is
important to find an alternative strategy that is as promising yet less labor-
intensive than growing and har vesting biofilms of bacteria.
In this research, liquid cultures of bacteria are applied to pro duce
hydrophobic concrete in a concentration-dependent method.
This greatly facilitates the application of bacteria as a supplement to
hydrophobize cements, because several labour-intensive and time-
consuming procedures, which are necessary to cultivate and harvest
bacterial biofilms, can be avoided. In addition, we show that hydrophilic
bacteria can also endow concrete with hydropho bic properties, which
greatly expands the repertoire of bacteria that can be used for creating
water-resistant concrete. Our research demonstrates that the hydrophobic
properties originated from a significant increase in the roughness of the
morphology of the concrete at nano-to-micro-meter length scales. These
microstructure alterations result from the structure changes of ettringite
likely due to biomineralization. Moreover, the addition of the bacteria has
an impact on the hydration and phase formation of the cement. All in all,
this research provides insights into the mechanism for the preparation of
microbe-modified hydrophobic
concrete.
2. Experimental
2.1. Materials
Cement used for the research is CEM I 52.5 R from ENCI. The
aggregates used to prepare the mortar samples are CEN standard sand.
The bacteria used in this study for the hydrophobic modifica tion are B.
subtilis 3610 and E. coli (BL21 (DE3)) from ATCC. Two
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Construction and Building Materials 297 (2021) 123744
kinds of culture medium (Luria/Miller LB-Medium and LBPlus from Sigma
Aldrich) are used to investigate the influence of the culture medium on the
hydrophobic performance of the bacteria.
2.2. Preparation and testing methods
2.2.1. Preparation of the biofilm hybrid mortars
Bacillus subtilis is a well-studied Gram-positive bacterium com monly
found in soil and the gastrointestinal tract of humans [32,33]. B. subtilis
cells are intrinsically hydrophobic due to the hydrophobin-like proteins
displayed on their surfaces [33,34]. In a recent study by Grumbein et al, it
is demonstrated that biofilms of B. subtilis grown on different media exhibit
hydrophilic (LB agar) and hydrophobic (LBPlus agar) behaviors,
respectively [22].
Remarkably, both hydrophilic and hydrophobic biofilms of B. sub tilis can
be used as an admixture to create concrete with water repellent
properties, suggesting that the innate hydrophobicity of the bacterium is
not required for creating hydrophobic concrete.
We therefore reason that a wide range of other inexpensive, and easy-to-
culture bacteria might be suitable as admixtures to create hydrophobic
concrete.
The process for the preparation of microbe-modified mortar is shown
in Fig. 1. Firstly, bacteria from a frozen glycerol stock are inoculated in 15
mL of liquid Luria/Miller LB-Medium or LBPlus - Medium to prepare the
bacterial cultures. Then, the cultures are stored in a shaking incubator
(250 rpm) overnight for growth. At last, the cultures are plated on the agar
plates for the generation of biofilm for 24 h. In control experiments, LB
medium (without bacteria) is added to mortar and the wetting behavior of
the sam ples is not changed. All mortar specimens are prepared in a
labora tory mixer. The mortar is prepared with a ratio of water: cement:
sand = 0.5: 1: 3.
2.2.2. Preparation of the bacteria hybrid mortars
Given the numerous culturing and harvesting steps involved in growing
biofilms, the production of biofilm-hybrid concrete is time-consuming and
labor-intensive [22,35,36]. To simplify the procedure to prepare bio-based
hydrophobic concrete, bacterial suspensions are cultured and applied as
an admixture instead. This new approach reduces the preparation time
by at least one day as this is the time it takes to grow and collect the
biofilms (Fig. 1).
Furthermore, the cost and waste are reduced by omitting the addi tional
materials such as agar and petri dishes required for growing biofilms.
The preparation route to prepare hybrid mortar from bacterial
suspensions is shown in Fig. 1. Two liters of LB medium are used as the
growth medium to expand the production of cultured bacte ria. The
centrifugation speed is set at 10,000 g (g = 9.81 m/s2 ) and the duration
is set at 10 min. After centrifugation, cell pellet is resuspended in lower
volumes of water to achieve bacterial sus pensions (as shown in Fig. 1).
This resuspended cell pellet is subse quently added to mortar at a
content of 1%, 2%, 5% and 10% by weight of pellet to cement, respectively.
The water used to prepare the bacteria suspensions has been included
into the total water consumption of the mortar preparation.
2.2.3. Characterization of the hybrid mortar samples
Static water contact angle measurements are performed on a
DataPhysics SCA20 (DataPhysics Germany) at ambient tempera ture.
Water with the volume of 5 ll is pipetted onto the outer sur face or the
inner surfaces of the mortar sample with curing age of 7 days. In control
experiments where LB or LBplus medium (without bacteria and biofilm) is
mixed with the hybrid mortars, no perceiv able change is found to the
wetting performance of the samples.
The phase composition of hydration products with curing age of 7 days is
investigated by X-ray diffraction (XRD) on a D2
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Z. Qu, S. Guo, Y. Zheng et al. Construction and Building Materials 297 (2021) 123744

Fig. 1. The process for the preparation of bio mortars.

Fig. 4. The water absorption of the bio mortars to which either B. Subtilis or E. Coli is
Fig. 2. Water contact angles of the biofilm modified mortar.
added at a 5% bacteria to cement ratio.

equipment from Bruker and the samples are ground and sieved,
and particles with diameter smaller than 40 lm are collected for
the test. The influence of the bacteria on the cement hydration
kinetics is assessed by calorimetry employing a TAM Air isothermal
calorimeter. The tests are carried out for 80 h at a constant temper
ature of 20 ÿ. The results are normalized by the mass of solid pow
ders. To analyze the microstructures of concrete, mortar samples
with curing age of 7 days sputter-coated with gold are analyzed
by scanning electron microscopy. The capillary water absorption
of the mortars is determined according to an adapted protocol of
EN 480–5 [37]. The experiments started by storing the samples
vertically in a chamber with a RH of about 65 ± 5% at room temper
ature (20 ± 1 C) after curing the samples for 7 days. The samples
are exposed to water with an immersion depth of about 3 mm
for a duration of 9 days and the mass of the samples was periodi
cally measured during the experiment. The fresh mortar is cast into
molds with dimensions of 40 mm 40 mm 160 mm. The prisms
are demolded 24 h after casting and cured at 100% RH at about
21 C. After curing for 1 and 7 days, the compressive strengths of
the specimens are tested according to EN 196–1 [38]. Three spec
imens for the compressive strength are tested at each age to com
Fig. 3. Water contact angles of the bacteria modified mortar. pute the average strength and standard deviation. The pore size
3
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Z. Qu, S. Guo, Y. Zheng et al.
Fig. 5. The microstructure and wetting property of the reference and bacteria-modified (5%wt) mortars determined by SEM and static water contact angle measurements. (a, c, e) Top surface and
(b, d, f) cross-section showing the inner layer of (a, b) the reference sample, (c, d) the B. subtilis hybrid mortar, and (e, f) the E. coli hybrid mortar.
distribution is measured using mercury intrusion porosimetry (MIP,
Autopore IV, Micromeretics), with a maximum applied mer cury
pressure of 228 MPa, contact angle of 130, and equilibration time of
20 s. The setting time of pastes is evaluated by using the Vicat
apparatus based on EN 196–3: 2005 [39].
3. Results and discussion
3.1. The influence of the biofilms to the hydrophobicity of the mortar
The influence of the biofilms on the hydrophobic performance of
the mortar is shown in Fig. 2. Four different kinds of biofilms
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Construction and Building Materials 297 (2021) 123744
are cultured to prepare the hydrophobic mortars at a fixed biofilm to
cement weight percentage of 5%. These are E. coli biofilms cul
tured with LB and LBplus, and B. subtilis biofilms cultured with LB
and LBplus. For the B. subtilis biofilms grown with different culture
media, the modified mortars present similar water contact angles,
which are 61 and 66 for LB and LBplus, respectively. Interestingly,
the mortar with the E. Coli biofilm also presents enhanced
hydrophobicity displaying a water contact angle of 64 and 63, which
is essentially identical to the B. subtilis biofilm- containing samples.
This result indicated that, in order to prepare hydropho bic concrete,
the innate hydrophobicity of the bacteria is not a requirement as the
cells of E. Coli are hydrophilic [32]. Thus,
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Z. Qu, S. Guo, Y. Zheng et al. Construction and Building Materials 297 (2021) 123744

elucidation of the mechanism underlying the creation of bacteria
based hybrid concrete with hydrophobic properties warrants fur ther
research.
3.2. The influence of bacterial liquid cultures on the water resistance of
the mortar
The effect of bacterial dose on the hydrophobicity of the hybrid
mortars was investigated as shown in Fig. 3. The cell concentra tions
are characterized by optical density values at 600 nm
(OD600nm). The OD600nm values remain stable at ~4 for both types
of bacteria after culturing overnight, which indicates that suspen sions
have reached the growth limit. To further increase the bacte rial
concentration, the bacteria are pelleted from the liquid
suspension by centrifugation. Then the cell pellets are dispersed
in water to prepare the bacteria-modified mortars with bacteria
to cement ratios of 1 wt%, 2%, 5% and 10%. The hydrophobicity
increases with increasing dosage. The 5% B. subtilis and E. coli mod
ified concrete display a contact angle of 96 and 89, respectively,
which shows a higher water contact angle values compared to
the samples directly made with 5% biofilms. A further increase in
the dosage to 10%, slightly raises the contact angles to 103 (B. sub
tilis) and 95 (E. coli) which in turn indicates 5% is an optimum
amount to prepared hydrophobic concrete taking both the
hydrophobic performance and costs into account. Remarkably,
even higher contact angles can be achieved on the interface created
upon cross-sectioning the samples with 5% bacteria addition,
which were as high as 150 for B. subtilis and 138.5 for E. coli. Both
of these samples demonstrated superhydrophobicity as water
drops rapidly rolled off the surface of slightly tilted mortar speci mens
(Video S1). Concomitantly, the increased hydrophobicity of
the 5 wt% specimens with either B. subtilis or E. coli resulted in
an enhanced resistance against water absorption, which is reduced
by approximately 60% as shown in Fig. 4. In sum, these results
demonstrate that the amount of bacteria added impacts the
hydrophobicity of the concrete, while the hydrophobicity of the
bacteria is unimportant. Furthermore, these findings strengthen
the hypothesis that neither hydrophobic bacteria nor biofilms are
required to create bio-based hydrophobic concrete. Without these
rigorous prerequisites, the design and application of microbe modified
hydrophobic concrete is considerably simplified. This
finding opens up many exciting opportunities, since a broad vari ety
of microorganisms may be applied, which could help gain other
useful functions to concrete, such as removal of odor or self healing.
carbon dioxide from the atmosphere for metabolic processes such
as respiration, their Ca2+-associated membranes can be ideal nucle
ation sites for calcite (CaCO3) deposition. These nanoscale calcites
and the micron-scale cell-like particles roughen the concrete sur face
on multiple length scales. We propose that it is this multi scale
roughness that contributes to the increased hydrophobicity
of concrete top surfaces. Intriguingly, clear differences in
microstructures were also found on the internal cross-section
(Fig. 5b, d, f). Both the inner sections of concrete modified by B.
subtilis (Fig. 5d) and E. coli (Fig. 5f) contained many spike-like
ettringite structures, which were much less abundant in the refer ence
mortars (Fig. 5b). It has been reported that the shape of ettrin gite in
concrete can be influenced by biomaterials such as bacteria
or biofilms [42–44]. It should be noted that these short spike-like
structures may increase the roughness of the mortars, thereby
increasing the hydrophobicity of the internal cross-section in a
manner resembling the ‘‘lotus-leaf effect” [45,46].
3.3. Impact of bacteria modification on the deposition of calcite
To determine whether the admixing of bacteria results in the
elevation of calcite, X-ray diffraction XRD measurements are per
formed on mortar modified with different levels of E. coli- and B.
subtilis, as well as on a reference sample (Fig. 6). The characteristic
Bragg reflections of calcite (CaCO3), quartz, calcium hydroxide (Ca
(OH)2) induced portlandite, C2S and C3S are clearly visible in all
samples. The quartz peaks (labeled with Q in Fig. 5)) can be attrib
uted to the standard sand. Additionally, as the concentration of
bacteria is increased, the calcite peak becomes more pronounced,
while the portlandite peak diminishes. The XRD patterns thus cor
roborate the SEM findings, providing further evidence that the bac
teria involving in the calcium carbonate precipitation and
consumption of calcium hydroxide. Interestingly, the C2S and C3S
Bragg reflections are considerably larger in the bacteria-modified
mortars than in the reference, which suggests a slower hardening.
3.4. Impact of bacterial suspensions on the setting time, hydration of
cement and mechanical property
To evaluate whether the hardening process of ordinary Portland
cement (OPC) is affected by the addition of bacterial suspensions,
cement hydration is investigated using calorimetry. Hererin, the

To gain mechanistic insight into the wetting properties dis played

by the bacteria-modified concrete samples, scanning elec tron
microscopy (SEM) is used to analyze their morphology at
nano-meter and micro-meter length scales as shown in Fig. 5. Sig
nificant differences in morphology are caused by the addition of B.
subtilis (Fig. 5c, d) or E. coli (Fig. 5e, f) both on the surface (Fig. 5a, c,
e) and internal cross-section (Fig. 5b, d, f) compared to concrete
specimens without added bacteria (Fig. 5a, b). Cubic crystals are
clearly visible on the outer surface of the B. subtilis modified con crete
(Fig. 5c). These crystals are under 1 mm in length rendering
the concrete surfaces rough. In view of the well-characterized
capabilities of B. subtilis to precipitate calcium carbonate, these
crystals are likely calcite (the most stable polymorph of CaCO3)
formed during the biomineralization process. Indeed, Willem
et al. [40] suggested biomineralization products could enhance
the water and gas penetration resistance of the bacteria containing
concrete. By contrast, no crystals nor other similar products were
found in E. coli-based samples. Instead, some cell-like structures
were observed (Fig. 5e). Anbu et al. [41] suggested that the net neg
ative charge on the bacterial membrane can act as scavengers for Fig. 6. X-ray diffraction patterns of the reference mortars and bacteria-modified
divalent cations, such as Ca2+ and Mg2+. Since bacteria sequester samples with 5% or 10% addition of liquid-cultured E. coli and B. subtilis.

5
Machine Translated by Google
Z. Qu, S. Guo, Y. Zheng et al.
Fig. 7. Hydration kinetics of the reference and 5% bacteria mixed cement paste at 20 C: a) Heat flow, b) Released heat. C) setting time of the cement paste, D) compressive strength of the paste
samples, pore structure of the cement paste (7d) e) and f).
exothermic heat flow is monitored over time as shown in Fig. 7 (a)
and (b).
In line with previous studies, the first calorimetric heat peak
appears in a few minutes after the start of the measurement, which
is due to the heat release upon contact with water and dissolution
of cement particles [47]. A single second peak is observed at round
10 h over the course of the hydration process of reference which is
due to the massive formation of reaction products [48]. Interest
ingly, two second peaks are observed for each of the bacteria
modified pastes: a minimal peak at 4 h and a second, more pro
6
Construction and Building Materials 297 (2021) 123744
nounced peak, reflecting the hydration kinetics at ~3 days. The
cumulative heat agrees well with the evolution of the cement
hydration as shown in Fig. 7b. In the first 70 h, the bacteria
containing samples present a much lower cumulative heat than the
reference. This is attributed to the retardation effect of bacteria on
cement hydration [47,49]. Furthermore, B. subtilis retards the
reaction more than E. coli. The peak in heat flow (Fig. 7a) and
upward inflection in the released heat (Fig. 7b) are shifted upward
by approximately 6 h. This is likely due to differences in their
biomineralization capabilities. Tentatively, we propose that
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Z. Qu, S. Guo, Y. Zheng et al. Construction and Building Materials 297 (2021) 123744

biomineralization-inducing encapsulate themselves in a shell of calcite.

This temporarily limits the available amount of Ca2+ in the cement pore
solution, as it is scavenged by the bacteria due to complexation with
the bacterial membrane and delays the hydration process. Fig. 7c
presents the initial and final setting time of reference and bacteria
contained pastes. In line with the influ ence to the cement hydration,
both bacteria increase the setting times. The early age (1d & 7d)
compressive strength is shown in Fig. 7d. Due to the retardation effect,
the bacteria results in a slower development of early-age strength of
the
mortar samples. At 1d, the compressive strength of bacteria
contained mortars is only about 65% of the reference. However, its
should be noted that at 7d, the compressive strength of the bacteria
contained mortars increases to 90% of the reference. This can be
attributed to the pore structure refinement by the bacteria as the bio
mineralized prod ucts can fill in the voids among cement matrix [50].
As shown in Fig. 7e and f, the bacterial contained pastes show more
distribution in the finer range of 10–40 nm and less pores in the range
around 1000 nm.

3.5. The influence of the bacterial membrane on the hydrophobicity of

the mortars

Our findings indicate that the hydrophobic performance of the

microbe-modified mortars originated from the impact of bacteria on the
biomineralization and morphology in the mortars. Yet, other factors may
also play a role of importance. It is conceivable that the innate
hydrophobicity of the phospholipid-based cell membrane of the bacteria
is influential. To investigate this possi bility, bacterial membranes were
collected from lysed cells to be used as admixture to prepare bacterial-
membrane-modified
mortars.
Interestingly, water contact angle measurements (Fig. 8) revealed
that the addition of 5% either B. subtilis or E. coli cell mem branes by
weight of cement significantly increased the wetting resistance of the
bacterial-membrane-modified mortar specimens compared to the
reference. This can be attributed to the hydropho bic nature of the
phospholipids, which are the main constituents of bacterial membranes.
The mortar with B. subtilis membranes pre sented a higher water
contact angle (91.4) than that of its E. coli counterparts (82.6). This is
likely due to the innate hydrophobicity of Bs1A, which is a hydrophobin-
like protein in the cell membrane of B. subtilis [28]. Intriguingly, the
addition of the bacterial mem branes does not generate spike-like
structures (Fig. 7b, c) seen in the specimens with living bacteria. A
comparison with the SEM micrographs of pristine mortars with neither
bacterial suspensions nor membranes (Fig. 4a and b), indicates that
lifeless cell mem branes do not significantly change the microstructures
of the cement matrix. This explains the lower hydrophobicity of the
bacterial-membrane-modified mortars compared to the bacterial
suspension-modified mortars. In sum, the addition of bacterial cell
membranes does render concrete more water-resistant, but these
samples do not exhibit the multiscale roughness observed in mor tars
with living bacteria. The latter modification is thus preferred, as it
generates more hydrophobic mortar through a simpler preparation.

3.6. Application of bacteria as a coating on concrete surface

Fig. 8. Morphology of the bacterial-membrane-containing mortars determined by SEM. a) Cell
In an attempt to further simplify the hydrophobization of con crete lysate of B. subtilis. b) Mortars with B. subtilis membranes. c) Mortars with E. coli membranes.
with bacteria, we further explored whether it is important to mix bacteria
with bulk concrete, or whether instead, hydropho bic mortar can be
achieved by simply coating the bacteria on the concrete surface. B.
subtilis and E. coli-coated mortar samples with 0.5 g cell pellet are pre-hydration (immediately after casting) and post-hydration (after three
prepared at two distinct time points, namely days of hydration). Remarkably, all samples,
7
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Z. Qu, S. Guo, Y. Zheng et al.
Fig. 9. Morphology and water contact angles of the mortars with bacterial coatings: a) Coating on pre-hydration mortar with B. subtilis b) Coating on pre-hydration mortar with E. coli, c) Coating on post-
hydration mortar with B. subtilis, d) Coating onpost-hydration mortar with E. coli.
irrespective of whether hydrophobic or hydrophilic bacteria are used,
exhibit hydrophobic properties with contact angles larger than 90
(Fig. 9). This facile coating route presents appealing appli cation
potential as the coating has little influence on the bulk prop erties
of the mortar, whilst water penetration is delayed by the bacterial
coating.
4. Conclusion and outlook
In conclusion, we herein present an effective and dramatically
simplified strategy to design biohybrid concrete with hydrophobic
properties by replacing admixed bacterial biofilms with bacteria
collected from liquid cultures. Both B. subtilis and E. coli are effec
tive, whether admixed in bulk or applied as coating. Hydrophobic
mortars with contact angles in excess of 90 are achieved. Impor
tantly, the choice of bacteria to prepare hydrophobic concrete does
not appear to be restricted to the range of inherently hydrophobic
bacteria. The concentration of bacteria is a critical determinant of
the wetting properties of the hydrophobized concrete. With higher
bacterial concentrations, the mortars present better water resis
tance and higher contact angles.
Interestingly, the mechanism underlying the hydrophobization by
living microbes is multi-faceted. The hydrophobicity of the bac terial
membrane contributes as well as alterations in the microstructure of
concrete at nano-to-micron length scales. The latter are likely
induced by biomineralization and other metabolic processes, which
appear to induce calcite precipitation during the hydration of the
cement. This results in elevated water resistance reminiscent of the
the lotus leaf effect, which in turn enhances
8
Construction and Building Materials 297 (2021) 123744
the durability of concrete. To fully comprehend the impact of
microorganisms on cementitious materials, future work should also
examine other physical and mechanical properties of microbe-
modified concrete, such as shrinkage, self-healing and chloride
permeability.
Declaration of Competing Interest
The authors declare that they have no known competing finan
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgements
The authors would like to acknowledge the financial support by
STW-foundation, the EPSRC-NSFC Joint Research Project (No.
51461135005), the European Union (ERC-2014-StG Contract No.
635928), the Dutch Science Foundation (NWO ECHO Grant No.
712.016.002), and the Dutch Ministry of Education, Culture and
Science (Gravity Program 024.001.035) for funding this research.
Mrs. ACA Delsing in the lab of Building Physics and Services at
Eindhoven University of Technology is acknowledged for providing
experimental support on the SEM analysis. Furthermore, the authors
wish to express their gratitude to the following sponsors of the
Building Materials research group at TU Eindhoven: Rijkswaterstaat
Major Projects and Maintenance; Granite Import Benelux; Kijlstra
concrete mortar; Struyk Verwo; Attero; Enci; Rijkswaterstaat Zee en
Delta-District Noord; Van Gansewinkel Minerals; BTE; V.d. Bosch
Concrete; Selor; GMB; Icopal; BN International; Eltomation; Knuaf
Gypsum; Hess AAC Systems; Kronos; Joma; CRH Europe
Machine Translated by Google

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