molecules

Review
Microbiologically Induced Carbonate Precipitation in
the Restoration and Conservation of Cultural
Heritage Materials
Erick Ortega-Villamagua 1 , Marco Gudiño-Gomezjurado 2 and Alex Palma-Cando 1, *
1 Grupo de Investigación Aplicada en Materiales y Procesos (GIAMP), School of Chemical Sciences and
Engineering, Yachay Tech University, Hda. San José s/n y Proyecto Yachay, Urcuquí 100119, Ecuador;
[email protected]
2 School of Biological Sciences and Engineering, Yachay Tech University, Hda. San José s/n y Proyecto Yachay,
Urcuquí 100119, Ecuador; [email protected]
* Correspondence: [email protected]

Academic Editor: Hortensia Rodriguez, Nelson Santiago Vispo and Luciana Dente





Received: 30 June 2020; Accepted: 19 July 2020; Published: 24 November 2020

Abstract: Microbiologically induced carbonate precipitation (MICP) is a well-known biogeochemical

process that allows the formation of calcium carbonate deposits in the extracellular environment.
The high concentration of carbonate and calcium ions on the bacterial surface, which serves as
nucleation sites, promotes the calcium carbonate precipitation filling and binding deteriorated
materials. Historic buildings and artwork, especially those present in open sites, are susceptible
to enhanced weathering resulting from environmental agents, interaction with physical-chemical
pollutants, and living organisms, among others. In this work, some published variations of a novel
and ecological surface treatment of heritage structures based on MICP are presented and compared.
This method has shown to be successful as a restoration, consolidation, and conservation tool for
improvement of mechanical properties and prevention of unwanted gas and fluid migration from
historical materials. The treatment has revealed best results on porous media matrixes; nevertheless,
it can also be applied on soil, marble, concrete, clay, rocks, and limestone. MICP is proposed as a
potentially safe and powerful procedure for efficient conservation of worldwide heritage structures.

Keywords: microbiologically induced carbonate precipitation (MICP); conservation; restoration;

cultural heritage; calcium carbonate

1. Introduction
Minerals precipitation by living organisms activity, so-called biomineralization, is a process
that occurs from bacteria to chordates [1]. This mineral formation occurs through two different
processes. The first takes place in various animals in a process where the organism produces
an organic framework to introduce ions for further crystallization and growth mediated by an
organic matrix. The second is distinguished by massive intracellular and/or extracellular mineral
formation commonly in the form of teeth, skeletons, shells, etc. [2,3]. The precipitation of minerals by
microorganisms is obtained by the modification of the local environment as a result of the metabolites
release. This releasing of molecules increases pH and elevates the supersaturation, resulting in
the precipitation of minerals. In addition, some macromolecules and cell structures can act as
heterogeneous crystallization nuclei, inducing the precipitation [4,5]. The different type of minerals
that bacteria are able to produce includes nitrates, silicates, calcium oxalates, halides, apatite, gypsum,
oxides, phosphates, and calcium carbonate [4,6]. The process of calcium carbonate precipitation is
present in nature, commonly in marine environments, freshwater, and soil (e.g., solid surfaces) [7,8].

Molecules 2020, 25, 5499; doi:10.3390/molecules25235499 www.mdpi.com/journal/molecules

Molecules 2020, 25, 5499 2 of 23

Calcium carbonate may precipitate through the attachment of the calcium ions to the microbial
cell walls or to the extracellular polymeric substances, which act as crystal nucleation sites [9,10].
Depending on the cell surface properties of bacteria, especially proteins and extracellular polymeric
substances, the morphology and mineralogy of calcium carbonate can be varied, e.g., rhombohedral
(calcite), hexagonal (vaterite), or needle-like crystal (aragonite) [9], being calcite the most stable
molecular structure [11]. Microbiologically induced carbonate precipitation (MICP) is mainly driven by
factors, such as pH, Ca2+ concentration, dissolved inorganic carbon concentration, and availability of
nucleation sites [12]. Microorganisms use different metabolic pathways to induce CaCO3 precipitation;
however, this process is not entirely defined yet. Genetics and physiology involved in the process are
quite a challenge to understand [13]. Some of the metabolic pathways involved in CaCO3 precipitation
are anaerobic sulfide oxidation, photosynthesis, methane oxidation, ammonification, denitrification,
sulfate reduction, and ureolysis [10].
On the other hand, chemical, physical, biological, and anthropogenic factors are the principal
perpetrators of monumental stone decay. Architectural structures and monuments begin to weaken
through progressive matrix dissolution and porosity increase [14], resulting in deteriorative effects,
such as inclination and discoloring [15], water retention, growth of heterotrophic and higher organisms,
patinas formation, corrosion, alkaline dissolution, among others (see Figure 1) [16]. Due to the common
social and historical value of these structures, conservation and restoration approaches should be
addressed by scientists and conservators in a joint interdisciplinary work [15]. Conventional organic
and inorganic conservation treatments show several drawbacks. Synthetic resins (e.g., silane, epoxy,
acrylic, polysiloxane) polymerize and plug the stone pores, retaining water and accelerating the internal
degradation [16]. External protecting coats tend to deteriorate, peels off, and requires maintenance [17],
while additional noxious solvents might be released by decomposition [18]. Limewater treatments
and inorganic solutions based on Ba(OH)2 [19] and Ca(OH)2 [20] usually lead to non-consolidating
calcite superficial aggregation [15,21]. These problems have motivated researchers to seek alternative
methodologies. Bacterial calcium carbonate precipitation was proposed as a method for the restoration
of calcareous stones as one of the most vulnerable materials against deterioration. This treatment,
patented in 1990 (expired in 2010), aims for the production of a superficial coating of calcium carbonate
by using living cultures of bacterial strains [22]. Calcium carbonate is involved in the restoration
process through the biological healing, that is, the production of calcium carbonate commonly through
urease producer bacteria (106 –108 colony-forming unit (CFU)), in an aerobic environment and in
the presence of a calcium source [11,23,24]. The carbonatogenesis helps the concrete micro-cracks
sealing, avoiding the penetration of water into the rock or cement matrices. Resistance and strength of
these materials [25,26] get boosted under an energy-efficient mechanism in an eco-friendly way [9,23].
This methodology has already been proved to reduce stone porosity, resulting in more consolidated
structures [14,21].
In this short review, we summarize techniques and strategies conducted by researchers in the field
of bacterially induced CaCO3 precipitation for the conservation of heritage materials. A short section
of biotic carbonate formation is presented, followed by a description of the most common mechanisms
of bacterial precipitation through ureolysis, tests in vitro and in situ with their respective results for
the conservation of calcareous historical stones, and finally recommendations and limiting factors for
this type of treatment. Microbiologically induced carbonate precipitation (MICP) has been reviewed
for its use in biotechnology [10], engineered applications [28], sand treatment [29], and environmental
problems [23], but, as far as we know, until now, a review with priority in its application for conservation
and restoration in cultural and historical heritage has not been addressed. The major purpose of
this review is to present MICP as one powerful consolidation and restoration technique for historical
building materials. There is not a consensus on the optimal conditions for the application of this type
of treatment; therefore, some branches of science (chemistry, biology, historical restoration, materials
science and engineering, geology) could be interested in the standardization and optimization of
this methodology.
that is, the production of calcium carbonate commonly through urease producer bacteria (106–108
colony-forming unit (CFU)), in an aerobic environment and in the presence of a calcium source
[11,23,24]. The carbonatogenesis helps the concrete micro-cracks sealing, avoiding the penetration of
water into the rock or cement matrices. Resistance and strength of these materials [25,26] get boosted
under an
Molecules energy-efficient
2020, 25, 5499 mechanism in an eco-friendly way [9,23]. This methodology has already
3 of 23
been proved to reduce stone porosity, resulting in more consolidated structures [14,21].

(b)
(a)

(c)

Figure 1. Degraded historic buildings by (a) a massive salt weathering in building stone, and (b) staining
and microbial growth. Modified with permission from [27]. 2017, Consejo Superior de Investigaciones
Científicas (CSIC). (c) Skull shape, completely lost with the presence of orange lichen. Modified with
permission from [16].

2. Biogenic Precipitation of Calcium Carbonates

Calcium carbonate comprises more than 4% of the earth’s crust. This compound is found in chalk,
marble, travertine, tufa and even is the principal component of shells and pearls [9]. Calcium carbonate
biomineralization can occur by two different mechanisms based on the degree of microbiological
control, named as microbiologically controlled carbonate precipitation (MCCP) and microbiologically
induced carbonate precipitation [30]. In MCCP, the cells of the organisms designate a site for mineral
formation, such as polymerized macromolecules, membranes, or vesicles, after that, ions are imported
in a regulated sequence. Crystal nucleation and growth control induced by organic matrices differ
from different phyla, but, in general, occur over solid surfaces, such as membranes, macromolecular
substrates, and extracellular polymeric substances (EPS) [2,3,30]. Mineral particles are formed
intracellularly under metabolic and genetic control, leading to specialized structures like exoskeletons,
teeth, and shells. The formation of these structures works independently from environmental
conditions [2,31]. In comparison, MICP is regulated by the combined physiological activities of
microorganisms. This process is carried out in open environments; therefore, external physical-chemical
parameters play an important role in the type of mineral produced [30]. MICP usually occurs in
the extracellular environment, commonly driving to the mineralization of the proper bacterial cells.
One of the most accepted hypotheses for CaCO3 precipitation considers that calcium ions are not used
by microbial metabolism; instead, they are aggregated and crystallized in the cell surface using EPS
as nuclei for crystallization [5,32–34]. In general, the CaCO3 precipitation rate is a linear function
dependent on Ca2+ and CO2− 3 ions concentration product, therefore, following second-order kinetics
or pseudo-first-order kinetics if there is an excess of one of the reagents [35]. Bacteria can influence the
reachable saturation and rate of carbonate precipitation by controlling the CaCO3 crystal polymorph
produced. Supersaturation (S) is only reached when the solubility product (Ksp ) is exceeded by the
h
concentration of [Ca2+ ] and CO2− 3 ] . Precipitation of CaCO3 is favored with higher supersaturation
level and defined by Equation (1) [35]:
h ih i
Ca2+ CO2−
3
S= (1)
Ksp

Calcium carbonate can precipitate as any of the six polymorphs. Listed in increasing
thermodynamic stability, CaCO3 polymorphs are (i) amorphous calcium carbonate, (ii) calcium
carbonate monohydrate, (iii) calcium carbonate hexahydrate, (iv) vaterite, (v) aragonite, and (vi)
calcite [36]. Although researches have reported the presence of almost all polymorphs in CaCO3
Molecules 2020, 25, 5499 4 of 23

bioprecipitation, calcite and vaterite are the most common precipitates [37]. Some parameters controlling
the type of precipitated polymorphs are the medium supersaturation level, presence of glycoproteins
and amino acids, the solubility of the possible phases [36], the specific bacterial strain, specific proteins
present in EPS, dissolved organic carbon [37], abiotic factors, complex interactions associated with
organic molecules, the order in the addition of reactants, among others [38]. This extended variety of
possible parameters, which influence the morphology and the formation of polymorphs by bacteria,
is the main reason for a no consensus on the main mechanism that affects the biomineralization of
polymorphs. The extracellular calcium carbonate synthesis occurs by autotrophic and heterotrophic
pathways. Algae and cyanobacteria are responsible for the autotrophic pathway through fixing
carbon dioxide to carbonate by means of (i) anoxygenic photosynthesis, (ii) non-methylotrophic
methanogenesis, and (iii) oxygenic photosynthesis [9,39]. On the other hand, Arthrobacter, Bacillus,
and Rhodococcus have been described as microorganisms capable of employing organic salts as an
energy source, producing carbonate minerals, such as magnesium carbonate or calcium carbonate,
in caves, marines, lakes, and soils [11]. In the presence of a calcium source, bacteria of the genera
Bacillus, Lysinibacillus, and Sporosarcina can produce calcium carbonate through urea hydrolysis.
Among these genera, the most common species described as healing crack microorganisms are Bacillus
amyloliquefaciens, Bacillus cereus, Lysinibacillus sphaericus, and Sporosarcina pasteurii [11].

3. Bacterial CaCO3 Precipitation Through Ureolysis

The most common method for CaCO3 bioprecipitation is urea hydrolysis [10]. When bacteria
with ureasic activity are in the presence of a medium that contains urea (CO(NH)2 )2 and Ca2+ ions,
they might produce calcium carbonate precipitates. Ureolytic bacteria can produce urease enzyme.
Bacterial urease (urea amidohydrolase E.C.3.5.1.5) is a multi-subunit nickel metalloenzyme composed
of two (αβ) or three (αβγ) subunits [40]. Jabri et al. described the crystal structure of Klebsiella aerogenes
urease, which consists of two nickel atoms linked by a carbamate group [41]. This metallic center acts as
a catalytic center for the reaction of urea to carbamic acid and ammonia [42]. An increase in pH by NH4+
production fosters the CaCO3 precipitation. The catalytic activity of this kind of enzymes depends on
the temperature and the bacterial species. For example, Anbu et al. reported the optimum temperature
for most ureases ranging from 20 ◦ C to 37 ◦ C [23]. On the other hand, the enzymatic activity of these
enzymes may differ among the species or even between the strains of the same species. For instance,
the enzymatic activity of Bacillus megaterium urease decreases at high temperature, while at low
temperature, surpasses the enzymatic activity of Sporosarcina pasteurii [43]. In fact, the genera Bacillus
and Sporosarcina are known for their high production levels of urease, which are the most frequently
used ureolytic bacteria for biotechnological applications [10,44,45]. Another factor that defines the
urease activity is the urea and calcium concentrations [46]. De Muynck et al. reported that the best
calcite production was achieved with 0.25 M and 0.5 M of calcium chloride and urea, respectively [47].
The availability of nucleation sites is another key factor that governs the rate of precipitation [36].
During the bioprecipitation process, particles in suspension [48], dust particles [36], and bacteria
themselves serve as active sites for calcite nucleation [49]. The bacterial cell surface is typically
negatively charged; hence, it is able to attach divalent cations like Ca2+ or Mg2+ [50]. In more detail,
the mechanism of precipitation begins with the urea hydrolysis, producing carbamic acid and ammonia
(Equation (2)). Spontaneous decomposition of carbamic acid produces carbonic acid and ammonia
(Equation (3)). Ammonia and carbonic acid equilibrate in their protonated and deprotonated form in
the aqueous medium, modifying the pH (Equations (4) and (5)). Actually, ammonia formation increases
the pH up to 9.2, which favors the calcium carbonate bio-mineralization [9,11,43]. Reaction continues
towards calcium carbonate precipitation by bonding Ca2+ ions to the bacteria cell surface (Equation (6)).
The high concentration of carbonate and calcium ions on the bacterial surface, which serves as
Molecules 2020, 25, 5499 5 of 23

nucleation sites, promotes calcium carbonate precipitation (Equations (7) and (8)) that could bind and
consolidate deteriorated materials in historical structures (see Figure 2) [35,51,52].
Molecules 2020, 25, x FOR PEER REVIEW 5 of 23
→
CO(NH2 )2 + H2 O (urease) NH3 + NH2 COOH (2)
CO(NH ) + H O (urease) ⃗ NH + NH COOH (2)
NH2 COOH + H2 O → NH3 + H2 CO3 (3)
NH COOH + H O → NH + H CO (3)
H2 CO3 HCO− + H+ (4)
H CO ⇌ HCO 3 + H (4)
2NH3 + 2H2 O 2NH+ + 2OH− (5)
2𝑁𝐻 + 2𝐻 𝑂 ⇌ 2𝑁𝐻 4 + 2𝑂𝐻 (5)
2+
Ca +
Ca cell →
+ cell cell −
→ cell Ca2+
− Ca (6) (6)
− + −
HCO
HCO 3 ++HH ++ 2OH CO2−
2OH ⇌ CO 3 ++
2H2HO2 O (7) (7)
2+ 2−
cell−−
cell CaCa ++CO
CO3 →→ cell
cell−− CaCO
CaCO3↓ ↓ (8) (8)

Figure2.2.A A
Figure general overview
general of chemical
overview processes
of chemical involvedinvolved
processes in ureolytic
in calcium
ureolyticcarbonate
calcium precipitation.
carbonate
precipitation.
4. Microbiologically Induced Carbonate Precipitation on Solid Samples in Laboratory
MicrobiologicallyInduced
4. Microbiologically inducedCarbonate
carbonatePrecipitation
precipitationonisSolid
a controlled
Samplesprocess that can be used for
in Laboratory
different applications, such as improvement of concrete mechanical properties and self-healing
Microbiologically induced carbonate precipitation is a controlled process that can be used for
of cracks [53], heavy metals removal [54], sand and clay biocementation [55–59], dust suppression,
different applications, such as improvement of concrete mechanical properties and self-healing of
radionuclide
cracks [53], remediation
heavy metals [28], CO2 sequestration
removal [54], sand and [60],clay
and biocementation
conservation and[55–59],restoration
dustofsuppression,
historical and
cultural objects [61]. In 1973, Boquet et al. isolated 210 microorganisms from the
radionuclide remediation [28], CO2 sequestration [60], and conservation and restoration of historical soil, which precipitate
calcite crystals in a suitable environment [62]. Since this pioneering work,
and cultural objects [61]. In 1973, Boquet et al. isolated 210 microorganisms from the soil, which various research groups
have been studying
precipitate and testing
calcite crystals microbial
in a suitable carbonate[62].
environment precipitation onto surfaces,
Since this pioneering work, commonly stone or
various research
marble slabs, changing conditions, such as calcifying bacteria strain, metabolic
groups have been studying and testing microbial carbonate precipitation onto surfaces, commonly pathways, nutrient
media, pH,
stone or among
marble others.
slabs, Calcium
changing carbonatesuch
conditions, bioprecipitation is gaining
as calcifying bacteria interest
strain, amongpathways,
metabolic researchers
over the last
nutrient decades
media, pH, for non-conventional
among others. Calcium applications
carbonate [10].
bioprecipitation is gaining interest among
There are different studies about the implementation
researchers over the last decades for non-conventional applications of new[10].strategies to improve the yield
of calcium
There carbonate
are different production,
studies about allowing further applications
the implementation in restoration
of new strategies and conservation.
to improve the yield of
Shirakawa et al. compared
calcium carbonate the efficiency
production, allowingoffurther
three different bacterial
applications strains (Lysinibacillus
in restoration sphaericus,
and conservation.
Pseudomonas
Shirakawa et putida, and Bacillus
al. compared thesubtilis) forof
efficiency calcium carbonatebacterial
three different precipitation
strains[7].(Lysinibacillus
Bacteria weresphaericus,
inoculated
inPseudomonas
two differentputida,
culture media
and withsubtilis)
Bacillus no pH adjustment,
for calcium(i)carbonate
modifiedprecipitation
B4 media with calcium
[7]. acetate
Bacteria wereas
Ca 2+ source,inand
inoculated two(ii)
different
295 mediaculture
with media withchloride
calcium as Ca2+ source.
no pH adjustment, (i) modified
The samplesB4 mediawerewith calciumin
incubated
acetate
static andasshaking
Ca2+ source, and (ii)at295
conditions 28 ◦media
C for 12with calcium
days. Afterchloride
a period asofCaincubation,
2+ source. The samples were
atomic absorption
incubated in static and shaking conditions at 28 °C for 12 days. After a
spectrometry [63] was used to measure the concentration of calcium ions remaining in the media period of incubation, atomicas
absorption spectrometry [63] was used to measure the concentration of calcium ions remaining in the
media as an indicator of calcium carbonate production. Bacteria in B4 modified the media-generated
greater CaCO3 precipitation compared to bacteria in 295 media. Average calcium consumption in B4
modified media was 96% for P. putida, 74% for L. sphaericus, and 28% for B. subtilis, in comparison
with sterile media. X-ray powder diffraction (XRD) [64] showed that B. subtilis and P. putida produced
Molecules 2020, 25, 5499 6 of 23

an indicator of calcium carbonate production. Bacteria in B4 modified the media-generated greater

CaCO3 precipitation compared to bacteria in 295 media. Average calcium consumption in B4 modified
media was 96% for P. putida, 74% for L. sphaericus, and 28% for B. subtilis, in comparison with sterile
Molecules 2020, 25, x FOR PEER REVIEW 6 of 23
media. X-ray powder diffraction (XRD) [64] showed that B. subtilis and P. putida produced vaterite
and calcite,
vaterite calcite,L.while
andwhile sphaericus produced
L. sphaericus the only
produced thevaterite in B4 in
only vaterite modified media
B4 modified (see Figure
media 3a).
(see Figure
Environmental
3a). Environmental scanning electron
scanning microscopy
electron (ESEM) (ESEM)
microscopy [65] showed,
[65] for all strains
showed, in shaking
for all strains conditions,
in shaking
the production of smaller calcium carbonate crystals than static conditions. Rectangle,
conditions, the production of smaller calcium carbonate crystals than static conditions. Rectangle, hemispherical,
spherical, rhombohedral,
hemispherical, spherical, and pinacoidal crystals
rhombohedral, were formed
and pinacoidal under
crystals werethese
formedconditions (see Figure
under these 3c).
conditions
Higher magnification showed holes inside crystals, which coincided with the cell size
(see Figure 3c). Higher magnification showed holes inside crystals, which coincided with the cell sizeof Bacillus strains,
pointing
of Bacillusout for bacteria
strains, cellout
pointing surface as nucleation
for bacteria sites (see
cell surface Figure 3b).sites
as nucleation This(see
study proved
Figure 3b).for thestudy
This first
time thatfor
proved static
the and
firstshaking
time thatconditions
static andatshaking
the same temperature
conditions could
at the samealter the shape and
temperature could size of the
alter the
CaCO bioprecipitated crystals.
shape3and size of the CaCO3 bioprecipitated crystals.

(a)

(b) (c)

Figure
Figure 3. (a) The
3. (a) The comparison
comparisonof ofx-ray
x-raypowder
powderdiffraction
diffraction(XRD)
(XRD)patterns
patternsbetween
between control
control samples,
samples, P.
P.putida,
putida, L. sphaericus, and B. subtilis. Environmental scanning electron microscopy (ESEM)
L. sphaericus, and B. subtilis. Environmental scanning electron microscopy (ESEM) images ofimages of
(b)
(b)calcium
calciumcarbonate
carbonatecrystals
crystalswith
withbacterial
bacterialpresence
presenceevidence,
evidence, and
and(c)
(c)spherical
sphericalcalcium
calciumcarbonate
carbonate
within a rhombohedral and pinacoidal crystal. Adapted with permission
within a rhombohedral and pinacoidal crystal. Adapted with permission from [7]. from [7].

Schwantes-Cezario et al. tested B. subtilis for calcium carbonate bioprecipitation [13]. B. cereus and
Schwantes-Cezario et al. tested B. subtilis for calcium carbonate bioprecipitation [13]. B. cereus
E. coli were used as a positive and negative control, respectively. B4 was used as culture media adjusted
and E. coli were used as a positive and negative control, respectively. B4 was used as culture media
at different pH conditions (non-buffered, basic buffered at pH = 8.2, and neutral buffered at pH = 7.0).
adjusted at different pH conditions (non-buffered, basic buffered at pH = 8.2, and neutral buffered at
Bacteria were incubated with constant agitation at 37 ◦ C for 7 days. After incubation, calcium carbonate
pH = 7.0). Bacteria were incubated with constant agitation at 37 °C for 7 days. After incubation,
precipitation was only observed in non-buffered and basic conditions. Scanning electron microscopy
calcium carbonate precipitation was only observed in non-buffered and basic conditions. Scanning
(SEM) [66] images indicated the same crystal morphology for B. subtilis and B. cereus as rounded
electron microscopy (SEM) [66] images indicated the same crystal morphology for B. subtilis and B.
cereus as rounded structures. Quantification of CaCO3 concentration exhibited no significant
. .
difference between B. subtilis and B. cereus . Large biofilms were
obtained, showing the potential to be used in crack filling purposes for increasing lifespan or
preventing early deterioration. Similar results were reported by Páramo Aguilera et al., who found
Molecules 2020, 25, 5499 7 of 23

structures. Quantification of CaCO3 concentration exhibited no significant difference between B. subtilis

 1.325 g CaCO   1.291 g CaCO 
3 3
1 mL B4 media and B. cereus 1 mL B4 media . Large biofilms were obtained, showing the potential to be
used in crack filling purposes for increasing lifespan or preventing early deterioration. Similar results
were reported by Páramo Aguilera et al., who found optimal biofilm formation by using B. subtilis and
B. cereus bacterial strains [67].
Marvasi et al. showed that buffering B4 media at pH values of 7.3 and 8.2 influenced the mineral
bioprecipitation [33]. After two weeks of incubation, crystal precipitation was observed from the
49 isolated strains at 39 ◦ C. Moreover, 73% of the isolates were able to produce crystals at pH = 8.2,
whereas media buffered to pH = 7.3 inhibited the crystal precipitation in 79% of the strains. Five strains
of Lysinibacillus sphaericus (strain numbers 55,56,57,58,59) and one strain of Bacillus lentus (strain number
60) were tested for the carbonate bioprecipitation on Euville limestone surface by Dick et al. [68].
Ureolytic calcium carbonate precipitation was proposed to be followed using a medium that contains
nutrient broth, sodium bicarbonate, urea, and CaCl2 as Ca2+ source. After 12 h of incubation for
B. lentus, slow CaCO3 precipitation was shown, and hence deficient CaCO3 deposition. Samples with
L. sphaericus strains (55,56,58) began precipitation after four hours of incubation. SEM images revealed
the deposition of calcite crystals on the sample surface. Strain 58 showed a heterogeneous distribution
of calcite crystal, whereas strain 59 produced rhombohedral homogenous calcite crystal deposition.
Treated limestone surface with strains 57 and 59 showed a reduced capillary water absorption [69] within
two days. The electrical charge at the shear plane (ζ-potential) [70] originated from (de)protonation or
(de)complexation of surface molecules was linked to homogeneous surface limestone colonization.
The highly negative ζ-potential at the surface of the bacteria cell and the positive ζ-potential of the
limestone set appropriate conditions for bacterial colonization and precipitation. Bacterial strain
colonization capacity depends on its negative ζ-potential. Strains 56, 57, and 59 presented the most
negative ζ-potential at pH 9. These strains had a significant impact, decreasing limestone capillary
absorption property.
Rodriguez-Navarro et al. used the theory behind bioprecipitation to test if this technique
generates good results in the consolidation and protection of limestone slabs [71]. Myxococcus xanthus
(strain number 422) was used as the calcifying bacteria in two different culture media of non-buffered
M-3 (with Ca(CH3 COO)2 as a source of Ca2+ ) and buffered M-3P (in phosphate buffer) at pH = 8.
The experiment was carried out by using test tubes for small slabs (0.5 cm3 ) under shaking conditions
and Erlenmeyers for larger slabs (5.63 cm3 ) under static conditions. Incubation was performed at
28 ◦ C for 30 days; however, only 5 to 10 days were required to give high yields of the precipitates.
Results showed that weight changes due to the precipitation of carbonates began around 2.5 days for
small slabs and 5 days for larger ones. XRD showed the formation of calcite and vaterite, with calcite
as the main precipitated phase (see Figure 4a). SEM analysis revealed sparitic and rhombohedra calcite
crystals and needle-shaped vaterite crystals. Epitaxial growth was observed over the pre-existing
crystals, resulting in the precipitation of carbonate, without blocking or plugging the pores in the slabs
(see Figure 4b). Sonication removed the biofilm formed, although calcified bacteria cells and vaterite
crystals were not removed in appreciable amounts for larger slabs. Slabs incubated in the M3-P media
showed lower weight loss. Newly formed crystals were strongly added to the surface and were more
resistant than the pre-existing ones.
In 2009, histological stains were used for the first-time by Zamarreño et al. to reveal polysaccharides
surrounding carbonate crystals [72]. Pseudomonas (D2 and F2) and Acinetobacter (B14) strains were both
isolated from freshwater. Bacterial strains were inoculated in B4 modified media before spreading
over 500 µm thick limestone slides and incubated over three weeks at 30 ◦ C. Bacterial distribution
was determined on crystals and limestone slides by staining live bacteria with 5-cyano-2,3-ditolyl
tetrazolium chloride (CTC) and polysaccharides with alcian blue–periodic acid-Schiff stain (AB-PAS).
After the incubation period, B14 and D2 samples precipitated light and dark brown crystals, respectively,
while the F2 sample precipitated light green crystals. The main polymorph distribution was calcite
for D2 and F2 and vaterite for B14, showing spheroidal crystal morphology. Histological staining
significant impact, decreasing limestone capillary absorption property.
Rodriguez-Navarro et al. used the theory behind bioprecipitation to test if this technique
generates good results in the consolidation and protection of limestone slabs [71]. Myxococcus xanthus
(strain number 422) was used as the calcifying bacteria in two different culture media of non-buffered
Molecules 2020, 25, 5499
M-3 (with Ca(CH3COO)2 as a source of Ca2+) and buffered M-3P (in phosphate buffer) at pH = 8.8 The of 23

experiment was carried out by using test tubes for small slabs (0.5 cm3) under shaking conditions and
Erlenmeyers
showed carbonate for larger slabs
crystals (5.63 cm3)by
surrounded under static
bacteria forconditions. Incubation
sample F2, with wasdistribution
a uniform performed between
at 28 °C
for 30 days; however, only 5 to 10 days were required to give high yields of
the inner and outer core. Thick sections of B14 precipitates revealed the presence of polysaccharides the precipitates. Results
showed that weight
in the center changesMoreover,
of the crystal. due to thestain
precipitation of carbonates
revealed that began around
multiple crystals 2.5 daysbyfor
were bonded small
neutral
slabs and 5 days for larger ones. XRD showed the formation of calcite and
and acid polysaccharides (see Figure 5a). Carbonate precipitates from sterile modified B4 media vaterite, with calcite as the
main
reducedprecipitated
the open pore phase (seebyFigure
area 19%. On 4a).the
SEM analysis
other hand, revealed
carbonatesparitic
depositsand rhombohedra
from calcite
inoculated media
crystals and needle-shaped vaterite crystals. Epitaxial growth was
for B14, F2, and D2 samples reduced open pore area by 43%, 46%, and 49%, respectively (see observed over the pre-existing
crystals,
Molecules
Figure resulting
2020,
5b,c). in the
25, x FOR
Bacterial PEERprecipitation
REVIEW
presence of carbonate,
at least duplicated the without blocking
pore reduction or After
area. plugging the pores
330 days, in23the
8 of
no presence
slabs (seecells
of viable Figure was 4b). Sonication
found removed
for crystal the biofilm
precipitates fromformed,
B14 andalthough
F2 samples, calcified bacteria
and only a fewcells and
viable
vateriteshowing
crystals preferred
were crystallographic
not removed in orientation.
appreciable Adapted
amountswith for
permission
larger from
slabs.[71] 2003,incubated
Slabs American in the
cells (~9 cell/mg) were found on crystals precipitated by isolate D2. These results contrast with
Society for Microbiology.
M3-P media
the findings showed lower weight loss.
of Rodriguez-Navarro et al.Newly
[4], who formed crystals
reported almostwerethestrongly added to thewithout
same consolidation surface
and wereinoculation.
bacteria more resistant than the pre-existing ones.
In 2009, histological stains were used for the first-time by Zamarreño et al. to reveal
polysaccharides surrounding carbonate crystals [72]. Pseudomonas (D2 and F2) and Acinetobacter (B14)
(a)
strains were both isolated from freshwater. Bacterial strains were inoculated in B4 modified media
before spreading over 500 µm thick limestone slides and (b) incubated over three weeks at 30 °C.
Bacterial distribution was determined on crystals and limestone slides by staining live bacteria with
5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and polysaccharides with alcian blue–periodic acid-
Schiff stain (AB-PAS). After the incubation period, B14 and D2 samples precipitated light and dark
brown crystals, respectively, while the F2 sample precipitated light green crystals. The main
polymorph distribution was calcite for D2 and F2 and vaterite for B14, showing spheroidal crystal
morphology. Histological staining showed carbonate crystals surrounded by bacteria for sample F2,
with a uniform distribution between the inner and outer core. Thick sections of B14 precipitates
revealed the presence of polysaccharides in the center of the crystal. Moreover, stain revealed that
multiple crystals were bonded by neutral and acid polysaccharides (see Figure 5a). Carbonate
precipitates from sterile modified B4 media reduced the open pore area by 19%. On the other hand,
carbonate deposits from inoculated media for B14, F2, and D2 samples reduced open pore area by
43%, 46%, and 49%, respectively (see Figure 5b,c). Bacterial presence at least duplicated the pore
Figure 4.
Figure (a) XDR
4.area.
(a) XDR patterns
patterns of slabs
ofno
slabs subjected
subjected to
to microbiologically
microbiologically induced carbonate precipitation
reduction After 330 days, presence of viable cells was foundinduced carbonate
for crystal precipitation
precipitates from B14
(MICP);
(MICP); (b) formed
(b) formedcalcite
calcitecrystals,
crystals,developing
developing epitaxially
epitaxially (cc’e)
(cc’e) on on pre-existing
pre-existing calcite
calcite crystals
crystals and
and F2 samples, and only a few viable cells (~9 cell/mg) were found on crystals precipitated by isolate
and showing preferred crystallographic orientation. Adapted with permission from [71] 2003,
D2. These results contrast with the findings of Rodriguez-Navarro et al. [4], who reported almost the
American Society for Microbiology.
same consolidation without bacteria inoculation.

(a) (b) (c)

Figure
Figure 5. 5.(a)(a)Carbonate
Carbonatecrystals
crystals precipitated
precipitated byby isolate
isolateB14
B14joined
joinedby
bya amixture
mixtureofofacid and
acid andneutral
neutral
polysaccharides. Magnification
polysaccharides. Magnification ×200. NPS,
×200. neutral
NPS, polysaccharides;
neutral APS, acid
polysaccharides; APS,polysaccharides; MPS,
acid polysaccharides;
mixture
MPS, mixtureof neutral and acid
of neutral andpolysaccharides. Isolate D2
acid polysaccharides. on limestone
Isolate (b) before(b)
D2 on limestone treatment and (c) after
before treatment and
MICP treatment. Dash line rectangles allow the visual comparison of pore size.
(c) after MICP treatment. Dash line rectangles allow the visual comparison of pore size. Adapted withAdapted with
permission
permission fromfrom [72]2009,
[72] 2009,American
AmericanSociety
Society for
for Microbiology.
Microbiology.

DeDe Muyncketetal.al.studied
Muynck studiedthethe effectiveness
effectiveness ofof the
the carbonatogenesis
carbonatogenesistreatment
treatment and
andthethe
difference
difference
in the protective performance between
in the protective performance between macro macro and microporous stones [73]. Five types
microporous stones [73]. Five types of of porous
porous
french
french limestonewere
limestone were treated:
treated: (i)(i)Avesnes
Avesnes (32.10%
(32.10% porosity), (ii) Savonnières
porosity), (30.90%
(ii) Savonnières porosity),
(30.90% (iii)
porosity),
(iii)Euville
Euville(17.24% porosity),
(17.24% porosity),(iv) Aubigny
(iv) Aubigny(14.12% porosity),
(14.12% and (v) Massangis
porosity), (9.98% porosity).
and (v) Massangis (9.98% Several
porosity).
specimens
Several were were
specimens cut into smallsmall
cut into pieces in the
pieces form
in the of prisms,
form of prisms, cubes, or or
cubes, cylinders.
cylinders.Biodeposition
Biodeposition
treatment was divided into two steps. First, stone pieces were immersed in media containing yeast
and urea (pH 9.45) inoculated with L. sphaericus culture under static and nonsterile conditions at 28
°C for 24 h. For the second step, stone pieces were immersed in a sterile medium containing urea and
calcium chloride for four days. SEM micrographs revealed crystal precipitate variations in
morphology and size (see Figure 6a,c). Energy-dispersive X-ray spectroscopy (EDX) [74] confirmed
Molecules 2020, 25, 5499 9 of 23

treatment was divided into two steps. First, stone pieces were immersed in media containing yeast
and urea (pH 9.45) inoculated with L. sphaericus culture under static and nonsterile conditions at 28 ◦ C
for 24 h. For the second step, stone pieces were immersed in a sterile medium containing urea and
calcium chloride for four days. SEM micrographs revealed crystal precipitate variations in morphology
and size2020,
Molecules (see25,Figure 6a,c).REVIEW
x FOR PEER Energy-dispersive X-ray spectroscopy (EDX) [74] confirmed that newly 9 of 23
precipitate crystals consisted of calcium carbonate. Depending on the type of stone, microtomographs
showed
biocoating large differences
(greater thanin2 the
mm degree of coating
for Euville andand penetration (see
Savonnières) depthFigure
of the 6b,d).
biocoating
MICP (greater than
treatment
2produced
mm for Euville
weightand gainSavonnières)
in all the stone (see Figure 6b,d).
samples, whichMICP
was moretreatment produced
noticeable in theweight gain instones.
most porous all the
stone samples, which
Spectrophotometric was more
analyses noticeable
exhibited in the most
significant color porous
changes, stones.
and the Spectrophotometric
overall degree coloranalyses
change
exhibited
(∆E) values significant
fluctuated color changes,
between 14.6andandthe7.3.
overall degree
Capillary colorabsorption
water change (∆E) values
tests fluctuated
showed, for allbetween
stones,
14.6 and 7.3. Capillary
the diminished wateruptake,
rate of water absorptionwithtests
a 20 showed, for all stones,
times reduction the diminished
for Savonnières samples. rate of water
Resistance
uptake, with atests
to sonication 20 times
showedreduction
around for50%
Savonnières
less weightsamples.
loss ofResistance to sonication
MICP-treated tests showed
stones compared to
around 50% less weight loss of MICP-treated stones compared to untreated
untreated ones. Cycles of sodium sulfate exposure revealed that MICP-treated stones increased the ones. Cycles of sodium
sulfate exposure
resistance to salt revealed that MICP-treated
attack, which was more noticeablestones increased
in the most theporous
resistance to salt
stones. attack,Savonnières
Treated which was
more noticeable in
and Massangis the most porous
limestones remained stones. Treated
almost Savonnières
unaffected afterand15 Massangis limestones
and 20 cycles remained
of salt attack,
almost unaffected
respectively. after 15 and
MICP-treated 20 cycles
stones of salt higher
displayed attack, respectively.
resistance toMICP-treated
freezing andstonesthawingdisplayed
cycles
higher
compared resistance to freezing
to untreated ones. and thawing
It seems that cycles
the pore compared
structuretoinfluences
untreated theones. It seems
coating that the pore
distribution and
structure
production influences
of newlythe coatingcrystals.
formed distribution
Greaterandcalcium
production of newly
carbonate formed crystals.
protective effect andGreater calcium
precipitation
carbonate
throughout protective
stones witheffectaand
largeprecipitation
number ofthroughout
macropores stones with a large
is explained by number
cell size.ofThe
macropores
size of L. is
explained by cell
sphaericus lies size. The
between 1 µmsize L. sphaericus
to 4ofµm, meaning lies thatbetween
pores bigger1 µmthan
to 4 2µm,
µmmeaning
of radiusthat
are pores bigger
necessary to
than
obtain2 µm of radius
maximum are necessary
microbial to obtain maximum microbial cell absorption.
cell absorption.

(a) (b)

(c) (d)

Figure 6. Scanning electron microscopy (SEM) images of MICP-treated

MICP-treated limestone: (a) Aubigny and
(c) Euville samples. 2D (left) and 3D (middle and
and right)
right) microtomograph
microtomograph ofof MICP-treated
MICP-treated limestone:
limestone:
(d) Savoniers
(b) Euville and (d) Savoniers samples.
samples. Newly formed carbonate crystals are yellow.
yellow. Adapted with
2011, American
permission from [73] 2011, American Society
Society for
for Microbiology.
Microbiology.

Limestone is
Limestone is not
not the
the unique
unique material
material able
able to
to be
be treated
treated via
via calcium
calcium carbonate
carbonate biomineralization.
biomineralization.
Calcium sulfate dihydrate (CaSO 2H O), known as gypsum, has been
Calcium sulfate dihydrate (CaSO44 2H22O), known as gypsum, has been used since 12,000 used since 12,000 BC
BC asas
decorative or building material worldwide. Jroundi et al. reported carbonate bioprecipitation
decorative or building material worldwide. Jroundi et al. reported carbonate bioprecipitation as a
as a consolidant
consolidant on archeological
on archeological gypsumgypsum plaster
plaster [75].
[75]. In thisIn this study,
study, fifteen fifteen historical
historical gypsum gypsum
pieces
pieces (dated from the fourteenth century) were obtained from “Alcazar Real
(dated from the fourteenth century) were obtained from “Alcazar Real de Guadalajara”, Spain. de Guadalajara”,
Spain. Calcifying
Calcifying bacterial bacterial
treatmenttreatment
was carriedwasout
carried out bysterile
by spraying spraying
M-3Psterile M-3P
nutrient nutrient
media twicemedia
a day
twice a day for 6 days to reactivate the bacterial strains already present in the gypsum
for 6 days to reactivate the bacterial strains already present in the gypsum samples. Conventional samples.
Conventional
consolidants ofconsolidants of tetraethyl(TEOS),
tetraethyl orthosilicate orthosilicate (TEOS),
polyvinyl polyvinyl
butyral (PVB),butyral (PVB), and
and poly(ethyl poly(ethyl
methacrylate-
methacrylate-co-methacrylate)
co-methacrylate) (PEMA/PMA)(PEMA/PMA)
were appliedwere applied pieces
on gypsum on gypsum
with pieces
a brush with a brush
every 24 h every 24 h
for 7 days
for comparison. After treatment, the drilling resistance (DR) test [76] was used for measuring the
consolidation performance. A cutting depth of 0.62 mm per revolution was set for gypsum plasters,
with an initial porosity of 29% to 38%, showing average values of 0.24–0.82 N mm−1 for untreated
pieces, up to ~2.4 N mm−1 for PEMA/PMA coating (~3 mm depth), up to ~1.25 N mm−1 for PVB coating
(~2 mm depth), and ~0.8 N mm−1 all over the depth for TEOS coating (see Figure 7a). For the
Molecules 2020, 25, 5499 10 of 23

for 7 days for comparison. After treatment, the drilling resistance (DR) test [76] was used for measuring
the consolidation performance. A cutting depth of 0.62 mm per revolution was set for gypsum plasters,
with an initial porosity of 29% to 38%, showing average values of 0.24–0.82 N mm−1 for untreated
pieces, up to ~2.4 N mm−1 for PEMA/PMA coating (~3 mm depth), up to ~1.25 N mm−1 for PVB
coating (~2 mm depth), and ~0.8 N mm−1 all over the depth for TEOS coating (see Figure 7a). For the
bioconsolidation treatment, ~1.7 N mm−1 was obtained throughout the whole depth of the treated piece.
Molecules 2020, 25, x FOR PEER REVIEW 10 of 23
Moreover, the bacterial treatment decreased gypsum porosity from 48% to 41%, increasing average DR
from 0.24 N −1 −1
average DRmm from up to N
0.24 1.38
mmN−1mmup toat1.38
~1.5Nmm mmdepth, withmm
−1 at ~1.5 a strengthening
depth, with aeffect extended ~6
strengthening mm
effect
depth (see Figure 7b). SEM micrographs and XRD showed newly precipitated
extended ~6 mm depth (see Figure 7b). SEM micrographs and XRD showed newly precipitated spherulites of vaterite,
with an average
spherulites crystallite
of vaterite, withsize
an of ~23 nm.
average These spherulites
crystallite size of ~23 nm.were surrounded
These by were
spherulites bacterial EPS and
surrounded
calcified bacterial
by bacterial EPS andcellscalcified
without bacterial
pore plugging. Cross-section
cells without analysisCross-section
pore plugging. showed bacterial precipitates
analysis showed
homogeneously distributed in the samples. SEM images for the conventional
bacterial precipitates homogeneously distributed in the samples. SEM images for the conventional consolidant coatings
showed the formation
consolidant of a surface
coatings showed the film, whichofblocked
formation a surfacethefilm,
pores (see Figure
which blocked7c–f). Colorimetry
the pores analysis
(see Figure 7c–
revealed total color
f). Colorimetry changerevealed
analysis values (∆E) of color
total ca. 5 for almost
change all the(∆E)
values treated samples,
of ca. which is
5 for almost allanthe
acceptable
treated
value according
samples, whichto is conservation
an acceptableguidelines.
value according to conservation guidelines.

(c) (d)
(a) (b)

(e) (f)

Figure7.7. (a)
Figure (a) Drilling resistance (DR)
Drilling resistance (DR)measured
measuredon ongypsum
gypsumplaster
plaster pieces,
pieces, with
with an an initial
initial porosity
porosity of
of29–38%
29–38%and andtreated
treatedwith
withthree
threeconventional
conventionalconsolidants
consolidantsand andM-3P
M-3Pculture
culture medium.
medium. (b) (b)DR
DR of
of
untreated and bacterially treated gypsum plaster with an initial porosity of 48%. Values
untreated and bacterially treated gypsum plaster with an initial porosity of 48%. Values are averagesare averages
ofofthree
threetotofive
fivedrill
drillholes.
holes.Samples
Samples treated
treated with
with (c)
(c) M-3P,
M-3P, showing
showing bacterial vaterite surrounded
bacterial vaterite surroundedby by
EPS and bacterial cells (inset shows a magnification), (d) poly(ethyl methacrylate-co-methacrylate)
EPS and bacterial cells (inset shows a magnification), (d) poly(ethyl methacrylate-co-methacrylate)
(PEMA/PMA),
(PEMA/PMA),(e); (e);polyvinyl
polyvinyl butyral
butyral (PVB),
(PVB), and
and (f)
(f) tetraethyl
tetraethyl orthosilicate (TEOS). Adapted
orthosilicate (TEOS). Adapted with
with
permission from [75] 2014, Elsevier.
permission from [75] 2014, Elsevier.

Bucci
Buccietet al. reported
al. reported MICP
MICP treatment
treatment forartificial
for an an artificial fractured
fractured sandstone
sandstone core specimen
core specimen with a
with a porosity of 12.25% [77]. The strategy carried out by the researchers involved
porosity of 12.25% [77]. The strategy carried out by the researchers involved the use of the ureolytic the use of the
ureolytic
bacteriabacteria Sporosarcina
Sporosarcina pasteurii.pasteurii.
Sandstone Sandstone was saturated
was saturated with deionized
with deionized water 24
water for about forh.about 24 h.
Bacterial
Bacterial incubation was performed in urea
incubation was performed in urea and CaCl2 containing and CaCl containing media (pH ~ 6). Treatment
2 media (pH ~ 6). Treatment was divided into was
divided
variousinto various
separate separate
injections injections
of ~25 of ~25 mLand
mL of inoculated of inoculated
sterile mediaand insterile media in
the saturated the saturated
sandstone core.
sandstone
Between each injection, a period of 24 h was left to accomplish bacteria surface attachment andsurface
core. Between each injection, a period of 24 h was left to accomplish bacteria calcite
attachment
precipitation.andResults
calciteshowed
precipitation. Results
that fracture showed that
permeability wasfracture
decreasedpermeability was decreased
by ~29%. Besides confirmed by
~29%.
visualBesides confirmed
precipitation, visual
X-Ray CTprecipitation, X-Ray CT allowed
allowed the comparison of three the comparison of
cross-sections of three cross-sections
the core, showing
ofeffective
the core,sealing
showing effective sealing
of the rock fracture. of the rock fracture.
Quality
Qualityimprovements
improvements of mixed
of mixed and ceramic recycledrecycled
and ceramic aggregates (RAs) obtained
aggregates (RAs)from construction
obtained from
demolition
construction waste were studied
demolition waste bywereGarcía-González et al. using MICP
studied by García-González et al.treatments
using MICP [78]. Ceramic[78].
treatments RAs
from different recycling plants were specially chosen by their ceramic composition
Ceramic RAs from different recycling plants were specially chosen by their ceramic composition as as follows: TEC-REC
(Tecnología y Reciclado
follows: TEC-REC S.L., Madrid-Spain,
(Tecnología y Reciclado 33.6% ceramic), ANTWERP
S.L., Madrid-Spain, (Antwerp
33.6% ceramic), Recycling(Antwerp
ANTWERP Company,
Antwerp-Belgium,
Recycling Company, 38.4% ceramic), and BIERZO
Antwerp-Belgium, (Bierzo Recicla
38.4% ceramic), S.L., Leon-Spain,
and BIERZO (Bierzo Recicla97.9% ceramic)
S.L., Leon-
samples. L. sphaericus LMG 222 57 was used as carbonatogenic bacteria.
Spain, 97.9% ceramic) samples. L. sphaericus LMG 222 57 was used as carbonatogenic bacteria. Biodeposition liquid culture
media consistedliquid
Biodeposition of yeast extract,
culture urea,consisted
media and calcium nitrate.
of yeast After urea,
extract, cleaning andRA samples
calcium in HCl
nitrate. and
After
cleaning RA samples in HCl and distilled water, samples were submerged for 24 h in a liquid culture
of L. sphaericus under non-sterile and static conditions at 20 °C. Then, RA samples were submerged
in the biodeposition liquid media for four days. Results showed weight gain between 16% and 46%
for small samples (4–12.5 mm) in comparison to the large samples (12.5–20 mm). Samples with
increased ceramic content gained more weight, which is attributed to greater roughness of the
Molecules 2020, 25, 5499 11 of 23

distilled water, samples were submerged for 24 h in a liquid culture of L. sphaericus under non-sterile
and static conditions at 20 ◦ C. Then, RA samples were submerged in the biodeposition liquid media
for four days. Results showed weight gain between 16% and 46% for small samples (4–12.5 mm) in
comparison to the large samples (12.5–20 mm). Samples with increased ceramic content gained more
weight, which is attributed to greater roughness of the ceramic surface. Moreover, continuous calcium
carbonate layers were present in less irregular ceramic surfaces. Water permeability reduction
Molecules 2020, 25, x FOR PEER REVIEW
was
11 of 23
observed in all tested samples. Comparison between small and large samples showed a difference
in
forwater permeability
TEC-REC, ANTWERP, of 46%,
and41%, and 16%
BIERZO, for TEC-REC,
respectively. ANTWERP,
Samples’ resistance and BIERZO,
against respectively.
ultrasonic attack
Samples’ resistance against ultrasonic attack exhibited different responses between
exhibited different responses between biotreated and untreated samples. TEC-REC large samples biotreated and
untreated samples. TEC-REC large samples registered 44% weight loss and small
registered 44% weight loss and small samples 6% weight loss. For samples with higher ceramic samples 6% weight
loss. For biotreated
content, samples with higherexhibited
samples ceramic content,
higher biotreated
weight loss samples exhibited higher
than untreated samples.weight
SEM loss than
analysis
untreated samples. SEM analysis revealed calcium carbonate uniform deposition over a
revealed calcium carbonate uniform deposition over a regular surface and partial pore surface filling regular surface
and partial pore
by globular surface filling
precipitates by globular
(see Figure precipitates
8a,b). EDX revealed(see Figure
that 8a,b).precipitates
globular EDX revealed
werethat globular
likely to be
precipitates were likely to be
calcium carbonate (see Figure 8c). calcium carbonate (see Figure 8c).

(a) (b)

(c)

Figure 8. SEM images

8. SEM images of
of (a) CaCO33 precipitated over recycled aggregates
aggregates surface,
surface, (b)
(b) CaCO
CaCO33
precipitated inside a pore
pore of
of recycled
recycled aggregates
aggregates (RA)
(RA) sample,
sample, (c)
(c)CaCO
CaCO33 globular precipitate with
Energy-dispersive X-ray spectroscopy (EDX) spectrum
spectrum showing
showing the
the composition
composition of
of the
the precipitate.
precipitate.
Adapted with
with permission
permission from
from [78]
[78] 2017,
2017, Elsevier.
Elsevier.

Minto
Minto etetal.al.studied
studiedthethe
restoration of degraded
restoration marblemarble
of degraded structures using MICP
structures usingby MICP
X-ray computed
by X-ray
tomography The carbonatogenic bacteria used was Sporosarcina
computed tomography (X-CT) [79,80]. The carbonatogenic bacteria used was Sporosarcina DSM-33.
(X-CT) [79,80]. pasteurii pasteurii
Biodeposition inducing inducing
DSM-33. Biodeposition media was composed
media of urea and
was composed CaCland
of urea pH =2 at
2 at CaCl 6.5.pHSamples of marble
= 6.5. Samples of
were obtained
marble by crushing
were obtained marble gravel
by crushing marbleuntil a particle
gravel until a size of 0.5–1.4
particle size ofmm andmm
0.5–1.4 filled
andinto a column.
filled into a
Bacteria
column. culture
Bacteriaand biodeposition
culture media were
and biodeposition injected
media wereevery day for
injected six day
every days.forAfter completion
six days. After
of MICP treatment, results revealed that the inlet surface had greater cementations
completion of MICP treatment, results revealed that the inlet surface had greater cementations compared to
outlet marble
compared grains.
to outlet Aftergrains.
marble each injection,
After eachmeasurements showed a gradual
injection, measurements showed permeability decrease.
a gradual permeability
Porosity was also reduced from 32.4% before MICP treatment to ca. 28%
decrease. Porosity was also reduced from 32.4% before MICP treatment to ca. 28% after MICP after MICP treatment.
At 4.5 mm At
treatment. depth
4.5 mm from the inlet,
depth from athe
minimum porosity porosity
inlet, a minimum of 7.2% was obtained.
of 7.2% From inlet
was obtained. From toinlet
outlet,
to
non-heterogeneous precipitation was observed by X-CT, resulting in a color gradient
outlet, non-heterogeneous precipitation was observed by X-CT, resulting in a color gradient (see (see Figure 9).
Figure 9). Different injection strategies should be applied for in situ restoration to reach more
homogeneous precipitation. MICP treatment was able to cement the marble crushed pieces, meaning
that the method allows restoration of the considerable size cracks.
Molecules 2020, 25, 5499 12 of 23

Different injection strategies should be applied for in situ restoration to reach more homogeneous
Molecules 2020, 25, x FOR PEER REVIEW 12 of 23
precipitation. MICP treatment was able to cement the marble crushed pieces, meaning that the method
allows restoration of the considerable size cracks.

(a)

(b)
(d)

(e)

(c)
Figure 9. X-ray computed tomography (X-CT) column scans, showing in yellow-white colors high
attenuation from computed
Figure 9. X-ray solid material. Purple-black
tomography colors
(X-CT) indicate
column low showing
scans, attenuation
in (e.g., air-filled pore
yellow-white space).
colors high
(a) Slice averaged X-ray attenuation vs. distance from inlet column; (b) Maximum average attenuation;
attenuation from solid material. Purple-black colors indicate low attenuation (e.g., air-filled pore
(c) Attenuation from the weakly cemented region; (d) Overall vertical profile attenuation; (e) Inverted
space). (a) Slice averaged X-ray attenuation vs. distance from inlet column; (b) Maximum average
column after non cemented sand removal. Adapted with permission from [79].
attenuation; (c) Attenuation from the weakly cemented region; (d) Overall vertical profile attenuation;
(e) Inverted column after non cemented sand removal. Adapted with permission from [79].
Hudyma et al. presented a study of MICP on coquina core specimens [81]. Coquina is a cemented
limestone composed of shells and quartz sand. Thus, calcite, phosphate, and siliciclastic material
Hudyma et al. presented a study of MICP on coquina core specimens [81]. Coquina is a cemented
are the main chemical components of coquina [82,83]. Core specimens were cut in ~50 mm diameter
limestone composed of shells and quartz sand. Thus, calcite, phosphate, and siliciclastic material are
from two blocks, labeled as J and K. Treatment began with the immersion of the coquina samples in a
the main chemical components of coquina [82,83]. Core specimens were cut in ~50 mm diameter from
Sporosarcina pasteurii solution as calcifying bacteria for one hour, allowing saturation and penetration
two blocks, labeled as J and K. Treatment began with the immersion of the coquina samples in a
of bacteria in the limestone. Then, coquina samples were immersed in biodeposition media containing
Sporosarcina pasteurii solution as calcifying bacteria for one hour, allowing saturation and penetration
an equimolar solution of CaCl2 and urea. Treatment with the biodeposition media was performed
of bacteria in the limestone. Then, coquina samples were immersed in biodeposition media
during 2, 10, 20, and 40 days. For coquina specimen JZ14-P2, evident crystal deposition was observed
containing an equimolar solution of CaCl2 and urea. Treatment with the biodeposition media was
as a protective surface, without the presence of calcite deposition inside the pores, while specimen
performed during 2, 10, 20, and 40 days. For coquina specimen JZ14-P2, evident crystal deposition
JX12-P2 showed spheroidal MICP deposition inside the stone pores. Measurements revealed a global
was observed as a protective surface, without the presence of calcite deposition inside the pores,
unit weight increase after treatment between 1.1% and 9.7%. Overall, the water absorption decrease
while specimen JX12-P2 showed spheroidal MICP deposition inside the stone pores. Measurements
was between 2.1% and 47.9% for the different samples. MICP treatment of coquina samples clearly
revealed a global unit weight increase after treatment between 1.1% and 9.7%. Overall, the water
enhanced the protective properties of the material.
absorption decrease was between 2.1% and 47.9% for the different samples. MICP treatment of
Jongvivatsakul et al. investigated the crack healing performance within cement mortars using
coquina samples clearly enhanced the protective properties of the material.
MICP [84]. L. sphaericus LMG 2257 was used as calcifying bacteria, while biodeposition media
Jongvivatsakul et al. investigated the crack healing performance within cement mortars using
consisted of nutrient broth, CaCl2 , and sodium bicarbonate (NaHCO3 ) at pH = 8. Mortar samples were
MICP [84]. L. sphaericus LMG 2257 was used as calcifying bacteria, while biodeposition media
prepared from Portland cement. Artificial cracks were prepared by placing a copper plate in fresh
consisted of nutrient broth, CaCl2, and sodium bicarbonate (NaHCO3) at pH = 8. Mortar samples were
samples. Each day inoculated biodeposition media and urea were applied to the mortars for 20 days.
prepared from Portland cement. Artificial cracks were prepared by placing a copper plate in fresh
MICP treatment was visually evaluated through 40× magnification photographs (see insets, Figure 10).
samples. Each day inoculated biodeposition media and urea were applied to the mortars for 20 days.
After six days of treatment, crack width was decreased by about 34%; after 16 days, remediation was
MICP treatment was visually evaluated through 40× magnification photographs (see insets, Figure
decreasing up to 84%. Non-significant changes were observed in the last 4 days. Samples were divided
10). After six days of treatment, crack width was decreased by about 34%; after 16 days, remediation
into treated and untreated zones. SEM images showed vaterite as the main phase in the treated zone.
was decreasing up to 84%. Non-significant changes were observed in the last 4 days. Samples were
EDS analyses revealed the presence mostly of calcium, carbon, and oxygen. Qualitative XDR analysis
divided into treated and untreated zones. SEM images showed vaterite as the main phase in the
identified the presence of calcite and vaterite in the treated zones. For the first 5 days, artificial crack
treated zone. EDS analyses revealed the presence mostly of calcium, carbon, and oxygen. Qualitative
XDR analysis identified the presence of calcite and vaterite in the treated zones. For the first 5 days,
artificial crack remained apparently non healed, pointing out for artificial crack healing that began
from inside to outside. Ultrasonic pulse velocity (UPV) [85] measurements demonstrated that the
pulse velocity increased linearly for the first 14 days in treated samples (see Figure 10). Compressive
 Molecules 2020, 25, 5499 13 of 23

Molecules 2020, 25, x FOR PEER REVIEW 13 of 23

remained apparently non healed, pointing out for artificial crack healing that began from inside to
outside. Ultrasonic pulse velocity (UPV) [85] measurements demonstrated that the pulse velocity
strengthincreased
increased fromfor
linearly 17.3
the MPa (cracked
first 14 mortar)samples
days in treated to 24.7(see
MPa (healed
Figure 10). mortar). Water
Compressive adsorption
strength
was measured, showing
increased from 17.3 MPa~72% lower
(cracked adsorption
mortar) for(healed
to 24.7 MPa healed mortars
mortar). compared
Water adsorptionto cracked
was samples.
measured,
showing ~72% lower adsorption for healed mortars compared to cracked samples.
Water penetration was 10.8 mm and 8.6 mm depth in the mortars before and after treatment, Water penetration
was 10.8
respectively. mm and
CaCO 8.6 mm depth in the mortars before and after treatment, respectively. CaCO3 formation
3 formation not only filled the artificial crack but also improved the quality of the
not only filled the artificial crack but also improved the quality of the mortars. Compressive strength
mortars. Compressive strength and UPV measurements confirmed that the MICP-treated mortars
and UPV measurements confirmed that the MICP-treated mortars became stronger materials compared
becametostronger materials compared to the original samples.
the original samples.

Figure 10. The plot of ultrasonic pulse velocity (UPV) value vs. crack width of MICP-treated sample
Figure 10. The plot of ultrasonic pulse velocity (UPV) value vs. crack width of MICP-treated sample
along 20 days of treatment. Adapted with permission from [84]. 2019, Elsevier.
along 20 days of treatment. Adapted with permission from [84]. 2019, Elsevier.
Liu et al. studied the protection and restoration of cracks in Tulou or earthen buildings
Liu(see
et Figure
al. studied
11a) madethe protection
of mainly tabiaand[86].
restoration of cracks
Tabia consists of sand,in limestone
Tulou or(CaCOearthen buildings
3 ), and clay, (see
whichmade
Figure 11a) usuallyofpossess
mainly a low tensile
tabia [86].strength.
Tabia Cracks
consists tend
oftosand,
occur limestone
in the wall of(CaCO
the Tulou as starting
3), and clay, which
points for damage propagation (see Figure 11b,c). Soil samples were collected nearby
usually possess a low tensile strength. Cracks tend to occur in the wall of the Tulou as starting points earthen buildings
for the preparation of probes. Soil analysis showed the presence of silica and kaolinite. After mixing
for damage propagation (see Figure 11b,c). Soil samples were collected nearby earthen buildings for
soil samples with sand and lime, mixtures were compacted in different shapes, such as cylinders,
the preparation
beams, and ofwallets.
probes. Soil analysis
Bacterial solutionshowed the presence
and cementation of silica
media were andevery
injected kaolinite.
day forAfter mixing soil
three days.
samplesBacillus
with sand and
pasteurii lime, mixtures
(DSM-33) was usedwere compacted
as calcifying in while
bacteria, different shapes,media
cementation such was
as cylinders,
composed beams,
and wallets.
of ureaBacterial
and CaClsolution and cementation
2 . Unconfined media were
compressive strength (UCS)injected
tests wereevery day
carried outfor
forthree days. Bacillus
the samples,
indicating that mixture samples after treatment increased the average
pasteurii (DSM-33) was used as calcifying bacteria, while cementation media was composed of urea UCS. SEM images revealed
and CaCl that MICP treatment was able to bind loose soil particles (see Figure 11d). XRD analysis showed the
2. Unconfined compressive strength (UCS) tests were carried out for the samples, indicating
presence of calcite, as the CaCO3 unique phase, and SiO2 peaks from the soil. Flexural strength (FS)
that mixture samples after treatment increased the average UCS. SEM images revealed that MICP
was measured before and after MICP treatment to determine the recovery ratio of FS. The repair rate of
treatment was able
FS average to bind
recovery loose soil particles
measurements indicated(see Figure
35.2%, 11d).
56.86%, andXRD79.92%analysis
for crackshowed
widths of the15presence
mm, of
calcite, 10
as mm,
the CaCO 3 unique
and 5 mm, phase,The
respectively. andrecovery
SiO2 peaks from
ratio of shearthe soil. Flexural
strength after MICPstrength
treatment (FS)
waswas measured
50.74%,
before and after
69.53%, andMICP
88.54%treatment to determine
for crack widths of 15 mm, the
10recovery
mm, and 5mm, ratio respectively.
of FS. The repair rate ofangle
Static contact FS average
after MICP treatment was found between 83.6 ◦ and 100◦ compared to a contact angle 0◦ for untreated
recovery measurements indicated 35.2%, 56.86%, and 79.92% for crack widths of 15 mm, 10 mm, and
samples, pointing out for an increased hydrophobicity of the tabia samples.
5 mm, respectively. The recovery ratio of shear strength after MICP treatment was 50.74%, 69.53%,
and 88.54% for crack widths of 15 mm, 10 mm, and 5mm, respectively. Static contact angle after MICP
treatment was found between 83.6° and 100° compared to a contact angle 0° for untreated samples,
pointing out for an increased hydrophobicity of the tabia samples.

(a) (b) (c) (d)

before and after MICP treatment to determine the recovery ratio of FS. The repair rate of FS average
recovery measurements indicated 35.2%, 56.86%, and 79.92% for crack widths of 15 mm, 10 mm, and
5 mm, respectively. The recovery ratio of shear strength after MICP treatment was 50.74%, 69.53%,
and 88.54% for crack widths of 15 mm, 10 mm, and 5mm, respectively. Static contact angle after MICP
treatment was
Molecules 2020, 25, found
5499 between 83.6° and 100° compared to a contact angle 0° for untreated samples,
14 of 23
pointing out for an increased hydrophobicity of the tabia samples.

(a) (b) (c) (d)

Molecules 2020, 25, x FOR PEER REVIEW 14 of 23

Figure 11. Photographs of (a) The King of Tulou – Chengqilou, (b) and (c) wall deteriorated cracks
Figure 11. Photographs of (a) The King of Tulou – Chengqilou, (b) and (c) wall deteriorated cracks
(width is approximately 1.5 cm, depth is up to 100 cm). (d) SEM images of samples after MICP treatment.
(width is approximately 1.5 cm, depth is up to 100 cm). (d) SEM images of samples after MICP
Adapted withAdapted
treatment. permission
withfrom [86] 2020,
permission fromElsevier.
[86] 2020, Elsevier.

LiuLiu
et al.etstudied the formation
al. studied the formationof an of
antierosion layer by
an antierosion means
layer of MICP
by means oftoMICP
reduceto the weathering
reduce the
caused by rain erosion on ancient clay roof tiles [87]. Clay samples were
weathering caused by rain erosion on ancient clay roof tiles [87]. Clay samples were obtained obtained from Shaoming
from
Lou,Shaoming
a Chinese Hakka
Lou, Tulou,
a Chinese built Tulou,
Hakka in 1915built
andinlocated
1915 and in located
Longyan in city. Calcifying
Longyan bacteriabacteria
city. Calcifying used were
Bacillus
usedpasteurii DSM pasteurii
were Bacillus 33. Consolidation media consisted
DSM 33. Consolidation media of consisted
calcium chloride
of calcium and urea. Clay
chloride samples
and urea.
were cleaned
Clay samples and cut cleaned
were into 5 cm anddiameter
cut into 5discs. Bacterial
cm diameter solution
discs. Bacterialand consolidation
solution media were
and consolidation
mediaover
brushed werethebrushed
sample over the sample
surface threesurface
times. three
Aftertimes. After samples
brushing, brushing,were
samplesleft were left in
in static static
conditions
at 30 ◦ C for 7atdays.
conditions 30 °C Measurements
for 7 days. Measurements of static contact
of static contact angle of angle of water
water showed showed contact
contact angles
angles up to
up
◦ to 101°, indicating an increased hydrophobicity of sample surface treated
101 , indicating an increased hydrophobicity of sample surface treated with MICP (see Figure 12a). with MICP (see Figure
SEM12a). SEM micrographs
micrographs showed different
showed different imperfections
imperfections in theof
in the surface surface of the samples
the original original (see
samples
Figure(see12b),
Figure 12b), which were almost completely disappeared after MICP treatment (see Figure 12c). The
which were almost completely disappeared after MICP treatment (see Figure 12c). The width of
width of cracks was significantly reduced, preserving the air permeability. EDS analysis of
cracks was significantly reduced, preserving the air permeability. EDS analysis of precipitated crystals
precipitated crystals indicated as main elements Ca, O, and C, supporting the presence of CaCO3. The
indicated as main elements Ca, O, and C, supporting the presence of CaCO3 . The thickness of the
thickness of the MICP surface layer increased with increasing concentration of the bacterial solution
MICP surface layer increased with increasing concentration of the bacterial solution and consolidation
and consolidation media. Capillary water absorption was reduced in all treated samples; however,
media.
higherCapillary water absorption
concentrations of bacteriawas reduced
solution (108incell/mL)
all treated
hadsamples; however, higher
poorer contributions, concentrations
reducing water
of bacteria solution (108 cell/mL) had poorer contributions, reducing water absorption. Coatings were
absorption. Coatings were resistant to acid corrosion tests, whose pH was higher than 1.0. The total
resistant to acid corrosion
color difference showedtests, whose(∆E
low values pH< was higher than
3), indicating that1.0.
the The
MICP total color difference
treatment showed
met standard colorlow
values (∆E < 3), indicating that the MICP treatment met standard color conditions.
conditions.

(a) (c)
(b)

Figure
Figure 12.12.
(a)(a)Water
Waterstatic
static contact
contactangles
anglesofofsamples before
samples (0◦ ) after
(0°) and
before and treatment (A3, B3,(A3,
after treatment C3). SEM
B3, C3).
SEM images of ancient tiles (b) before and (c) after MICP treatments. Adapted withfrom
images of ancient tiles (b) before and (c) after MICP treatments. Adapted with permission [87]
permission
2020, Elsevier.
from [87] 2020, Elsevier.

5. Microbiologically
5. Microbiologically Induced
Induced Carbonate
Carbonate Precipitation
Precipitation for for In Situ
In Situ Restoration
Restoration of of Historical
Historical Structures
Structures
Métayer-Levrel
Métayer-Levrel et et
al.al.went
wentfurther
furtherwith
with induced
induced bacterial
bacterialprecipitation
precipitationbyby
testing over
testing miniature
over miniature
walls.
walls. Limestonesused
Limestones used ininthis
thisstudy
studywere Tuffeau
were (small
Tuffeau pores),
(small Saint-Maximin,
pores), and Saint-Vast
Saint-Maximin, (large
and Saint-Vast
pores) [88]. Patented bacteria and culture media met industrial and financial requirements. Treatment
media inoculated with a bacterial strain was sprayed over the limestone surface, followed by
spraying of sterile media every 24 h for 5 days and then every 48 h for 10 days. After treatment, a
biocalcin coating of several micrometers was observed. Mineral particles filled the voids moderately
by getting engrained in the structure. In situ tests were carried out on the SE tower of Saint Médard
Church, located in Thouars, France. Tuffeau limestone was used to build the church. Treatment began
Molecules 2020, 25, 5499 15 of 23

(large pores) [88]. Patented bacteria and culture media met industrial and financial requirements.
Treatment media inoculated with a bacterial strain was sprayed over the limestone surface, followed by
spraying of sterile media every 24 h for 5 days and then every 48 h for 10 days. After treatment,
a biocalcin coating of several micrometers was observed. Mineral particles filled the voids moderately by
getting engrained in the structure. In situ tests were carried out on the SE tower of Saint Médard Church,
located in Thouars, France. Tuffeau limestone was used to build the church. Treatment began in June
1993 over an area of 50 m2 . The bioprecipitated produce was exposed to normal weather variations.
Treatment effects were evaluated after 6 months and 1 year by measuring surficial permeability
(a measurement of the standard time of water absorption using a water pipe). MICP treatment
increased the time of water absorption from ~70 s (before treatment) to ~250 s after 6 months of
treatment but then decreased to ~130 s after one year of treatment. In any case, the surficial permeability
after treatment showed lower values if compared to before treatment values. No appreciable changes
for gas permeability, color, or aesthetic appreciation were observed if compared to the original samples.
Bioprecipitate guaranteed the preservation of the limestone by reducing the exchange of degrading
agents and environmental pollutants. Moreover, calcifying bacteria bulk population interfered with the
development of acidifying bacteria, which might be a threat to historical buildings. Finally, no changes
in the external aspect of the tower were observed after 3.5 years.
Perito et al. reported a bacterial CaCO3 mineralization treatment on the Andera Cathedral,
Italy [14]. First, stone samples (10 cm × 10 cm × 4 cm) obtained from the main façade of the Pietra
d’Angera lithotype were tested in the laboratory. B. subtilis 168 strain was the calcifying bacteria,
and B4 was used as the precipitation medium. After bacterial incubation in B4 media at 37 ◦ C,
cells were collected at a 108 cells/mL concentration, centrifugated, autoclaved, stripped of metal cations
through deionized water, rinsed, and stored in physiological solution (PS) at −20 ◦ C. After dilutions of
PS in calcium chloride solution, ammonium carbonate vapors induced the precipitation of calcium
carbonate. Besides crystallization, no visible changes were observed after the seventh day at room
temperature. Then, the frozen cell mixture was ground with alumina, then unbroken cells and alumina
were eliminated through centrifugation. Cytosol, membranes, and cell walls were stored at −20 ◦ C and
labeled as Bacillus cell fraction (BCF). Finally, BCF was lyophilized and resuspended in CaCl2 solution
mixed with supersaturated calcium bicarbonate solution (Super C) and enriched with 20 nm calcite
nanoparticles (2% w/v). Around 30 mL per application was sprayed on the stone samples surface twice
a day for 3 days. A stone sample was sprayed only on Super C as reference. In vitro tests showed
an overall color change ∆E < 3, meaning acceptable and no detectable changes. Water absorption
decreased by 16.7%. Cohesion profiles from DR did not show remarkable differences. In situ test
was implemented on the main façade of the Angera Cathedral, which is dated from the sixth century.
Selected areas sized 0.29 m2 and 0.28 m2 , with a 0.04 m2 area as a control. Approximately 1 L m−2
per spray application of BCF (8.5 g L−1 ) in Super C solution and bacteria-free super C solution was
sprayed for the first day. BCF (0.032 g L−1 ) in Super C, enriched with nanoparticles, and bacteria-free
Super C were sprayed for the second day. On the third day, only a bacteria-free Super C enriched
solution was sprayed on the chosen areas. After four months, evaluation tests were performed, such as
water absorption, surface color change, and cohesion profiles. Small cores from the chosen areas were
taken to the laboratory to analyze new calcite penetration. CaCO3 precipitated crystals were collected
and analyzed with FT-IR and XRD, indicating the presence of calcite. In situ color test measurements
showed ∆E < 3, meaning no detectable changes to the human eye. Water absorption decreased by 6.8%
for in situ experiments. Cohesion profiles indicated an increased hardness of the treated areas for the
first 3 mm.
Rodriguez-Navarro et al. reported in situ restoration using MICP over stone monuments dated
from the sixteen century in Granada, Spain [4]. Heavily degraded areas from three different test
sites were treated: (i) Hospital Real, (ii) San Jeronimo’s Monastery Apse, and (iii) Capilla Real.
These three buildings were constructed using porous calcarenite, which is a material with high water
absorption and porosity. Treatments were performed with M-3P medium inoculated with M. xanthus.
Molecules 2020, 25, 5499 16 of 23

The inoculated medium was sprayed twice on the treated area, followed by one spray of sterile medium.
Treatment was applied twice a day for six consecutive days. After each application, the treated area
was wrapped with aluminum/plastic foil to avoid possible pigmentation by sunlight effect and to
reduce media evaporation. The binding capacity of treatments, surface strengthening, and chromatic
changes were tested for four years after initial treatment. Peeling tape test showed weight loss
reduction after inoculated media treatment for all the treated areas, which is related to the bacterial
CaCO3 precipitation (calcite and vaterite, according to XDR). SEM analysis showed that new crystals
were precipitated aligned to the pore system without plugging them. Moreover, loose calcarenite
grains were connected via EPS with new bacterial calcium carbonate. Results with sterile M-3P media
showed an identical degree of consolidation for San Jeronimo and Hospital Real treated areas. In some
cases, sufficient consolidation was obtained with the activation of the original carbonatogenic bacteria
present, thus reducing treatment cost. Moreover, spectrophotometric ∆E measurements were below 5,
meaning acceptable value for treatment.
Self-inoculation of indigenous calcifying bacteria present in stones prior to MICP treatment is
another viable way that Jroundi et al. designed for in situ treatment of salt weathered stones [89].
The cloister entrance of San Jeronimo Monastery, Spain, presented granular disintegration and surface
loss. This weathering and erosion were
 attributed to the crystallization of salts, such as magnesium
sulfate hexahydrate MgSO4 ·6H2 O , niter (KNO3 ), halite (NaCl), among others. Fifty-five bacterial
isolates were obtained from selected areas. Incubation of M-3P inoculated medium led to high
CaCO3 precipitation in all bacterial isolates after 48 h. XDR analysis identified calcite as the main
phase and <10 wt% of vaterite. To evaluate the applicability of the activated carbonatogenic bacteria,
the sterile calcite substrate was immersed in an inoculated M3-P medium. Field emission scanning
electron microscopy (FESEM) [90] and transmission scanning electron microscopy (TSEM) [91] showed
dense bacterial colony formation and CaCO3 embedded in EPS after 20 h treatment (see Figure 13b).
Spheroidal or rhombohedral calcite structures fully covered the calcite substrate growth controlled on
a self-epitaxy way after 48 h treatment (see Figure 13c). In situ treatments were applied on stone blocks
with a similar degree of exposure and decay. Three different treatment approaches were performed
using M. xanthus, sterile M-3P media, and a self-inoculation biotreatment. M. xanthus and sterile
M-3P treatment results showed limited consolidation. The peeling tape test revealed modest surface
consolidation for both treatments after 5 months. Drilling resistance values were slightly higher in
the first ~5 mm depth for treated areas after 24 months. SEM showed almost no presence of calcium
carbonate or EPS. Chromatic changes revealed, for both treatments, the acceptable values of ∆E (<5).
On the other hand, self-inoculated bacteria treatment showed remarkable consolidation by a peeling
tape test along with a 24 months study. DR measurements showed about 4 times higher values than
untreated stone (see Figure 13d–f). The maximum DR value achieved was 10 ± 6 N for the first 3~5 mm
depth. Color changes after treatment were not significant, showing an ∆E = 3.8 ± 1.7. XRD analyses of
samples from treated areas showed calcite as the unique calcium carbonate phase (see Figure 13a).
Strengthening of the stone was associated with the abundant presence of newly precipitated calcium
carbonate crystals and EPS. Mercury intrusion porosimetry (MIP) showed a reduction in porosity
from 27 ± 1% to 25 ± 1%, resulting from the formation of 30–100 nm size nanocrystals that cemented
the stone without plugging the pores. No presence of material loss or granular disaggregation was
observed 24 months after the treatment.
1.7. XRD analyses of samples from treated areas showed calcite as the unique calcium carbonate
phase (see Figure 13a). Strengthening of the stone was associated with the abundant presence of
newly precipitated calcium carbonate crystals and EPS. Mercury intrusion porosimetry (MIP)
showed a reduction in porosity from 27 ± 1% to 25 ± 1%, resulting from the formation of 30–100 nm
size nanocrystals that cemented the stone without plugging the pores. No presence of material loss
Molecules 2020, 25, 5499 17 of 23
or granular disaggregation was observed 24 months after the treatment.

(a)
(b) (c)

(d) (e) (f)

Figure 13.(a)
Figure13. (a)XRD
XRDpattern
patternbefore
before(blue)
(blue)and
andafter
after(red)
(red)MICP
MICPtreatment.
treatment. SEM
SEMimages
imagesof
of(b)
(b)newly
newly
formed CaCO 3 ; (c) calcite substrate covered by bacterial calcite. Drilling resistance depth profile:
formed CaCO3; (c) calcite substrate covered by bacterial calcite. Drilling resistance depth profile: (d)
(d) M. xanthus treatment (blue-line); (e) M-3P treatment (blue-line); (f) self-inoculated treatment (blue
M. xanthus treatment (blue-line); (e) M-3P treatment (blue-line); (f) self-inoculated treatment (blue
line). Untreated stones (red line) are presented for comparison. Shaded areas represent s.d. (±1σ).
Adapted with permission from [89].

6. Conclusions and Perspectives

Highly ureolytic bacteria are the main strains of microorganisms used in MICP-treatment for the
restoration of historic buildings. Despite higher calcium carbonate production by some microorganisms,
the usage of human pathogenic bacterial strains (e.g., P. putida, B. cereus) is not recommended because
restoration in situ lacks the biosafety that a microbiology laboratory could provide. MICP treatment
has been applied in different ways, such as immersion, spraying, injection, and brushing. This is an
important parameter, necessarily to be taken, depending on the in-situ circumstances and requirements.
Optimization of the treatment, such as lowering reagents costs, high yields of carbonate production,
shorter treatment time, avoiding unnecessary tests or treatment steps, could allow researchers to save
some time, money, and resources.
Although, until now, the method has not been standardized, a good approach for this is the usage
of the ζ-potential because it has been proven that bacteria with lower ζ-potential possess higher surface
colonization capacity. A modification in the MICP treatment to obtain CaCO3 crystals using a dead cell
fraction instead of the application of live calcifying bacteria becomes interesting for the case in which
certain bacterial strains, like nitrifying or sulfur-oxidizing bacteria, are needed, e.g., Nitrosomonas spp,
Nitrobacter spp, Thiobacillus spp. These bacteria species are capable of excreting acids [92] (if nitric acid is
produced, it can react with calcite, thus forming highly soluble calcium nitrate), leading to biocorrosion
of the material [23]. These problems were addressed by Ganendra et al. [93], using Methylocystis parvus
as calcifying bacteria to induce calcium carbonate precipitation from different calcium formate
concentrations. Although culture-dependent methods have been useful to isolate carbonatogenic
microorganisms, new genomic microbiological analyses have allowed us to study the epilithic microbial
biodiversity in monuments and historical buildings [94]. For example, Chimenti et al. carried out a
metagenomic analysis of the bacterial communities, colonizing the walls of the medieval church of
San Leonardo di Sponto, in Italy [95]. The microbiome of the wall samples was composed not only of
possible deteriorated microorganisms but also carbonatogenic bacteria corresponding to the phylum
Actinobacteria, specially Arthrobacter spp. In fact, in brick samples, this phylum may be predominant,
depending on the environmental conditions [96]. DNA next-generation sequencing studies have
focused on the microbial diversity in bricks of the former Auschwitz II-Birkenau concentration,
Molecules 2020, 25, 5499 18 of 23

and extermination camp in Poland has shown the presence of the genus Arthrobacter as part of the
microbiota [97]. Although these microorganisms can colonize these surfaces, the carbonatogenic
bacteria distribution may differ in relation to the collection site. For instance, Andrei et al. found that
the diversity of epilithic bacterial communities on Saint Donatus statue, Romania, varied depending on
the monument sampling site [98]. Microbiologically induced carbonate precipitation has been shown
to be an efficient strategy to consolidate, protect, restore, and enhance the mechanical properties of a
broad variety of materials used in historical buildings.

Author Contributions: E.O.-V. wrote the draft manuscript. M.G.-G. and A.P.-C. revised the manuscript. A.P.-C.
supervised the writing process and edited the manuscript. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: E.O.-V. thanks to Dra. Martha Romero and Carlos Vasquez from the Instituto Nacional del
Patrimonio Cultural (National Institute for the Cultural Heritage, Ecuador) for the research stay. A.P.-C. thanks to
the University Yachay Tech for the internal grants No. Chem19-08 and Chem19-17.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
AB-PAS Alcian blue-periodic acid-Schiff stain
BCF Bacillus cell fraction
CTC 5-Cyano-2,3-ditolyl tetrazolium chloride
DR Drilling resistance
EDX Energy-dispersive X-ray spectroscopy
EPS Extracellular polymeric substances
ESEM Environmental scanning electron microscopy
FESEM Field emission scanning electron microscopy
FS Flexural strength
MICP Microbiologically induced carbonate precipitation
MIP Mercury intrusion porosimetry
PEMA/PMA Poly(ethyl methacrylate-co-methacrylate)
PS Physiological solution
PVB Polyvinyl butyral
RA Recycled aggregates
SEM Scanning electron microscopy
Super C Supersaturated calcium bicarbonate solution
TEOS Tetraethyl orthosilicate
TSEM Transmission scanning electron microscopy
UCS Unconfined compressive strength
UPV Ultrasonic pulse velocity
X-CT X-ray computed tomography
XRD X-ray powder diffraction
ζ-potential Electrical charge at the shear plane
∆E Overall degree color change

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