Ecological Engineering 36 (2010) 118136

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Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Review

Microbial carbonate precipitation in construction materials: A review

Willem De Muynck a,b , Nele De Belie a, , Willy Verstraete b,1
a
Magnel Laboratory for Concrete Research, Dept. of Structural Engineering, Ghent University, Technologiepark Zwijnaarde 904, B-9052 Gent, Belgium
b
Laboratory of Microbial Ecology and Technology (LabMET), Dept. of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Gent, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Evidence of microbial involvement in carbonate precipitation has led to the exploration of this process
Received 12 August 2008 in the eld of construction materials. One of the rst patented applications concerned the protection of
Received in revised form 11 February 2009 ornamental stone by means of a microbially deposited carbonate layer, i.e. biodeposition. The promising
Accepted 13 February 2009
results of this technique encouraged different research groups to evaluate alternative approaches, each
group commenting on the original patent and promoting its bacterial strain or method as the best per-
forming. The goal of this review is to provide an in-depth comparison of these different approaches. Special
Keywords:
attention was paid to the research background that could account for the choice of the microorganism and
Bacteria
Stone
the metabolic pathway proposed. In addition, evaluation of the various methodologies allowed for a clear
Biomineralization interpretation of the differences observed in effectiveness. Furthermore, recommendations to improve
Biodeposition the in situ feasibility of the biodeposition method are postulated. In the second part of this paper, the use
Biomortar of microbially induced carbonates as a binder material, i.e. biocementation, is discussed. Bacteria have
Biocement been added to concrete for the improvement of compressive strength and the remediation of cracks. Cur-
Bioconcrete rent studies are evaluating the potential of bacteria as self-healing agents for the autonomous decrease
Calcite of permeability of concrete upon crack formation.
Conservation
2009 Elsevier B.V. All rights reserved.
MICP

1. Introduction of problems related to incompatibility with the stone, both water

Construction materials such as stone and concrete are sub-
jected to the weathering action of several physical, chemical and
biological factors (Saiz-Jimenez, 1997; Le Metayer-Levrel et al.,
1999; Warscheid and Braams, 2000). Because of their composition
and textural characteristics, carbonate stones (limestones, dolo-
stones and marbles) are particularly susceptible to weathering.
Progressive dissolution of the mineral matrix as a consequence of
weathering leads to an increase of the porosity, and as a result, a
decrease of the mechanical features (Tiano et al., 1999). In order
to decrease the susceptibility to decay, many conservation treat-
ments have been applied with the aim of modifying some of the
stone characteristics. Water repellents have been applied to pro-
tect stone from the ingress of water and other weathering agents.
The use of stone consolidants aims at re-establishing the cohesion
between grains of deteriorated stone. However, both conservation
treatments are subject to frequent controversy due to their non-
reversible action and their limited long-term performance. Because
Corresponding author. Tel.: +32 092645522; fax: +32 092645845.
E-mail addresses: Willem.DeMuynck@UGent.be (W. De Muynck),
Nele.DeBelie@UGent.be (N. De Belie), Willy.Verstraete@UGent.be (W. Verstraete).
1
Tel.: +32 092645976; fax: +32 092646248.
repellents and consolidants have often been reported to acceler-
ate stone decay. (Clifton and Frohnsdorff, 1982; Delgado Rodrigues,
2001; Moropoulou et al., 2003).
Organic treatments commonly result in the formation of incom-
patible and often harmful surface lms. Additionally, because large
quantities of organic solvents are used, they contribute to pollution
(Camaiti et al., 1988; Rodriguez-Navarro et al., 2003). Inorganic con-
solidation may be preferable since stone materials and protective
or consolidating materials share some physico-chemical afnity
(Rodriguez-Navarro et al., 2003). Some researchers have tried to
develop methods based on the reintroduction of calcite into the
pores of limestone. The lime-water technique, i.e. application of a
saturated solution of calcium hydroxide, has been proposed and
experimented both for wall painting mortars and for some deterio-
rated calcareous stones, in order to impart a slight water repellent
and consolidating effect (Tiano et al., 1999). As of yet, little success
has been achieved in consolidating stone with inorganic materials.
Some of the reasons for the poor performance of inorganic con-
solidants are their tendencies to produce shallow and hard crusts
because of their poor penetration abilities, the formation of soluble
salts as reaction by-products, growth of precipitated crystals and
the questionable ability of some of them to bind stone particles
together (Clifton and Frohnsdorff, 1982). In the case of the calcite
reintroduction methods, the latter is attributable to the production

0925-8574/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ecoleng.2009.02.006
 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
of many small crystallites, which are not chemically bound to the
internal surface of the pore and which are not able to bridge the
pores (Tiano et al., 2006).
Recently, bacterially induced carbonate precipitation has been
proposed as an environmentally friendly method to protect
decayed ornamental stone. The method relies on the bacterially
induced formation of a compatible carbonate precipitate on lime-
stone, and unlike the lime-water treatment, the carbonate cement
appears to be highly coherent (Le Metayer-Levrel et al., 1999).
In addition, this technique has been explored for the improve-
ment of the durability of cementitious materials (Ramachandran et
al., 2001; Ramakrishnan et al., 2001; De Muynck et al., 2008a,b).
2. Microbially induced carbonate precipitation (MICP)
Like other biomineralization processes, calcium carbonate
(CaCO3 ) precipitation can occur by two different mechanisms:
biologically controlled or induced (Lowenstan and Weiner, 1988).
In biologically controlled mineralization, the organism controls
the process, i.e. nucleation and growth of the mineral particles,
to a high degree. The organism synthesizes minerals in a form
that is unique to that species, independently of environmental
conditions. Examples of controlled mineralization are magnetite
formation in magnetotactic bacteria (Bazylinski et al., 2007)
and silica deposition in the unicellular algae coccolithophores
and diatoms, respectively (Barabesi et al., 2007). However, cal-
cium carbonate production by bacteria is generally regarded as
induced, as the type of mineral produced is largely dependent
on the environmental conditions (Rivadeneyra et al., 1994) and
no specialized structures or specic molecular mechanism are
thought to be involved (Barabesi et al., 2007). Different types
of bacteria, as well as abiotic factors (salinity and composi-
tion of the medium) seem to contribute in a variety of ways
to calcium carbonate precipitation in a wide range of different
environments (Knorre and Krumbein, 2000; Rivadeneyra et al.,
2004).
Calcium carbonate precipitation is a rather straightforward
chemical process governed mainly by four key factors: (1) the cal-
cium concentration, (2) the concentration of dissolved inorganic
carbon (DIC), (3) the pH and (4) the availability of nucleation sites
(Hammes and Verstraete, 2002). CaCO3 precipitation requires suf-
cient calcium and carbonate ions so that the ion activity product
(IAP) exceeds the solubility constant (Kso ) (Eqs. (1) and (2)). From
the comparison of the IAP with the Kso the saturation state () of
the system can be dened; if > 1 the system is oversaturated and
precipitation is likely (Morse, 1983):
Ca2+ + CO3 2 CaCO3
2+ 2 9
= a(Ca )a(CO3 )/K so with K so calcite, 25 = 4.8 10
The concentration of carbonate ions is related to the concentra-
tion of DIC and the pH of a given aquatic system. In addition, the
concentration of DIC depends on several environmental parameters
such as temperature and the partial pressure of carbon dioxide (for
systems exposed to the atmosphere). The equilibrium reactions and
constants governing the dissolution of CO2 in aqueous media (25 C
and 1 atm) are given in Eqs. (3)(6) (Stumm and Morgan, 1981):
CO2(g) CO2(aq.) (pK H = 1.468)
CO2(aq.) + H2 O H2 CO3
(pK = 2.84)
+
H2 CO3 H + HCO3 (pK 1 = 6.352)
2 +
HCO3 CO3 +H (pK 2 = 10.329)
With H2 CO3 = CO2(aq.) + H2 CO3 .
119
Microorganisms can inuence precipitation by altering almost
any of the precipitation parameters described above, either sepa-
rately or in various combinations with one another (Hammes and
Verstraete, 2002). However, the primary role has been ascribed
to their ability to create an alkaline environment through various
physiological activities. Both autotrophic and heterotrophic path-
ways are involved in the creation of such an alkaline environment
(for an extensive review, see Castanier et al., 1999). While the
environmental conditions of heterotrophic pathways are diverse
(aerobiosis, anaerobiosis and microaerophily), carbonate precipita-
tion always appears to be a response of the heterotrophic bacterial
communities to an enrichment of the environment in organic mat-
ter (Castanier et al., 1999). A rst heterotrophic pathway involves
the sulphur cycle, in particular the dissimilatory sulphate reduc-
tion, which is carried out by sulphate reducing bacteria under
anoxic conditions. A second heterotrophic pathway involves the
nitrogen cycle, and more specically, (1) the oxidative deamina-
tion of amino acids in aerobiosis, (2) the dissimilatory reduction of
nitrate in anaerobiosis or microaerophily and (3) the degradation
of urea or uric acid in aerobiosis. Another microbial process that
leads to an increase of both the pH and the concentration of dis-
solved inorganic carbon is the utilization of organic acids (Braissant
et al., 2002), a process which has been commonly used in microbial
carbonate precipitation experiments. The precipitation pathways
described above are general in nature, which accounts for the com-
mon occurrence of microbial carbonate precipitation (MCP) and
validates the statement by Boquet et al. (1973) that under suitable
conditions, most bacteria are capable of inducing carbonate pre-
cipitation. In addition, carbonate particles can also be produced by
ion exchange through the cell membrane (Rivadeneyra et al., 1994;
Castanier et al., 1999).
Besides changes induced in the macro-environment, bacteria
have also been reported to inuence calcium carbonate precipita-
tion by acting as sites of nucleation or calcium enrichment (Morita,
1980). Due to the presence of several negatively charged groups on
the cell wall, at a neutral pH, positively charged metal ions can be
bound on bacterial surfaces (Douglas and Beveridge, 1998; Ehrlich,
1998). Such bound metal ions (e.g. calcium) may subsequently react
with anions (e.g. carbonate) to form an insoluble salt (e.g. calcium
carbonate). In the case of a sufcient excess of the required cations
and anions, the metal salt on the cell surface initiates mineral for-
mation by acting as a nucleation site. The anion (e.g. carbonate)
in this reaction may be a product of the bacterial metabolism, or
it may have an abiotic origin (Ehrlich, 1998). Furthermore, it has
been demonstrated that specic bacterial outer structures (glyco-
calyx and parietal polymers) consisting of exopolysaccharides and
(1) amino acids play an essential role in the morphology and mineral-
ogy of bacterially induced carbonate precipitation (Braissant et al.,
(2) 2003; Ercole et al., 2007).
The actual role of the bacterial precipitation remains, however,
a matter of debate. Some authors believe this precipitation to be
an unwanted and accidental by-product of the metabolism (Knorre
and Krumbein, 2000) while others think that it is a specic process
with ecological benets for the precipitating organisms (Ehrlich,
1996; McConnaughey and Whelan, 1997).
The evidence of microbial involvement in carbonate precipi-
tation has subsequently led to the exploration of this process in
(3) a variety of elds. A rst series of applications is situated in the
(4) eld of bioremediation. In addition to conventional bioremediation
strategies which rely on the biodegradation of organic pollutants
(5) (Chaturvedi et al., 2006; Simon et al., 2004), the use of MICP has
been proposed for the removal of metal ions. Applications include
(6)
the treatment of groundwater contaminated with heavy metals
(Warren et al., 2001) and radionucleotides (Fujita et al., 2004),
the removal of calcium from wastewater (Hammes et al., 2003).
120 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136

Table 1
Characteristics of the various biodeposition processes.

Characteristic Process
Biodeposition

Denition Biologically induced deposition of a carbonate layer on the surface of building materials
Type Surface treatment
Goals Improvement of the durability, consolidation and decrease of water absorption
Where/when Applied on the surface of building materials such as stone, bricks and concrete
Mediator Microorganisms Organic matrix molecules (OMM) Activator medium
How Spraying of bacteria and nutrients Spraying of OMM and carbonate rich solution Application of nutrient media
Application of poultice
Research group Calcite Bioconcept, Granada University, Ghent Bioreinforce consortium Granada University
University Biobrush consortium
Added value Ecological, environmentally friendly, compatibility
Possibility of including pigments
(Current) Limitations Costs of bacteria and nutrients Costs of chemicals
Rather limited efcacy Long activation period required
Status of use The Calcite Bioconcept: laboratory and in situ Laboratory and in situ experiments Laboratory and in situ experiments
experiments
Several research groups: laboratory experiments

Market Niche Restoration sector

Limestone, concrete Marble statues Limestone

Another series of applications aims at modifying the properties preceding the application of microbially induced carbonate pro-
of soil, i.e. for the enhancement of oil recovery from oil reservoirs duction to building materials.
(Nemati and Voordouw, 2003; Nemati et al., 2005), plugging (Ferris
and Stehmeier, 1992) and strengthening of sand columns (DeJong
3.1.1. From carbonate precipitation in natural environments and
et al., 2006; Whifn et al., 2007). Moreover, microbially induced
laboratory conditions to applications in situ
precipitation has been investigated for its potential to improve the
When exposed to atmospheric conditions, soft limestone
durability of construction materials such as limestone and cemen-
quickly acquires a protective skin (calcin) through dissolution of
titious materials. The latter is dealt with in this review paper and
carbonates within the pore water, evaporation and precipitation of
can be divided into processes for the deposition of a protective sur-
calcite at or near the exposed surface (Dreesen and Dusar, 2004).
face layer with consolidating and/or waterproong properties, i.e.
This layer demonstrates a higher hardness and density compared
biodeposition (Tables 1 and 2), and processes for the generation of
to the underlying layers. As a result of atmospheric pollutants,
a biologically induced binder, i.e. biocementation (Tables 3 and 4).
however, this layer slowly degrades, losing its protective role. The
discovery that bacteria contribute to the formation of limestone has
3. Biodeposition led to the suggestion to use bacteria for the re-establishment of this
calcin.
Adolphe et al. (1990) were among the rst to consider the use
Boquet et al. (1973) were among the rst to demonstrate the
of microbially induced carbonate precipitation (MICP) for the pro-
ability of soil bacteria to precipitate calcium carbonate under labo-
tection of ornamental stone. They applied for a patent regarding
ratory conditions. While previous research only concerned marine
the use of calcinogenic bacteria on stone surfaces, as is discussed in
bacteria in liquid media (Drew, 1911; Shinano, 1972), the authors
Section 3.1.2. The promising results of this so-called Calcite Biocon-
investigated crystal formation by soil bacteria on solid media.
cept technique encouraged different research groups to evaluate
The authors obtained the best results with B4 medium (Table 5).
alternative approaches for the biomediated carbonate precipita-
Among the organisms tested, several Bacillus strains (incl. Bacillus
tion on limestone. These approaches can be mainly divided into
cereus) and Pseudomonas aeruginosa were observed to form crys-
those falling within and those falling outside the specications
tals. The authors concluded that crystal formation is a function of
of the patent by Adolphe et al. (1990), i.e. the application of cal-
the medium, and that under suitable conditions most bacteria can
cinogenic bacteria to a stone surface. The rst series of approaches
form crystals.
(Sections 3.1.33.1.6), those falling within the patent specications,
In parallel with the work done by this Spanish research group,
are characterized by the use of different microorganisms, metabolic
Adolphe and Billy (1974) succeeded in the formation of calcite in the
pathways or delivery systems to overcome some of the poten-
laboratory by bacteria isolated from tuff and travertine. Between
tial limitations of the Calcite Bioconcept technique. The selection
1983 and 1987, Castanier et al. (1999) investigated the different
of a microorganism by the different research groups was often
mechanisms responsible for the microbial formation of calcium
based on their experiences from previous studies on microbially
carbonate, evidencing the microbial origin of limestone. Adolphe
induced mineral precipitation. In the second series of approaches,
et al. (1989) further demonstrated the bacterial origin of the calcite
no microorganisms are applied to the surface. These approaches
crusts in extreme climates, such as Greenland and the Sahara desert.
can be divided into studies where inducing macromolecules are
In addition, the team observed the great resistance of these lay-
supplied to the stone together with a supersaturated solution of
ers towards erosion. From the above ndings, Adolphe et al. (1990)
calcium carbonate (Section 3.2) and studies which obtain carbon-
applied for a patent for the treatment of articial surfaces by virtue
ate precipitation by the microbiota inhabiting the stone (Section
of a surface coating produced by microorganisms. In addition, a
3.3). In the latter, only nutrients are added to the stone.
company, Calcite Bioconcept, was created.

3.1. Application of calcinogenic bacteria

3.1.2. Procedure according to Calcite Bioconcept (France)
Before going into detail on the different methodologies pro- Although the ability of bacteria to precipitate calcium carbonate
posed, a short chronological overview is given on the work had been proven in the laboratory, further tests were necessary
Table 2
Overview of the different methodologies used for the deposition of a layer of calcium carbonate on stone and concrete (biodeposition).

Application Mediator Authors Organism/molecule Metabolisma Solutionb Stone type (porosity)

Limestone Microorganisms Calcite Bioconcept (Le Metayer-Levrel et al., 1999) Bacillus cereus ODAA Growth medium (CB) Tuffeau (40%), Saint Maximin
and Nutrical (30%)
Tiano et al. (1999) Micrococcus sp. Bacillus subtilis ODAA OAU B4 Pietra di Lecce, bioclastic
limestone (40%)
Rodriguez-Navarro et al. (2003) Myxococcus xanthus ODAA OAU M-3, M-3P Bioclastic calcarenite (2432%)
Dick et al. (2006) Bacillus sphaericus HU SF Euville, crinoidal limestone
(16%)
Biobrush (May, 2005) Pseudomonas putida ODAA OAU B4 AW Portland oolitic limestone
(20%)

Organic matrix Tiano (1995) and Tiano et al. (2006) Mytilus californianus shell extracts n.a. Ammonium carbonate Sound and articially aged
molecules method or marble (Gioia calcitic marble,
supersaturated 13.5%), limestone (Pietra di
bicarbonate solution Lecce, 40%) and dolostone
(Pietra dAngera, bioclastic,
20%)

W. De Muynck et al. / Ecological Engineering 36 (2010) 118136

Aspartic acid
Bacillus cell fragments

Activator medium Jimenez-Lopez et al. (2007) Microbiota inhabiting the stone ODAA OAU M-3, M-3P, CC Decayed limestone and quarry
calcarenite stone from La
Escribania (2432%)

Cementitious materials Microorganisms De Muynck et al. (2008a,b) Bacillus sphaericus HU SF Concrete/mortar with CEM I
52.5 N (1520%); w/c 0.5, 0.6
and 0.7

Authors Experimental methods Evaluation procedures

Inoculum Application procedure Septicity conditions

Bacteria Nutrients/chemicals Stone N/C Appl.

Calcite bioconcept Culture in exponential Spraying Spraying (5 times) NS NS NS Water absorption (Karsten
phase: 107 to pipe), SEM analysis, surface
109 cells mL1 roughness (imprint moulding),
colorimetry and Plate count
Tiano et al. Overnight culture: Brushing on water Wetting every day for S S NS Water absorption (contact
106 cells cm2 saturated specimens 15 days sponge), colorimetric
measurements, stone
cohesion(drilling resistance
measuring system)
Rodriguez-Navarro et al. 2% inoculum Immersion in growing bacterial culture (shaking or S S S Stone cohesion (sonicator
stationary conditions) for 30 days bath), weight increase, XRD
and SEM analysis, porosimetry
analysis
Dick et al. 1% inoculum Immersion in growing bacterial culture (intermediate S S S Water absorption (immersion
wetting) for 28 days test) SEM analysis
Biobrush 108 cells mL1 Spraying In Carbogel NS NS NS Water absorption and drying
due to evaporation
Tiano et al. n.a. n.a. Immersion in test NS NS NS Water absorption (contact
solution or spraying (in sponge), colorimetric
situ tests) measurements, stone cohesion
(drilling resistance measuring
system, peeling tape test),
staining of newly formed
calcite with Alizarin Red S and
Calcein

121
122 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136

to investigate the viability and performance of these bacteria in

and SEM analysis, porosimetry

absorption gas, permeability,

thawing, Thin sections, SEM,

situ. The technique was further optimized and industrialized as a

bath), weight increase, XRD

Carbonation, Freezing and

Stone cohesion (sonicator
result of the collaboration between the University of Nantes, the

Weight increase, Water

Evaluation procedures

Laboratory for the research of historic monuments (LRMH) and the

chloride migration,
company Calcite Bioconcept (Le Metayer-Levrel et al., 1999).
The rst step comprised the search for suitable microorganisms.

XRD analysis
Bacteria were isolated from natural carbonate producing environ-

analysis
ments and screened for their carbonatogenic yield, i.e. ratio of the
weight of calcium carbonate produced to the weight of organic
matter input (OM). The highest performance was obtained with B.
cereus, which showed a carbonatogenic yield of 0.6 g CaCO3 g OM1
(Castanier et al., 1999). Furthermore, since B. cereus could be easily
produced on an industrial scale, this organism was selected for in
Appl.

situ applications (Orial, 2000).

NS
S

The second step comprised the optimization of a nutrient

medium and the frequency of feeding to meet industrial economi-
cal constraints. The nutritional medium was designed to stimulate
the production of carbonate through the nitrogen cycle metabolic
N/C

NS

pathways, which are the only pathways to be activated in oper-

S
Septicity conditions

ational conditions, i.e. in aerobiosis and microaerophily. More

specically, the media contain a source of proteins for the oxida-
tive deamination of amino acids in aerobiosis and a source of
S and NS

nitrate for the dissimilatory reduction of nitrate in anaerobiosis or

Stone

microaerophily. In addition, a fungicide was added to prevent the

NS

unwanted growth of fungi present on the stone, or deposited from

the air (Orial et al., 2002).
From preliminary experiments in the laboratory, a treatment
procedure for in situ applications was proposed. The treatment
consists of rst spraying the entire surface to be protected with
Immersion for 4 days
Nutrients/chemicals

a suitable bacterial suspension culture (Tables 1 and 2). Subse-

Immersion in a growing bacterial culture for 30 days

quently, the deposited culture is fed daily or every 2 days with the
suitable medium in order to create a surcial calcareous coating
scale, the biocalcin. Usual industrial and economical constraints
restrict the number of feeding applications to ve, but treatment of
historic patrimony may be less restrictive. The frequency of feed-
ODAA: oxidative deamination of amino acids; OAU: organic acid utilization, HU: hydrolysis of urea.

ing was shown to be dependent on the stone type, with a daily

frequency more suitable for ne-grained limestone and the 2-day
frequence for coarse grained limestone (Le Metayer-Levrel et al.,
n.a.: not applicable, N/C: nutrients/chemicals, Appl.: application, NS: non-sterile and S: sterile

1999).
Application procedure

The rst application in situ was carried out in 1993 in Thouars on

Immersion for 1 day

the tower of the Saint Mdard Church. The treatment was applied
on an area of 50 m2 of Tuffeau limestone. The protective effect of
the treatment was evaluated by means of macro- and microscopic
Bacteria

investigations, such as measurements of the permeability, evalua-

tion of the roughness and colorimetry and SEM examination. SEM
images indicated the abundant development of calcinogenic bac-
terial populations, illustrating the viability of B. cereus on stone
surfaces. The presence of the biocalcin decreased the water absorp-
tion rate to a signicant extent (5 times lower) while retaining the
Experimental methods

Overnight culture: 107

permeability for gas. Furthermore, no inuence on the aesthetic

Microbiota inhabiting

appearance could be observed (Le Metayer-Levrel et al., 1999).

to 109 cells mL1

Long-term evaluation of the biocalcin layer has shown differ-

ences in the durability behaviour related to the orientation of the
Inoculum

the stone

facade and the micro-relief of the stone. The densest layers could
be observed inside the pores, while cracking of the biocalcin was
observed at crystoballite protrusions. From these observations, it
was concluded that every 10 years a new treatment is needed to
Composition see Table 5.

restore the protective effect of the biocalcin (Orial, 2000).

Similar experiments were applied on limestone statuaries
Jimenez-Lopez et al.

which had been placed in different climatic environments. Exper-

Table 2 (Continued)

De Muynck et al.

iments were performed on two types of limestone, Tuffeau and

Saint-Maximim. The former is a ne-grained limestone character-
ized by a high porosity and small (<10 m) pores. The latter belongs
Authors

to a group of limestones of variable porosity formed of both larger

a

grains and pores (>10 m) (Le Metayer-Levrel et al., 1999). The rural
 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 123

Table 3
Characteristics of the various biocementation processes.

Characteristic Process
Biocementation

Remediation of cracks Bacterial concrete Self-healing concrete Biomortar

Denition Generation of a biologically induced binder

Type Surface treatment Admixture Admixture Binder

Goals Improvement of the durability

External crack repair
Where/when Applied in cracks on the
surface of concrete and mortar
How Application of bacteria with a
carrier/binder material
immersion in nutrient solution
Strength improvement
Applied in the concrete
mixture
Application of bacteria
in the mixture, lower
amount of bacteria
(1 wt% admixture)
Self-healing of cracks
Application of mortar mixture to
repair broken limestone fragments
or to ll cavities in stone
Application of bacteria and
nutrients in the mixture, lower
amount of bacteria
(1 wt% admixture)
Strength development
formation of a
carbonate-binder-based
mortar
Application of bacteria and
nutrients in the mixture,
higher amount of bacteria
(25 vol.% binder)

Added value Ecological, environmentally friendly

Changes in Self-healing capacity Compatibility
microstructure
Status of use Laboratory experiments Laboratory Laboratory experiments Small scale applications in
experiments practice

(current) Limitations Costs of bacteria and nutrients

None or low bacterial activity at high pH of cementitious materials immobilization is necessary -
Difcult to apply in practice Long-term viability of spores
Market niche Eco-friendly repair of cracks Repair of difcult to reach concrete Restoration sector
eco-friendly reuse of brick

and maritime environment appeared to be very aggressive, with
almost complete loss of the biocalcin after 4 years of exposure.
However, in the urban environment, the biocalcin retained its pro-
tective effect, with treated statues showing little damage compared
to untreated ones. Furthermore, after 4 years of exposure to urban
conditions, no undesired biological colonisation could be observed
(Orial, 2000).
By adding natural pigments into the nutritional medium, it is
also possible to create a surcial patina with the biodeposition
treatment. The pigments are integrated into the biocalcin and thus
give a persistent light colouring to the stone. This technique has
been proposed to conceal some newly replaced stones on a monu-
ment facade (Le Metayer-Levrel et al., 1999).
3.1.3. Procedure according to the University of Granada (Spain)
Rodriguez-Navarro et al. (2003) addressed two important
limitations of the calcite method. As the thickness of the biocon-
solidating cement was limited to only a few microns, this method
seemed to be ineffective for in-depth consolidation. Moreover, the
formation of a supercial lm consisting of a mixture of biolog-
ical remains, plugged stone pores and provided no consolidation
(Rodriguez-Navarro et al., 2003). Besides, the authors commented
on the potential drawback of the use of Bacillus in stone conser-
vation. According to these authors, the formation of endospores
may lead to germination and uncontrolled biolm growth under
appropriate conditions (i.e. temperature, humidity and nutrient
availability).
The authors, therefore proposed the use of Myxococcus xanthus
for the creation of a consolidating carbonate matrix in the porous
system of limestone. Their research group previously demonstrated
the ability of this species to induce the precipitation of carbon-
ates, phosphates and sulfates in a wide range of solid and liquid
media (Gonzlez-Munoz et al., 1993, 1996; Ben Omar et al., 1995,
1998; Ben Chekroun et al., 2004; Rodriguez-Navarro et al., 2007),
being the rst to describe struvite ((NH4 )MgPO4 6H2 O) formation
by myxobacteria (Ben Omar et al., 1994). Furthermore, they were
able to obtain the crystallization of struvite and calcite by dead cells
and cellular fractions of M. xanthus (Gonzlez-Munoz et al., 1996).
The latter is an abundant Gram-negative, non-pathogenic aerobic
soil bacterium which belongs to a peculiar microbial group whose
complex life cycle involves a remarkable process of morphogene-
sis and differentiation. In the tested culture media, no formation
of a dormant stage was observed. Additionally, when applied on
stone specimens, no fruiting bodies were observed upon drying.
As a result of this cell death, no uncontrolled bacterial growth was
observed.

Table 4
Overview of the different applications in which biocementation has been used in building materials.

Application Author Organism Metabolisma Solutionb

Biological mortar Calcite bioconcept Bacillus cereus ODAA Nutrical

Remediation of cracks in Ramachandran et al. Bacillus pasteurii HU SF

concrete De Belie et al. Bacillus sphaericus HU Growth and biocementation medium (DB)

Bacterial concrete Ramachandran et al. Bacillus pasteurii HU SF

Ghosh et al. Shewanella
Self-healing Jonkers et al. Bacillus pseudormus
Bacillus cohnii OAU Calcium lactate

a
ODAA: oxidative deamination of amino acids; OAU: organic acid utilization, HU: hydrolysis of urea.
b
Composition see Table 5; : not available.
124 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136

Table 5
Overview of the different media used for the bacterially induced precipitation of calcium carbonate.

Name Composition pHa References

Nutrients Conc.

Growth medium (CB) Peptone Orial (2000)

Yeast extract
KNO3
NaCl

Nutrical Growth medium + CaCl2 2H2 O Orial (2000)

Actical
Natamycine

B4 Calcium acetate 0.25 wt% 8 Boquet et al.

Yeast extract 0.4 wt%
Glucose 1 wt%

M-3 BactoCasitone 1 wt% 8 Rodriguez-Navarro et

Ca(CH2 COO)2 4H2 O 1 wt% al. (2003)
K2 CO3 (1/2)H2 O 0.2 wt%

M-3P M-3 + phosphate buffer 10 mM 8 Rodriguez-Navarro et al. (2003)

CC BactoCasitone 0.3 wt% 8 Jimenez-Lopez et al.

Ca(CH2 COO)2 4H2 O 0.4 wt% (2008)
CaCl2 2H2 O 0.1 wt%
NaHCO3 0.3 wt%
Yeast extract 0.1 wt%

SFb , c Nutrient broth 3 g L1 6 Stocks-Fischer et al.

Urea 20 g L1 (1999)
CaCl2 2H2 O 1.45.6 g L1
NH4 Cl 10 g L1
NaHCO3 2.12 g L1

Growth medium (DB) Yeast extract 20 g L1 7 Whifn (2004)

Urea 20 g L1

Biodeposition (DB) Urea 20 g L1 7 De Belie and De

CaCl2 2H2 O 50 g L1 Muynck (2008)

a
pH was adjusted with HCl or NaOH.
b
Dick et al. used 7.5 g L1 CaCl2 2H2 O.
c
De Muynck et al. used 25 g L1 CaCl2 2H2 O or 26 g L1 Ca(CH2 COO)2 4H2 O; : not available.

For the production of carbonate ions, the authors proposed a production was observed in stones submerged in M-3 and M-3P
medium containing a pancreatic digest of casein as the nitrogen media under static conditions. These ndings are in sharp contrast
source. Also, the effect of a phosphate buffer on the carbon- with the abundant production of EPS by M. xanthus described by
ate production was investigated. Biodeposition experiments were Sutherland and Thomson (1975). The latter could be attributed to
performed both under static and non-static conditions. Sterilized differences in culture medium composition and culture conditions,
calcarenite samples were submerged into a certain volume of M-3 as was suggested by the authors.
or M-3P (Table 5) which was subsequently inoculated (1%, v/v) with
M. xanthus. All experiments were performed at 28 C under sterile
3.1.4. Procedure according to the University of Ghent (Belgium)
conditions.
3.1.4.1. Euville limestone. Dick et al. (2006), a Ghent University
The phosphate buffer had a profound effect on the bacterial cell
research team, proposed the microbial hydrolysis of urea as a strat-
yield and the carbonate productivity, as well as on the supersat-
egy to obtain a restoring and protective calcite layer on degraded
uration preceding the nucleation of carbonate crystals. A greater
limestone. The hydrolysis of urea (Eqs. (1)(5)) presents several
bacterial production also led to a higher yield in calcite crystals.
advantages over the other carbonate generating pathways, as it
Furthermore, the buffering effect of the phosphate prevented rapid
can be easily controlled and it has the potential to produce high
local pH variations and, concomitantly the occurrence of a high
amounts of carbonate within a short period of time.
supersaturation. As a result, the deposited carbonate crystals were
The hydrolysis of urea is catalyzed by means of urease. As a con-
shown to be strongly adhered to the surface of the pores, since
sequence, urea is degraded to carbonate and ammonium, resulting
the newly formed carbonates were more resistant to mechani-
in an increase of the pH and carbonate concentration in the bacte-
cal stress in the form of sonication than the calcite crystals in
rial environment (Stocks-Fischer et al., 1999). One mole of urea is
the stone. The authors attributed this to their epitaxial growth
hydrolyzed intracellularly to one mole of ammonia and one mole of
on pre-existing calcite crystals and to the incorporation of organic
carbamate (Eq. (7)), which spontaneously hydrolyzes to one mole of
molecules. Apparently, the presence of organic molecules causes a
ammonia and carbonic acid (Eq. (8)). These products subsequently
misalignment of different domains within a single crystal.
equilibrate in water to form bicarbonate and two moles of ammo-
The authors observed carbonate cementation to a depth of sev-
nium and hydroxide ions (Eqs. (9) and (10)):
eral hundred micrometers (>500 m) without the occurrence of
any plugging or blocking of the pores. Plugging is mainly a conse- CO(NH2 )2 + H2 O H2 COOH + NH3 (7)
quence of extracellular polymeric substance (EPS) lm formation
(Tiano et al., 1999). In accordance with this, only limited EPS NH2 COOH + H2 O NH3 + H2 CO3 (8)
 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
2NH3 + 2H2 O 2NH4 + + 2OH (9)
2 week. All experiments were performed at 28 C under sterile con-
2OH + H2 CO3 CO3 + 2H2 O (10)
The global reaction can be written as follows:
CO(NH2 )2 + 2H2 O 2NH4 + + CO3 2 (11)
In the presence of calcium ions, this will result in calcium
carbonate precipitation (Eq. (12)), once a certain level of supersat-
uration is reached:
2 2+
CO3 + Ca CaCO3 (12)
As calcium ions are bound to the cell wall as a result of the neg-
ative charge of the latter, this can result in the formation of crystals
on the bacterial cell. In addition, precipitation can also occur in the
bulk phase of the liquid. A schematic overview, of the ureolytic car-
bonate precipitation occurring at the microbial cell wall is given in
Fig. 1.
The purpose of the study by Dick et al. (2006) was to identify
the microbial key factors which contribute to the performance
of the biodeposition treatment. For the evaluation of the per-
formance, the authors investigated the water absorption rate of
treated and untreated Euville limestone. The key factors had to
be easy in use and applicable for quick screening. The following
parameters have been examined: calcite deposition on limestone
cubes, pH increase, urea degrading capacity, EPS production,
biolm formation, -potential and deposition of dense crystal
layers. Of these parameters, the -potential proved to be the factor
with the greatest predictive power to screen microorganisms for
good limestone restoration, reecting the effect on the initial
water absorption. The -potential is a measure of the potential of
the electric layer at the surface of the cells, and is, therefore, an
important parameter in the adhesion and surface colonization by
bacteria. Due to the positive -potential of the limestone, bacteria
with a highly negative -potential will be more easily retained. The
second important key factor was the specic urea degradation rate.
Bacteria with a high initial specic urea degradation rate show a
high afnity for urea. This allows for a high substrate turnover for
a limited amount of cells.
The bacteria which had been selected for screening were iso-
lated from an ureolytic calcication reactor. This type of reactor
had been previously developed by the same research group for the
removal of calcium from calcium-rich wastewater (Hammes et al.,
2003). Calcifying sludge was obtained through the stimulation of
autochthonous ureolytic organisms, by means of repeated addi-
tions of urea. From this sludge, bacteria were isolated and screened
for their ability to precipitate calcite on agar plates containing
urea and calcium chloride. Although urease activity is widespread
among different groups of microorganisms, it was mainly microor-
ganisms closely related to the Bacillus sphaericus group which
were shown to proliferate and express the urease gene under the
given cultivation conditions (Hammes et al., 2003). The ability of
B. sphaericus to precipitate calcium carbonate had been previously
described by Cacchio et al. (2003). Among several strains isolated
from a limestone cave, a B. sphaericus strain was shown to rapidly
precipitate CaCO3 in B4 medium (Table 5), even at low tempera-
tures such as 4 C. Furthermore, calcifying bacteria were found not
to solubilise carbonates.
For the deposition of a layer of carbonate on the surface, Dick et
al. (2006) rst proposed the establishment of a biolm. For that pur-
pose, limestone cubes were immersed for 2 weeks in liquid medium
inoculated with 1% of the different strains. The surface was rewet-
ted each 2 h for 5 min by shaking. After the 2 weeks ended, calcium
chloride was added to the medium in order to precipitate calcium
carbonate. In the third week, the specimens were suspended in
fresh medium in order to have a second phase of biolm growth.
125
Finally, calcium chloride was added for a second time in the fourth
ditions.
From the screening procedure described above, 2 strains of B.
sphaericus were selected for further experiments. These strains
were shown to decrease the initial water absorption rate by approx-
imately 50%.
3.1.4.2. Cementitious materials. As an extension to the above
mentioned study, De Muynck et al. (2008a,b) investigated the
biodeposition with B. sphaericus as a surface treatment for cementi-
tious materials (Portland cement mortar) with different porosities.
In contrast with the treatment procedure described by Dick et al.
(2006), all experiments were performed at 28 C under non-sterile
conditions. Due to the alkaline pH of cementitious materials, the
contamination was expected to be very low. Furthermore, the treat-
ment procedure was altered: the mortar specimens were immersed
for 24 h in a 1 day old culture of B. sphaericus containing ca.
107 cells mL1 , after which they were transferred to fresh medium
containing a calcium source (Table 5). The specimens were removed
from the solution after 3 days.
The authors demonstrated that the biodeposition treatment
resulted in an increased resistance of mortar specimens towards
carbonation, chloride penetration and freezing and thawing, espe-
cially for more porous mortars with higher water to cement ratios
(w/c). Moreover, the biodeposition treatment showed a similar pro-
tection towards degradation processes as some of the conventional
surface treatments under investigation (silanes, siloxanes, silicates
and acrylates).
According to the authors, the biodeposition treatment on
cementitious materials should be regarded as a coating system. This
could be attributed to the fact that the carbonate precipitation was
mainly a surface phenomenon due to the limited penetration of
the bacteria in the porous matrix. From thin section analyses, the
authors observed that the majority of the surface was covered with
a layer of crystals with thicknesses within the range of 1040 m,
in which often larger crystals (up to 110 m) could be found. The
morphology of the crystals was observed to be highly dependent
on the medium composition. Much research on bacterial induced
precipitation has been conducted with calcium chloride as the cal-
cium source (Adolphe et al., 1990; Ferris and Stehmeier, 1992; Bang
et al., 2001). As chloride ions are detrimental to the reinforcement
in concrete structures, the use of calcium acetate as an alternative
calcium source was investigated. In the event calcium chloride was
used as the calcium source, rhombohedral carbonate crystals were
obtained. In the presence of calcium acetate, spherulitic crystals
were observed (Fig. 2). However, no differences in the protective
effect were observed between biodeposition treatments with a dif-
ferent calcium source. Therefore the authors concluded that from
these two salts, calcium acetate should be used for biodeposition
on cementitious materials.
3.1.5. Procedure according to the Biobrush consortium (United
Kingdom)
The basic aim of the Biobrush (BIOremediation for Building
Restoration of the Urban Stone Heritage) project was to integrate
the existing knowledge on the application of microorganisms for
the remediation of damaged stone into a conservation practice. Fur-
thermore, the goal was to sequentially link the processes of salt
removal to the processes of consolidation (May, 2005).
The use of microorganisms has been investigated for the removal
of nitrates, sulphates and organic matter present on the surface
of artworks (Gauri et al., 1992; Ranalli et al., 1999). In addition
to the elimination of black crusts, microbial sulphate removal also
results in the conversion of gypsum to calcite. As such, this method
126 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136

Fig. 1. Simplied representation of the events occurring during the ureolytic induced carbonate precipitation. Calcium ions in the solution are attracted to the bacterial
cell wall due to the negative charge of the latter. Upon addition of urea to the bacteria, dissolved inorganic carbon (DIC) and ammonium (AMM) are released in the micro-
environment of the bacteria (A). In the presence of calcium ions, this can result in a local supersaturation and hence heterogeneous precipitation of calcium carbonate on
the bacterial cell wall (B). After a while, the whole cell becomes encapsulated (C), limiting nutrient transfer, resulting in cell death. Image (D) shows the imprints of bacterial
cells involved in carbonate precipitation. A more in-depth representation can be found in Hammes and Verstraete (2002).

could be considered as a special kind of biodeposition treatment.
Heselmeyer et al. (1991) obtained the complete removal of gyp-
sum crusts from marble samples in laboratory conditions using
a strain of Desulfovibrio vulgaris. The procedure was further opti-
mized by Ranalli et al. (1997), who used sepiolite as a carrier
material for D. vulgaris and Desulfovibrio desulfuricans. The use of
sepiolite not only provided anaerobic conditions and humidity, but
also enabled the authors to shorten the treatment time. Additional
improvements were made by Cappitelli et al. (2006) who reported
on the superiority of Carbogel as a delivery system for the bacte-
ria. The use of Carbogel allowed for a higher retention of viable
bacteria and signicantly decreased the time needed for entrap-
ment of the microorganisms as compared to the use of sepiolite.
In addition, methods were presented to avoid the precipitation
of black iron sulde. The optimized methodology appeared to be
superior to chemical treatments involving the use of ethylenedi-
aminetetraacetic acid (EDTA), since no sodium sulphate was formed
(Cappitelli et al., 2007).
As Cappitelli et al. (2006, 2007) were among the members of
the Biobrush consortium, the use of Carbogel was subsequently
introduced into the eld of biodeposition. According to the consor-
tium, these delivery systems could be used to control the possible
harmful side effects of bacteria to stone. In addition, it was noticed
that the application of calcinogenic bacteria by spraying alone only
resulted in a limited change of the capillary water uptake of Port-
land stone. According to the consortium, the latter is attributable
to the limited colonization of the stone as a result of drying
out.
Within the framework of the Biobrush project regarding biode-
position, bacteria isolated from a stream in Somerset (UK), and
bacteria from culture collections that had been reported to have
calcifying activity, were screened for their ability to deposit calcite
in solid and liquid modied B4 media (Table 5). From the 10 isolates
that were retained and assessed for their ability to deposit calcite
on stone surfaces, Pseudomonas putida was chosen for further study
in eld trials. The latter has a low risk to humans and is sensitive
to most of the tested antibiotics and precipitated calcite in a wide
temperature range (May, 2005).
In these eld trials, bacteria were applied to the stone by
brushing. Subsequently, the bacteria were covered with moistened
Japanese paper, above which a 11.5 cm thick layer of Carbogel pre-
pared with modied B4 was applied. TrisHCl buffer was added to
the Carbogel to adjust the low pH of this carrier. Finally, the gel
was covered with a polyethylene sheet. As a result of this treat-
ment a decrease of the water absorption and open porosity by 1%
and 5%, respectively, was obtained. In order for this treatment to be
effective as a consolidant, a 2 weeks treatment was observed to be
necessary.
3.2. Application of organic matrix molecules; procedure
according to the Bioreinforce consortium (Italy)
Tiano et al. (1999) commented on the use of viable cells for the
formation of new minerals inside the stone. This was the result
of their experiments with Micrococcus spp. and Bacillus subtilis
strains on Pietra di Lecce bioclastic limestone. In these experi-
ments, bacteria were applied by brushing sterilized specimens that
were soaked with distilled water, reaching a nal concentration of
106 cells cm2 . Subsequently, the bacteria were fed daily by wetting
with a small amount of B4 medium (Table 5) for a period of 15 days.
Experiments were performed at 28 C under non-sterile conditions.
According to the authors, the decrease in water absorption after
a biodeposition treatment is for a large part attributable to the
physical obstruction of pores, rather than to the stable presence of
newly precipitated calcite. Furthermore, the authors commented
on some possible negative consequences, such as (1) the pres-
ence of products of new formation, due to the chemical reactions
between the stone minerals and some by-products originating from
the metabolism of viable heterotrophic bacteria and (2) the forma-
tion of stained patches, due to the growth of air-borne micro-fungi
related to the presence of organic nutrients necessary for bacterial
development.
To avoid some of these problems, the authors proposed the use
of natural and synthetic polypeptides to control the growth of cal-
cite crystals in the pores. The rst suggestions in this direction
already date from the time at which the Calcite Bioconcept treat-
ment was developed. Tiano et al. (1992) and Tiano (1995) proposed
the use of organic matrix macromolecules (OMM) extracted from
Mytilus californianus shells to induce the precipitation of calcium
carbonate within the pores of the stone. The organic matrix was
shown to produce a more relevant and durable carbonate precipi-
tation compared to the single use of calcium chloride or hydroxide.
This precipitation resulted in a slight decrease in porosity and water
absorption by capillarity (Tiano, 1995).
However, the practical application was hindered by the com-
plexity of the extraction procedure and the very low yield of usable
product (Tiano et al., 1999). Given this, the authors searched for
alternative starting materials by changing the nature of the organic
macromolecules involved. As these bio inducing macromolecules
(BIM) are usually rich in aspartic acid groups, Tiano et al. (2006)
proposed the alternative use of acid functionalized proteins such
as polyaspartic acid. Calcium and carbonate ions for crystal growth
were supplied by means of an ammonium carbonate and calcium
chloride solution or a saturated solution of bicarbonate, and were
supplemented in some cases by calcite nanoparticles, in order
to maintain a saturated carbonate solution in the pore over a
prolonged period. Proteins, calcium ions and nanoparticles were
 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
Fig. 2. Scanning electron micrographs of untreated (A) and biodeposition treated
(B and C) CEM I mortar specimens. Notice the differences in crystal morphology
obtained with different calcium sources: predominantly rhombohedral crystals in
the case of calcium chloride (A) and spherulitic crystals with calcium acetate (B).
introduced in the stone by means of spraying. According to the
authors, the method is most suitable for the use on marble statues
and objects of high aesthetic value where conservation is required
with the minimum change in the chemistry of the object. Field test
results, however, indicate that the effects of the BIM treatment were
rather small. The consolidating effect and the decrease in water
uptake were very low compared to the use of ethylsilicates, i.e.
15% over 12 mm depth compared to 30% up to 10 mm depth (as
measured with the drilling resistance measuring system) and 17%
compared to 60%, respectively (Tiano et al., 2006).
Elucidation of the genetic background of crystal formation in
bacteria has been proposed as an alternative way for the produc-
tion of inducing macromolecules. This was one of the objectives
of the European Bioreinforce (BIOmediated calcite precipitation
for monumental stones REINFORCEment) project. According to
127
the consortium, the genes responsible for crystal formation could
be cloned and transferred to an appropriate expression vector,
enabling the overproduction of the molecules inducing crystal for-
mation (http://www.ub.es/rpat/bioreinforce/bioreinforce.htm).
Initially, the consortium searched for bacterial cell structures or
molecules able to induce and control the carbonate precipitation
process. In this way, living cells would no longer be needed for the
biodeposition treatment. The authors demonstrated the ability of
autoclaved cells and cell fragments to induce calcite crystallization
in liquid media. Furthermore, they observed that dead cells from
active calcinogenic strains (B. cereus and B. subtilis) showed a much
higher and/or faster production of CaCO3 crystals than dead cells
from less active strains (Escherichia coli). This led the authors to con-
clude that calcinogenic strains might have a subcellular structure,
resistant to the methods used to kill cells (sonication, autoclav-
ing), able to promote CaCO3 precipitation. The crystals induced by
dead cells and Bacillus cell fragments (BCF) had a more complex
shape compared to the crystals induced by the control solution.
After application of the BCF to stone surfaces, a slight decrease in
the water absorption was noticed; the effect was more pronounced
on high porosity stones such as Tuffeau. Again, this method only
appeared to be useful for very delicate small calcareous stone
objects, rather than for a monumental facade (Mastromei et al.,
2008).
In addition, Barabesi et al. (2007) reported on a gene cluster
of B. subtilis involved in calcium carbonate precipitation. From UV
mutagenesis experiments, six mutants impaired in calcite crystal
formation were isolated. Sequence analysis of the mutated genes
revealed that in many cases their putative function was linked to
the fatty acid metabolism (Perito et al., 2000; Barabesi et al., 2007).
Further experiments are ongoing to investigate the link between
this kind of metabolism and calcium precipitation.
3.3. Application of an activator medium (Spain)
Concerning possible changes of the activity and composition of
the autochthonous microbiota upon addition of an inoculated cul-
ture media to ornamental stone, Rodriguez-Navarro et al. (2003)
pointed out the possibility of a synergetic contribution of the for-
mer to the overall biodeposition process. In fact, Urzi et al. (1999)
previously demonstrated that the majority of bacteria isolated from
building materials are able to induce carbonate precipitation under
laboratory conditions.
From the above, Jimenez-Lopez et al. (2007) proposed the
application of a culture medium, able to activate the calcinogenic
bacteria from the microbial community of the stone, as a more
user friendly method for the in situ consolidation of ornamental
stone. In addition to their work on decayed limestone fragments
(Jimenez-Lopez et al., 2007), this technique was recently proposed
for the treatment of new stones used for replacement purposes
(Jimenez-Lopez et al., 2008).
Upon comparison of the microbial community identied in non-
treated quarry stone and that identied in the non-treated decayed
stone, the authors observed for the latter the presence of microor-
ganisms related to the quarry from which the stone was extracted
and microorganisms related to the environment and contamination
to which the stone was exposed.
Some of the identied bacteria, Pseudomonas and Bacillus, had
already been reported to produce calcium carbonate both in
laboratory conditions and in nature. These chemoorganotrophic
organisms are able to grow in culture media containing amino acids
such as nutrient agar and tryptic soy agar (Jimenez-Lopez et al.,
2007).
From these ndings, the authors proposed the use of bacto-
casitone as a way to activate the calcinogenic bacteria from the
128 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
stone microbial community. Bacto-casitone is a source of carbon
and nitrogen, which favours alkalinisation due to the oxidative
deamination of amino acids. Furthermore, as no carbohydrates
were supplied, the probability of acid production, which is detri-
mental to the stone, was believed to be minimal. According to the
authors, this procedure is much easier than the use of bacterially
inoculated media, since difculties linked to the need of specialized
persons and equipment to work with microorganisms or tech-
nical requirements to ensure optimal growth conditions would
be avoided (Jimenez-Lopez et al., 2007). However, the fact that
microorganisms do not need to be introduced not only leads to a
decrease of the overall cost, but also ensures that this method is not
covered by the claims of the patent by Adolphe et al. (1990). Conse-
quently, Gonzlez-Munoz et al. (2008) applied for a new patent for
the protection and reinforcement of construction and ornamental
materials by means of the application of an activator medium able
to induce the formation of calcium carbonate.
Sonication test results demonstrated that the new cement cre-
ated by the microbial community and/or the combined action of
the microbial community and M. xanthus was more resistant than
that created by the sole action of either M. xanthus or the culture
media. Furthermore, the authors did not observe any changes in the
porosity of the stone. In addition, limited exopolysaccharide pro-
duction was observed (Jimenez-Lopez et al., 2007). The latter could
be somewhat expected as Rodriguez-Navarro et al. (2003) noted
that organic lms are unable to attach to the stones under shak-
ing conditions, as was the case in the work by Jimenez-Lopez et al.
(2007, 2008).
Due to the time required for the activation of the microbial com-
munity, Jimenez-Lopez et al. (2007) proposed the additional use of
M. xanthus for those restoration interventions in which time is an
issue and fast formation of calcium carbonate is required.
Very recently, the application of an activator medium has been
successfully applied in situ on calcarenite stone (Monasterio de San
Jeronimo and Hospital Real, Granada). Preliminary results show the
effectiveness of the treatment in terms of colour changes (negligi-
ble) and surface resistance by means of a peeling test (Personal
communication by Rodriguez-Navarro, 2008).
Although the formation of endospores was previously consid-
ered a potential drawback for the use of Bacillus in stone conserva-
tion (Rodriguez-Navarro et al., 2003), spore forming bacteria, able
to germinate upon the application of the culture media, contribute
in large extent to the precipitation of carbonate by the method
described by Jimenez-Lopez et al. (2007). Drawbacks to the use of
spore forming bacteria were related to the possible uncontrolled
growth of bacteria upon germination. However, Le Metayer-Levrel
et al. (1999) found that no increases in the microbial activity or
changes in the autochthonous microbiota were observed immedi-
ately or 4 years after the application of calcinogenic bacteria.
The long activation times of this technique inspired De Muynck
et al. (2008c) to develop an in situ enrichment of carbonate pro-
ducing bacteria. For that purpose, different media were developed
which allowed a rapid growth of carbonate producing strains upon
exposure to the surrounding air. Among the different metabolic
pathways under investigation, conditions optimal for carbonate
precipitation were most rapidly obtained upon the hydrolysis of
urea. Nevertheless, the rate of urea hydrolysis and biodeposition
remained low compared to pure cultures of B. sphaericus.
4. Biocementation
Besides the deposition of a layer of carbonate on the surface of
building materials, MICP has also been used for the generation of
binder-based materials. Initial developments were mainly situated
in the eld of geotechnical engineering, i.e. plugging, strengthen-
ing and improvement of soils (Ferris and Stehmeier, 1992; Zhong
and Islam, 1995; Nemati and Voordouw, 2003; Whifn et al., 2007).
Recent advances, however, indicate the potential use of this tech-
nique for the remediation of cracks in building materials, strength
improvement and self-healing of cementitious materials.
4.1. Biological mortar (France)
The knowledge and experiences obtained with the Calcite Bio-
concept treatment for limestone, have resulted in the development
of a biological mortar for the remediation of small cavities on
limestone surfaces. The aim of the biological mortar was to avoid
some of the problems related to chemical and physical incom-
patibilities of commonly used repair mortars with the underlying
material, especially in the case of brittle materials (Castanier,
1995; Le Metayer-Levrel et al., 1999; Orial et al., 2002, Personal
communication by Loubire (Chief of Calcite Bioconcept), 2008;
http://www.calcitebioconcept.com/).
In general, a mortar refers to a workable paste consisting of a
binder, aggregates and water to bind building materials together
and to ll the gaps between them. In particular, a biological mortar
refers to a mixture of bacteria, nely ground limestone and a nutri-
tional medium containing a calcium salt. The term biological refers
to the microbial origin of the binder, i.e. microbiologically produced
calcium carbonate. Similar to lime mortars, the produced calcium
carbonate cements the aggregates together. Cementation occurs as
a result of the nucleation and growth of carbonate crystals at the
surface of the aggregates, especially at the contact areas between
them.
The optimization of the mortar composition encompassed the
dosage and composition of the three main components, i.e. lime-
stone powder, nutrients and bacterial paste. The mortars were
evaluated based upon their appearance (cohesion and colour),
the presence of micro-cracks and the resistance towards fractur-
ing. Concerning the medium composition, some adjustments were
made to the initial method, as was used for biodeposition purposes.
The amount of nutrient solution introduced during the fabrication
of the mortar was sufcient to support bacterial activity. Repeated
external applications of the nutrient solution were unable to com-
pletely wet the mortar. Furthermore, they resulted in discolorations
at the surface and were, therefore, rapidly omitted. Additionally, the
biological mortars necessitated the use of larger amounts of bacte-
ria and as a result the composition of the nutrient medium had to
be altered.
Based on the different evaluation parameters, best results
were obtained with one part of bacterial paste (containing
109 cells mL1 ), one part of nutritional medium and two parts
of limestone powder. Limestone powder with a granulometry
between 40 and 160 m was observed to be the most suited. The
technique has already been successfully tested on a small scale on
sculptures of the Amiens Cathedral and on a portal of the church
of Argenton-Chteau (France). Visual observations 2 years after
the treatment indicated a satisfactory appearance of the repaired
zones. (Le Metayer-Levrel et al., 1999; Orial et al., 2002).
4.2. Remediation of cracks in concrete (USA, Belgium)
In the recovery of heavy oil from oil elds, where water is more
readily removed than the viscous oil, the ability to selectively plug
porous rock to focus pumping energy in oil rich zones is highly
desirable (Hart et al., 1960; Lappin-Scott et al., 1988). Because of
the cost and unsatisfactory performance of some of the chemi-
cally cross-linked polymers, many workers suggested that insoluble
biopolymers and biomass generated by injection of indigenous
 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
microorganisms can be used to selectively plug off zones of high
water permeability (Lappin-Scott et al., 1988; Jack et al., 1991;
Gollapudi et al., 1995). In addition, the use of a microbial mineral
plugging system based on the precipitation of carbonates was sug-
gested (Ferris and Stehmeier, 1992; Zhong and Islam, 1995). While
initial research on MICP in sand columns was mainly focused on
the decrease of porosity and permeability as a result of the physi-
cal presence of the newly formed carbonates (Ferris and Stehmeier,
1992), recent investigations focus on the improvement of strength
as a result of the cementation of sand particles (Whifn, 2004;
Kucharski et al., 2006). The latter is due to the particle binding
properties of the microbially produced carbonates.
The hydrolysis of urea was selected as a very suitable pathway
for the production of carbonate ions due to its ability to alkalinize
the environment. Furthermore, urea is an important organic nitro-
gen carrier in natural environments and is commonly used as an
agricultural fertilizer (Nielsen et al., 1998). Moreover, the ability to
hydrolyze urea is widely distributed among indigenous bacteria in
soils and groundwater systems (Mobley and Hausinger, 1989; Fujita
et al., 2000). Urea-utilizing bacteria such as Sporosarcina pasteurii
and Sporosarcina ureae are commonly isolated from soil, water,
sewage and incrustations on urinals.
The participation of S. pasteurii in sand consolidation has been
demonstrated by Kantzas et al. (1992). Gollapudi et al. (1995) fur-
ther investigated the use of S. pasteurii for the plugging of sand
columns. Although the bacteria were mixed with the sand slurry,
consolidation mainly occurred near the surface. Stocks-Fischer et
al. (1999) showed that microorganisms directly participated in the
calcite precipitation by providing a nucleation site and by creating
an alkaline environment which favoured the precipitation of calcite.
Zhong and Islam (1995) used the consolidation of sand mixtures for
the remediation of cracks in granite. Cracks in granite were packed
with a mixture of bacteria, nutrients and a ller material. Among
the different materials that were mixed with S. pasteurii, the silica
fume (10%) and sand (90%) mixture lead to the highest compressive
strength and lowest permeability.
As a further extension to this research, Ramachandran et al.
(2001) investigated the microbiological remediation of cracks in
concrete. The authors proposed MICP as an effective way to seal
cracks. The appearance of cracks and ssures is an inevitable phe-
nomenon during the ageing process of concrete structures upon
exposure to weather changes. If left untreated, cracks tend to
expand further and eventually lead to costly repair. Specimens with
cracks lled with bacteria, nutrients and sand demonstrated a sig-
nicant increase in compressive strength and stiffness values when
compared with those without cells. The presence of calcite was,
however, limited to the surface areas of the crack. The authors
attributed this to the fact that S. pasteurii grows more actively in
the presence of oxygen. Still, the highly alkaline pH (1213) of con-
crete was a major hindering factor to the growth of the moderate
alkaliphile S. pasteurii, whose growth optimum is around a pH of
9. In order to protect the cells from the high pH, Day et al. (2003)
investigated the effect of different ller materials on the effective-
ness of the crack remediation. Beams treated with bacteria and
polyurethane showed a higher improvement in stiffness compared
to ller materials such as lime, silica, y ash and sand. According
to the authors, the porous nature of the polyurethane minimizes
transfer limitations to substrates and supports the growth of bac-
teria more efciently than other lling materials, enabling an
accumulation of calcite in deeper areas of the crack. No differ-
ences could be observed between the overall performances of free
or polyurethane immobilized cells in the precipitation of carbon-
ate (Bang et al., 2001). In addition to this research, Bachmeier et al.
(2002) investigated the precipitation of calcium carbonate with the
urease enzyme immobilized on polyurethane. The immobilization
129
was shown to protect the enzyme from environmental changes,
as the immobilized urease retained higher enzymatic activities at
high temperatures and in the presence of high concentrations of
pronase. While the rate of calcite precipitation of the immobilized
enzyme was slower compared to that of the free enzyme, lower
concentrations of the former where needed to obtain the theoreti-
cal maximum precipitation in a period of 24 h. Although the authors
mentioned ongoing research on the use of immobilized urease in
the remediation of surface cracks in concrete, to our knowledge no
published results are available at the moment.
As an extension to their research on biodeposition on cementi-
tious materials, De Belie and De Muynck (2008) further investigated
the use of microbially induced carbonate precipitation for the repair
of cracks in concrete. For the protection of B. sphaericus from the
alkaline pH conditions, bacteria were immobilized in a silica sol.
Upon the addition of a salt, a bioceramic material (biocer) was
formed, which was able to bridge the crack. Subsequent addition
of a urea and calcium chloride solution resulted in the formation of
carbonate crystals inside the pores of the biocer and concomitantly
sealing of the crack. As a result, a decrease of the water permeabil-
ity, similar to that obtained with traditional epoxy injections, was
observed.
4.3. Bacterial concrete (USA, India)
Besides external application of bacteria in the case of reme-
diation of cracks, microorganisms have also been applied in the
concrete mixture. Until now, research has mainly focused on the
consequences of this addition on the material properties of con-
crete, i.e. strength and durability. Both properties depend on the
microstructure of the concrete. However, the effects of the presence
of the microorganisms and/or the microbially induced carbonates
on the microstructure still need to be elucidated, especially the
interaction between the biomass and the cement matrix.
Ramachandran et al. (2001) investigated the use of microbio-
logically induced mineral precipitation for the improvement of the
compressive strength of Portland cement mortar cubes. This study
identied the effect of the buffer solution and type and amount of
microorganisms, i.e. S. pasteurii and P. aeruginosa, used. Further-
more, in order to study the effect of the biomass, the inuence
of both living and dead cells was investigated. Before addition to
the mortar mixture, bacteria were centrifuged and washed twice.
The nal pellets were then suspended in either saline or phos-
phate buffer, which was subsequently added to the mixture. After
demolding, the mortar specimens were stored in a solution con-
taining urea and calcium chloride for 7 days. Subsequently, the
specimens were cured in air until the measurement of the com-
pressive strength.
At lower concentrations, the presence of S. pasteurii was shown
to increase the compressive strength of mortar cubes. While the 28-
day compressive strength of the control cubes amounted to about
55 1 MPa, specimens treated with 103 cells cm3 had a compres-
sive strength of about 65 1 MPa. The contribution of P. aeruginosa
to the strength was found to be insignicant. From the X-ray diffrac-
tion (XRD) analysis, no signicant increased amounts of calcite
could be found in mortar specimens treated with bacteria. This
could be attributed to the inhibition of the microorganisms by the
high pH and the lack of oxygen inside the mortar mixture. The over-
all increase of strength, therefore, resulted from the presence of an
adequate amount of organic substances in the matrix due to the
microbial biomass. However, an increase of the biomass, as dead
cells in particular, resulted in a decreased strength. According to the
authors, this could be attributed to the disintegration of the organic
matter with time, making the matrix more porous (Ramachandran
et al., 2001).
130 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
Fig. 3. Schematic drawing of conventional concrete (AC) versus bacteria-based self-healing concrete (DF). Crack ingress chemicals degrade the material matrix and
accelerate corrosion of the reinforcement (AC). Incorporated bacteria-based healing agent activated by ingress water seals and prevents further cracking (DF) (courtesy of
Jonkers).
Table 6
Approximate costs of surface treatments.
Treatment Price D /unit Dosage unit/m2
Calcite bioconcept
Growth medium 1 D g1 23 g
Nutrical 0.2 D g1 816 g(5)
Water repellents 2.54 D L1 0.51 L
Consolidants 1015 D L1 >1 L
a
Unaltered stone.
b
Sculptured and degraded stone.
Ramakrishnan et al. (2001) investigated the effect of this tech-
nique on the durability of concrete. The presence of bacteria was
observed to increase the resistance of concrete towards alkali, sul-
fate, freeze thaw attack and drying shrinkage; the effect being more
pronounced with increasing concentrations of bacterial cells. The
authors attributed this to the presence of a calcite layer on the sur-
face, as conrmed by XRD analysis, lowering the permeability of
the specimens. The best results were obtained with the phosphate
buffer.
Ghosh et al. (2005) demonstrated the positive effect of the
addition of Shewanella on the compressive strength of mortar spec-
imens. Contrary to the aforementioned research, these authors did
not intend mineral precipitation, as these specimens were cured in
air and not in a nutrient containing medium. An increase of 25%
of the 28 days compressive strength was obtained for a cell con-
centration of about 105 cells mL1 and a water to cement ratio of
0.4. For these samples, the presence of a brous material inside the
pores could be noticed. As a result, a modication of the pore size
distribution was observed. The positive effect of the addition of She-
wanella improved with increasing curing times. For a concentration
of 105 cells mL1 , an increase of the compressive strength of 17% and
25% was observed after 7 and 28 days, respectively. However, no
increase of the compressive strength was observed with additions
of Escherichia coli to the mortar mixture. This led the authors to sug-
gest that the choice of the microorganism plays an important role
in the improvement of the compressive strength. More specically,
the production of EPS by the bacteria seemed to be of importance.
4.4. Self-healing concrete (the Netherlands)
As an extension to the aforementioned research, Jonkers (2007)
and Jonkers and Schlangen (2007) investigated the use of bac-
teria as self-healing agents for the autonomous remediation of
cracks in concrete (Fig. 3). In contrast with previous studies, such
an approach necessitated the presence of all the reaction com-
Product D /m2 No. of applic. Prod. + applic. D /m2
2328a
3540b
23 1
24(5) 5
1.254 1 1525
>10 1 >30
ponents, microorganisms and nutrients, in the matrix to ensure
minimal externally needed triggers. Therefore, the authors inves-
tigated the compatibility of different organic compounds with the
cement matrix. Moreover, suitable bacteria should be able to sur-
vive concrete incorporation for prolonged periods of time. For that
purpose, alkali-resistant spore forming bacteria related to the genus
Bacillus, Bacillus pseudormus DSM 8715 and Bacillus cohnii DSM
6307, were selected. In addition, the bacteria were added as spores,
as these are known for their ability to endure extreme mechanical
and chemical stress. On top of this, the authors decided to choose
a pathway different from the hydrolysis of urea for the production
of carbonate ions. In this way, possible negative effects of the pro-
duced ammonia on the reinforcement corrosion and degradation
of the concrete matrix (when further oxidized by bacteria to yield
nitric acid) could be avoided. Among the components selected, cal-
cium lactate did not substantially affect the compressive strength
values. Furthermore, the addition of a high number of bacterial
spores (108 cm3 ) resulted in a decrease of strength of less than 10%.
For the evaluation of the mineral producing capacity, healing
agent-incorporated specimens and control specimens were broken
to pieces after 7 or 28 days curing, immersed in tap water for 8 days
and subsequently analyzed by ESEM. While a massive production of
larger-sized CaCO3 precipitates was observed for the 7 days cured
specimens, no differences could be observed between the healing
agent incorporated specimens and control specimens after 28 days.
The authors related this to a decrease of the viability of the spores
upon incorporation in the cement matrix. The decrease in viabil-
ity appears to be linked with a decrease of the matrix pores size
diameter (Jonkers and Schlangen, 2007; Jonkers et al., 2008).
5. Cost evaluation
5.1. Biodeposition
Table 6 gives an overview of the costs related to the application
of surface treatments to building materials (Personal communi-
 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
cation by an employee of the Belgian company FTB remmers,
2008; http://www.ftbremmers.com/). The costs of the biodeposi-
tion treatment are attributable both to the price of the product
and the number of applications required. The theoretic price of the
product depends on the price of the microorganisms and the price
of the nutrients. One kilogram of lyophilized bacteria (1011 CFU g1 )
costs around 1100 D kg1 . As bacteria are applied in a concentra-
tion of 23 g m2 this results in a cost of 2.23.3 D m2 . The costs
of the nutrients are estimated to be about 180 D kg1 . Depending
on the porosity of the stone, the dosage ranges between 0.04 and
0.08 kg m2 , bringing the cost of the nutrients to 715 D m2 . This
brings the total product cost around 1017 D m2 . In addition, the
total price of the treatment amounts to about 2328 D m2 . The
latter includes the costs of application and the added value of the
product. In case heavily degraded surfaces need to be treated, the
cost of the treatment will be between 35 and 40 D m2 (Personal
communication by Loubire, 2008).
In case a carrier material is applied, such as proposed by
the Biobrush consortium (May, 2005), the biodeposition treat-
ment becomes even more costly. The price of high density
polyethylene sheets, as used on external wall assemblies, amounts
to about 2.3 D m2 (http://order.americanmicroinc.com/cgi-
bin/americanmicroinc/VB10X25X6.html). For the formation of
a gel, 10 g L1 of Carbogel (27.6 D kg1 , Carbogel, Brazil, 2008;
http://www.carbogel.com.br/) is required. Considering a proposed
thickness of the gel of about 1 cm, this will bring the cost per m2
to about 2.8 D m2 . The Japanese paper brings about an extra cost
of about 12.3 D m2 (http://japanesepaperplace.com/retail/retail-
products/conservation-papers.htm). As a result of the carrier
material, however, less applications of moisture (and hence nutri-
ents) will be necessary. The resulting decrease in cost will be
marginal compared to the costs of the carrier. Additionally, the
disposal of the carrier material will also present an extra cost.
As mentioned before, the method proposed by Jimenez-Lopez
et al. (2007) could offer an economical advantage over the Cal-
cite Bioconcept treatment, as no bacteria need to be added during
the treatment. However, the fact that the microbiota of the stone
needs to be rst activated, might necessitate an increased num-
ber of applications of nutrients, and hence, loss of the economical
advantage.
Due to the price of its constituents, the biodeposition treatment
will never be able to compete with some of the traditional surface
treatments on a pure economical basis. The focus of this kind of
treatment should, therefore, be on the added value compared to
other treatments.
The biodeposition treatment presents an ecological, envi-
ronmentally friendly alternative over the other treatments.
Furthermore, the application of a layer of calcium carbonate to
limestone ts in the current restoration concept of compatibility.
Some of the traditional surface treatments, typically organic resins,
have shown long-term incompatibilities with the stone. The latter
resulted in a much more intense damage to the stone than would
have occurred without restoration, necessitating replacement and
costly repair.
In addition, the biological deposited crystals show some unique
properties. Due to their growth on pre-existing calcite crystals and
the incorporation of organic molecules, these crystals are strongly
attached to the surface, exerting a consolidating effect (Rodriguez-
Navarro et al., 2003). Furthermore, by adding pigments to the
medium, it is also possible to create a surcial patina, allowing the
concealment of new replacement stone (Le Metayer-Levrel et al.,
1999).
Until now, practical applications of the biodeposition treatment
have been mainly limited to France. The treatment has been applied
on several historic monuments across the country, including a part
131
of the Notre Dame de Paris. The treatment has also been applied on
the facade of warehouses (ex. Galeries Lafayette, Paris), hotels and
apartments in and around Paris. As a consequence, the company
Calcite Bioconcept has an estimated annual turnover of the order of
100,000150,000 D (Personal communication by Loubire, 2008).
5.2. Biocementation
In contrast with the biodeposition treatment, the added value
of the biocementation treatment in building materials is less pro-
nounced. As a consequence, more difculties in competing with
traditional treatments can be expected.
In the case of the biological mortar, a similar performance could
be obtained with traditional lime mortars, also being compatible
with limestone. As mortars consist of a mixture of sand, water and
a binder, it is the latter which will make up the cost when com-
paring the two kinds of mortars. For non-hydraulic lime mortars,
the cost of the binder amounts to about 0.6 D kg1 (Carmeuse, Bel-
gium, 2008). For biological mortars, however, the binder consists
of a mixture of nutrients (about 180 D kg1 ) and a bacterial paste
(1100 D kg1 ) (Personal communication by Loubire, 2008).
Especially in the case of crack repair of cementitious materials,
at the moment, little or no added value is obtained. Because of the
fact that (organic) carrier materials are needed to protect the bac-
teria from the alkaline environment, the ecological aspect of the
treatment has been largely reduced. Furthermore, the method cur-
rently seems unfeasible to be readily applicable in practice due to
the large amount of specialist work needed.
Only in the case of self-healing building materials can a sig-
nicant added value be expected. The latter is, however, largely
attributable to the concept of self-healing materials, decreasing the
needs for manual inspection and repair. The research is still in its
infancy, and it will be largely questionable whether bacteria will be
able to remain viable for a prolonged time and upon activation be
able to seal the cracks.
6. Considerations
In our opinion, the feasibility of the biodeposition treatment in
practice largely depends on the time required for carbonate produc-
tion, and hence, precipitation to occur. The latter has also important
consequences on the economical aspect of the treatment.
In the case of the application of calcinogenic bacteria, longer
times required for precipitation to occur necessitate longer periods
during which the building material has to remain wet. This is due to
the fact that microorganisms require a minimum amount of water
to remain active. With increasing times for precipitation, increasing
amounts of EPS production, biolm formation and hence plugging
can be expected. In order to ensure the presence of a sufcient
amount of water, multiple applications of nutrients over several
days (Le Metayer-Levrel et al., 1999) or the application of a carrier
material (May, 2005) have been proposed. However, both measures
have a signicant inuence on the total cost of the treatment, as
can be seen from the previous paragraphs. Increasing the number
of applications of nutrients increases the cost of the treatment due
to the extra man hours needed, while the use of a carrier material
has a major inuence on the product cost.
From the different microbial metabolic pathways proposed for
biodeposition, the hydrolysis of urea results, without any doubt in
the fastest production of carbonate ions and hence precipitation
of calcium carbonate. This is due to the fact that the hydrolysis
of urea is a very rapid process and depends on only one enzyme.
As a consequence, no additional nutrients are necessary for the
long-term maintenance of the bacterial activity. And additionally,
132 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
the time of wetting and thus the number of nutrient applications
could be drastically decreased. Consequently, the outgrowth of
other microorganisms (e.g. fungi and heterotrophic bacteria) will
be highly unlikely. This makes the hydrolysis of urea a very feasible
pathway for applications in situ. From the above, it is clear that the
hydrolysis of urea could present some economical advantages over
the other pathways.
In addition to the feasibility, which is governed by the time
required for production of carbonates to occur (see previous para-
graphs), the efciency of the biodeposition treatment has been
observed to depend on the speed of precipitation.
Rodriguez-Navarro et al. (2003) reported on the importance
of the type and structure of the precipitated CaCO3 polymorphs
(vaterite or calcite) on the efciency of the biodeposition treatment.
The presence of well developed rhombohedral calcite crystals
resulted in a more pronounced consolidating effect compared to
the presence of tiny acicular vaterite crystals.
Differences in size and morphology of the crystals can be
attributed to differences in the saturation state of a system pre-
ceding nucleation, with large rhombohedral calcite crystals being
formed at relatively low supersaturation and vaterite crystals being
produced under highly supersaturated conditions. From the above,
the authors concluded that fast precipitation could result in a lower
efciency of the biodeposition treatment.
According to Rodriguez-Navarro et al. (2003), the presence of
a phosphate buffer could explain for the occurrence of rhombo-
hedral crystals. They attributed this to the buffering effect of the
phosphate, preventing rapid local pH variations, and hence, rapid
changes in the saturation state of the system. However, from the
papers by Jimenez-Lopez et al. (2007, 2008), it appears that the
phosphate buffer is not the only compositional difference between
the M-3 and M-3P media (composition see Table 5). From the
graphs indicating the removal of calcium ions from solution, it can
be clearly observed that the initial concentration of calcium ions
was much higher in the M-3 medium (50 mM Ca2+ ) compared
to that of the M-3P medium (35 mM Ca2+ ). The latter could be
attributed to the precipitation of calcium phosphate which was
removed before the start of the experiment (Personal communi-
cation by Jimenez-Lopez et al., 2008). As a result, the M-3 medium
showed initially a higher saturation state compared to the M-3P
medium.
In spite of the high speed of carbonate formation and calcium
dosages used, De Muynck et al. (2009) obtained an excellent water-
proong and consolidating effect with an ureolytic biodeposition
treatment on Euville limestone. From SEM examinations, the pres-
ence of rhombohedral crystals could be clearly observed.
Besides the hydrolysis of urea, most MICP treatments rely on
the production of ammonia for the alkalinization of the culture
medium. Because of the fact that atmospheric ammonia is being
recognized as a pollutant, the in situ use of such treatments might
raise some issues of environmental concern. Atmospheric ammo-
nia is known to contribute to several environmental problems,
including direct toxic effects on vegetation, atmospheric nitro-
gen deposition, leading to the eutrophication and acidication of
sensitive ecosystems, and to the formation of secondary partic-
ulate matter in the atmosphere, with effects on human health,
atmospheric visibility and global radiative balance (Sutton et al.,
2008).
However, when the concentration of ammonia generating com-
pounds does not exceed the concentration of the calcium salt, it is
possible to decrease the emission of ammonia to a great extent. De
Muynck et al. (2009) observed in their biodeposition experiments
that the pH of the solution remained about 7. At these pH values,
ammonium will be the predominating compound. The neutral pH
could be attributed to the fact that the precipitation of calcium
carbonate, resulting in a decrease of the pH, counteracts the pH
increase as a result of the release of ammonia.
Nonetheless, even in the case of the ureolytic biodeposition
treatment the production of ammonium will be rather low com-
pared to conventional sources of nitrogen pollution, i.e. agriculture
and domestic waste water. The treatment of 1 m2 of building mate-
rial with 1 L of a biodeposition medium containing 10 g L1 urea,
results in the production of 4.7 g N. For comparison, from waste
water treatment plants it can be calculated that one person pro-
duces between 6 and 16 g of N per day (DeCuyper and Loutz, 1992).
The presence of ammonium might also present some risks to
the stone itself. First of all, the presence of an ammonium salt might
present some risks related to salt damage. Depending on the type
of calcium salt used, ammonium acetate or ammonium chloride
will be present in the stone after treatment. To our knowledge, no
reports are available on the effect of these salts on stone. Therefore,
future investigations should investigate the retention of these salts
in the stone.
Secondly, ammonium can be converted to nitric acid by the
activity of nitrifying bacteria, resulting in damage to the stone.
However, Mansch and Bock (1998) observed that the initial col-
onization of natural stone by nitrifying bacteria takes several years.
In addition, the extent of colonization is mainly governed by the pH
of the pore solution, with a pH between 7 and 9 being optimal for
growth. As the initial pH of the biodeposition liquid is around 9.3,
the activity of the nitrifying bacteria will be suppressed. Moreover,
the applied chemoorganotrophic carbonate producing bacteria will
outcompete the nitrifying bacteria for oxygen during the precipita-
tion process. As a result of the precipitation, however, the pH will
drop to a value of about seven. Therefore, in order to avoid nitrica-
tion in the long-term, the presence of large amounts of ammonium
salts should be avoided. From long-term observations on the ef-
ciency of the Calcite Bioconcept treatment, however, no damages
to the stone have been reported.
If higher concentrations of ammonium should be produced, as
might be the case for the hydrolysis of urea, the use of a paste
might offer an attractive solution. The latter is one of the most
commonly applied methods for the removal of salts from build-
ing materials (Wooltt and Abrey, 2008; Carretero et al., 2006).
Upon wet application, the paste facilitates the dissolution of salts
within stones and migration of ions to the outside, where they
recrystallize and are retained. Once dry, the paste can be easily
removed. Different types of pastes or combinations thereof have
been applied for such purposes: paper pulp, clay materials (sepio-
lite, bentonite) and cellulose derivatives. As a result of their unique
properties, many of these materials have also been used for the
immobilization of microorganisms in a variety of elds. A com-
bination of these two applications has already been applied for
the removal of black crusts on stone artworks (Ranalli et al., 1997;
Cappitelli et al., 2006). While Carbogel was observed to remove
about 42% of the calcium ions from a black crust, the combina-
tion of the former with sulphate reducing bacteria led to a total
removal efciency of about 95%. Besides removing the produced
ammonium, the use of a paste will also protect the bacteria from
drying out, enhancing the overall biodeposition treatment, as was
observed by the Biobrush consortium (May, 2005). As seen before,
however, the use of a paste results in a higher cost for the treat-
ment.
In their search for alternative approaches towards the Calcite
Bioconcept method, most researchers have focused on the use of
different organisms or metabolic pathways. Little attention, how-
ever, has been paid to the inuence of the dosage (g m2 ) or
concentration (g L1 ) of the calcium salt and the nutrients (i.e.
carbonate precursor components such as urea or amino acids)
on the global effectiveness of the treatment. In many cases, the
 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
authors just applied the medium which had been used to illustrate
the carbonate precipitation potential of the strain. An overview of
the different concentrations of calcium salts used can be seen in
Table 5.
The importance of the calcium dosage on the overall effective-
ness of the biodeposition treatment can be easily demonstrated
by the following example. In the Calcite Bioconcept method, the
total calcium dosage amounts to about 5.5 g m2 calcium chlo-
ride (MW 147 g mol1 ) or 1.5 g m2 calcium. Theoretically, this will
result in an overall precipitation of about 3.74 g calcium carbonate
(MW 100 g mol1 ) per square meter of stone surface. Assuming a
density of calcium carbonate of 2.71 g cm3 and a homogenous pre-
cipitation over 1 m2 of a non-porous stone, this corresponds with
a layer of calcium carbonate of about 1.38 m in thickness. In the
case of a porous stone, a smaller thickness is expected due to the
high surface area of the pores. In practice, however, bacteria will
be mainly retained in the pores of the stone, especially those pores
with a diameter larger than 1 m. Consequently, precipitation will
mainly occur around large pores, and layer thicknesses greater than
1 m can be observed. This is in agreement with the ndings of Le
Metayer-Levrel et al. (1999) and Orial (2000), who observed layer
thicknesses of about 23 m and 45 m, respectively.
Let us now consider the methodology as proposed by Rodriguez-
Navarro et al. (2003). In one of their experiments, the authors
submerged limestone prisms of 2.5 by 4.5 by 0.5 cm in an Erlen-
meyer ask containing 100 mL of M-3 solution. As such, the
theoretical calcium dosage amounts to about 339 g m2 calcium
acetate (MW 230 g mol1 ) or 59 g m2 calcium, corresponding with
an overall precipitation of about 147 g calcium carbonate per square
meter. This will in theory result in a layer of calcium carbonate with
a thickness of about 54 m on the surface of the limestone prisms.
Although the authors did not report on the thickness of the carbon-
ate layer, cementation was found up to depths larger than 500 m
(Rodriguez-Navarro et al., 2003).
As a result of the more pronounced precipitation of calcium
carbonate in the case of the methodology proposed by Rodriguez-
Navarro et al. (2003), a larger consolidating effect can be observed
compared to the Calcite Bioconcept treatment. In their work on con-
solidation of sand columns by means of biocementation, Whifn
et al. (2007) observed that a minimum amount of carbonate
precipitation per m3 of sand was required in order to obtain a sig-
nicant consolidating effect. DeJong et al. (2006) observed that
the cementing effect occurred as a result of the precipitated cal-
cite forming bonds at the particle-particle contacts of sand grains.
With increasing concentrations of precipitated carbonate, increas-
ing bond formation and hence consolidation can be obtained.
Therefore, increasing amounts of carbonate precipitates could
result in an increased protective effect of the biodeposition treat-
ment. This was indeed observed by De Muynck et al. (2009), who
noticed an increased waterproong with increasing numbers of
treatments or increasing the concentration of the crystal precur-
sors in one treatment. The latter is also known to play a role in the
speed, and hence, the type of crystals that are formed, affecting the
global effectiveness of a treatment (Whifn et al., 2007).
An increase in the dosage of the calcium salt could, however, lead
to an accumulation of salts in the stone, which could depending on
the anion give rise to eforescence or damage related to crystal-
lization. As mentioned earlier, the use of a paste could prevent this
from happening Regarding the Calcite Bioconcept treatment, sev-
eral tests did not reveal any problems related to salt damage. The
chlorides are rapidly washed away as a result of raining (Personal
communication, Loubire, 2008).
Besides the dosage of the calcium salt used, the type of stone
will also have a major impact on the global performance of the
treatment. The porosity, and more specically the pore size dis-
133
tribution could be considered as one of the most determining
factors. Samonin and Elikova (2004) reported that for a maxi-
mum adsorption of microbial cells, the adsorbent pores must be
25 times larger than the cells. Therefore, the amount of bacte-
ria retained in high macroporosity stones will be higher than in
high microporosity stones. As a consequence, carbonate precip-
itation can occur at higher depths in macroporous stone. From
SEM analyses, precipitation has been observed at depths of about
100 m for the Calcite Bioconcept treatment (Personal communi-
cation by Loubire, 2008). As mentioned earlier, Rodriguez-Navarro
et al. (2003) observed precipitation at depths greater than 500 m
in a bioclastic calcarenite. De Muynck et al. (2008b) observed an
increased amount of biomass adsorption in mortar specimens with
increasing water to cement ratio (w/c). The authors attributed this
to the increasing amount of pores with a diameter larger than 1 m
in specimens with increasing w/c. Since the amount of capillary
pores between 2 and 10 m is rather limited in cementitious mate-
rials, the authors concluded that for these types of materials, the
biodeposition treatment is mainly a surface phenomenon. This was
also observed from thin sections, where a layer of crystals within
the range of 1040 m on the surface was found, correspond-
ing with the theoretical thickness calculated from the calcium
dosage.
From Table 1 it is clear that the differences between the various
methodologies are not limited to the mediator used for precipita-
tion. In addition, different research groups used different dosages
of calcium salts and different application procedures on different
types of stone. Besides the different metabolic pathways and bacte-
ria proposed, the difference in inoculum size could also account for
the differences in time required for precipitation to occur. Further-
more, many experiments were performed under sterile conditions.
However, for applications in situ growth and activity are required
under non-sterile conditions. This could potentially inuence on
the microbial activity. From the above mentioned it should be clear
that this will hamper any quantitative comparison between the dif-
ferent treatments. Additionally, such a comparison is even more
difcult due to the fact that different authors used different eval-
uation parameters and procedures (Table 1). Some authors mainly
focused on the waterproong effect (Dick et al., 2006), while others
mainly investigated the strengthening effect (Rodriguez-Navarro
et al., 2003; Jimenez-Lopez et al., 2007). In addition to these two
effects, Tiano et al. (1999) further proposed the evaluation of the
visual aspect before and after treatment by means of colorimetric
analysis.
Therefore, the next step in research regarding the application of
calcinogenic bacteria should be a qualitative and quantitative eval-
uation of the different methodologies under identical conditions.
Besides the evaluation of the protective performance (strength-
ening and waterproong (incl. porosity)), the inuence of the
treatment on the visual aspect should be investigated. From this,
the exact role of the microorganism and the metabolic pathway can
be distinguished among the other parameters contributing to the
overall effectiveness. An expanded knowledge on these factors will
no doubt contribute to the added value of the biodeposition treat-
ment, which is an ecological, compatible surface treatment with a
high protective effect.
7. Future perspectives
In 2010, the patent of Adolphe et al. (1990) will expire.
This will certainly lead to further explorations of the biodepo-
sition technique by the different research groups. As a result,
reports on experiences from life size experiments can be soon
expected.
134 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
In addition, promising results on the use of microorganisms for
the improvement of the durability of building materials have drawn
the attention of research groups all over the world. Until now, work
on biodeposition was mainly concentrated in Europe, while much
of the work on remediation of cracks in concrete has been done
in the USA. Recently, results from preliminary studies on bacteri-
ally induced carbonate precipitation from other research groups
have come to our knowledge. In China, researchers are investigat-
ing the use of microbially induced carbonate precipitation for the
restoration of ancient masonry buildings (Shen and Cheng, 2008)
and the protection of concrete surfaces (Chunxiang et al., 2009).
Furthermore, the method of producing CaCO3 by bacterial biomin-
eralization has been patented in China by Qian et al. (2007). The
latter was possible, as the patent by Adolphe et al. (1990) was
limited to European countries. In Brazil, Shirakawa et al. (2008)
are working on biodeposition on ber cement roof tiles. In India,
researchers have applied for a patent in which the use of Shewanella
for the improvement of the strength of concrete was described
(Saroj, 2006).
It is clear that the work done by several research groups, focus-
ing on different materials, can only improve our understanding
on the possibilities and limitations of biotechnological applica-
tions on building materials. However, as was already indicated by
Webster and May (2006), the challenge for the immediate future
is to translate some of the promising results obtained in the eld
of bioremediation of building materials into practical applications.
In accordance with the authors, we agree that the acceptance and
satisfactory use of biotechnological applications by conservators
requires knowledge on the risk factors, in particular the long-
term effects of the applied bacteria and their nutrient media. In
order to avoid any undesirable secondary effects, Gonzlez-Munoz
(2008) already stressed the importance of investigations on the
effects of the medium composition on the growth of autochthonous
bacteria. By excluding the use of carbohydrates in the medium,
Jimenez-Lopez et al. (2007) already indicated that the growth of
acid producing bacteria could be avoided.
Future investigations should also focus on the retention of nutri-
ents and metabolic products in the stone, as they have an inuence
on the survival, growth and biolm formation of the microorgan-
isms inside the stone.
Finally, research is needed on biodeposition on heavily degraded
stone. Experiments have shown that ethylsilicates are unable
to consolidate large grains (1.53 mm) of Euville limestone (KIK
Testrapport, 1997). Such particles can be often found as a result
of natural weathering. Biodeposition or more specically the con-
cept of biological mortars could be used for the cementation of such
coarse grains.
8. Summary
The knowledge about the microbial origin of limestone has
resulted in research concerning MICP for the protection of orna-
mental stone. The rst patent in which this method has been
described already dates from almost two decades ago. Since then,
different research groups have searched for alternative approaches
to obtain a protective layer of calcium carbonate on the surface of
building materials (biodeposition). Some authors suggested the use
of alternative microorganisms or metabolic pathways, while oth-
ers obtained precipitation without the application of calcinogenic
bacteria, consequently falling outside the scope of the claims of the
Calcite Bioconcept Patent.
The goal of this review is to provide an in-depth overview of
the different methodologies, allowing for a qualitative compari-
son of their performance. A quantitative evaluation however, has
been hampered as a result of the differences in experimental pro-
cedures between the different research groups. In our opinion, too
little attention has been paid to the calcium dosage and the type
of stone, which could largely attribute for the differences observed
between the various treatments. In this review, some recommen-
dations have been made to improve the in situ feasibility of this
type of treatment, both from an economical and practical point of
view. In addition to the biodeposition treatment, the use of bacte-
rially induced carbonates as a binder, i.e. biocementation, has been
addressed. An overview has been given of the different elds of
applications and their future prospects.
Acknowledgements
This research was funded by a BOF grant from Ghent Univer-
sity. The authors would like to thank Nico Boon, Bart De Gusseme,
Melissa Dunkle and Siegfried E. Vlaeminck for critically reading the
manuscript.
References
Adolphe, J.P., Billy, C., 1974. Biosynthse de calcite par une association bactrienne
arobie. C. R. Acad. Sci. Paris 278, 28732875.
Adolphe, J.P., Hourimche, A., Loubire, J.F., Paradas, J., Soleilhavoup, F., 1989. Les for-
mations carbonates dorigine bactrienne. Formations continentales dAfrique
du Nord. Bull. Soc. Geol. Fr. 8 (5), 5562.
Adolphe, J.P., Loubire, J.F., Paradas, J., Soleilhavoup, F., 1990. Procd de traitement
biologique dune surface articielle. European patent 90400G97.0. (after French
patent 8903517, 1989).
Bachmeier, K.L., Williams, A.E., Warmington, J.R., Bang, S.S., 2002. Urease activity
in microbiologically-induced calcite precipitation. J. Biotechnol. 93 (2), 171
181.
Bang, S.S., Galinat, J.K., Ramakrishnan, V., 2001. Calcite precipitation induced by
polyurethane-immobilized Sporosarcina pasteurii. Enzyme Microb. Technol. 28
(45), 404409.
Barabesi, C., Galizzi, A., Mastromei, G., Rossi, M., Tamburini, E., Perito, B., 2007.
Bacillus subtilis gene cluster involved in calcium carbonate biomineralization. J.
Bacteriol. 189 (1), 228235.
Bazylinski, D.A., Frankel, R.B., Konhauser, K.O., 2007. Modes of biomineralization of
magnetite by microbes. Geomicrobiol. J. 24, 465475.
Ben Chekroun, K., Rodriguez-Navarro, C., Gonzlez-Munoz, M.T., Arias, J.M., Cultrone,
G., Rodriguez-Gallego, M., 2004. Precipitation and growth morphology of cal-
cium carbonate induced by Myxococcus xanthus: implications for recognition of
bacterial carbonates. J. Sediment. Res. 74 (6), 868876.
Ben Omar, N., Entrena, M., Gonzlez-Munoz, M.T., Arias, J.M., 1994. Effects of Ph and
phosphate on the production of struvite by Myxococcus xanthus. Geomicrobiol.
J. 12 (2), 8190.
Ben Omar, N., Gonzlez-Munoz,
M.T., Penalver, J.M.A., 1998. Struvite crystallization
on Myxococcus cells. Chemosphere 36 (3), 475481.
Ben Omar, N., Martnez-Canamero, M., Gonzlez-Munoz, M.T., Maria Arias, J.,
Huertas, F., 1995. Myxococcus xanthus killed cells as inducers of struvite crys-
tallization. Its possible role in the biomineralization processes. Chemosphere
30 (12), 23872396.
Boquet, E., Boronat, A., Ramos-Cormenzana, A., 1973. Production of calcite (calcium
carbonate) crystals by soil bacteria is a general phenomenon. Nature 246 (5434),
527529.
Braissant, O., Cailleau, G., Dupraz, C., Verrecchia, E., 2003. Bacterially induced
mineralization of calcium carbonate in terrestrial environments: the role
of exopolysaccharides and amino acids. J. Sediment. Res. 73 (3), 485
490.
Braissant, O., Verrecchia, E., Aragno, M., 2002. Is the contribution of bacteria to
terrestrial carbon budget greatly underestimated? Naturwissenschaften 89 (8),
366370.
Cacchio, P., Ercole, C., Cappuccio, G., Lepidi, A., 2003. Calcium carbonate precipita-
tion by bacterial strains isolated from a limestone cave and from a loamy soil.
Geomicrobiol. J. 20 (2), 8598.
Camaiti, M., Borselli, G., Matteol, U., 1988. Prodotti consalidanti impiegati nelle oper-
azioni di restauro. Edilizia 10, 125134.
Cappitelli, F., Toniolo, L., Sansonetti, A., Gulotta, D., Ranalli, G., Zanardini, E., Sorlini, C.,
2007. Advantages of using microbial technology over traditional chemical tech-
nology in removal of black crusts from stone surfaces of historical monuments.
Appl. Environ. Microbiol. 73 (17), 56715675.
Cappitelli, F., Zanardini, E., Ranalli, G., Mello, E., Daffonchio, D., Sorlini, C., 2006.
Improved methodology for bioremoval of black crusts on historical stone art-
works by use of sulfate-reducing bacteria. Appl. Environ. Microbiol. 72 (5),
37333737.
Carretero, M.I., Bernab, J.M., Galan, E., 2006. Application of sepiolite-cellulose pastes
for the removal of salts from building stones. Appl. Clay Sci. 33, 4351.
 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
Castanier, S. 1995. Nouvelles compositions pour mortier biologique, procd de
recourvrement dun surface ou de comblement dune cavit laide des compo-
sitions. French patent. No. 95 05861.
Castanier, S., Le Metayer-Levrel, G., Perthuisot, J.-P., 1999. Ca-carbonates precipi-
tation and limestone genesisthe microbiogeologist point of view. Sediment.
Geol. 126 (14), 923.
Chaturvedi, S., Chandra, R., Rai, V., 2006. Isolation and characterization of Phragmites
australis (L.) rhizosphere bacteria from contaminated site for bioremediation of
colored distillery efuent. Ecol. Eng. 27 (3), 202207.
Chunxiang, Q., Jianyun, W., Ruixing, W., Liang, C., 2009. Corrosion protection of
cement-based building materials by surface deposition of CaCO3 by Bacillus
pasteurii. Mater. Sci. Eng. 29 (4), 12731280.
Clifton, J.R., Frohnsdorff, G.J.C., 1982. Stone consolidating materials: a status report.
In: Conservation of Historic Stone Buildings and Monuments. National Academy
Press, Washington, DC, pp. 287311.
Day, J.L., Ramakrishnan, V., Bang, S.S., 2003. Microbiologically induced sealant for
concrete crack remediation. In: Proc. of 16th Engineering Mechanics Conference,
Seattle.
DeCuyper, K., Loutz, S., 1992. Les caractristiques des eaux uses domestiques. Tri-
bune Cebedeau 45 (560), 719.
De Belie, N., De Muynck, W., 2008. Crack repair in concrete using biodeposition. In:
Proc. of ICCRR Cape Town, South Africa.
De Muynck, W., Cox, K., De Belie, N., Verstraete, W., 2008a. Bacterial carbonate pre-
cipitation as an alternative surface treatment for concrete. Constr. Build. Mater.
22 (5), 875885.
De Muynck, W., Debrouwer, D., De Belie, N., Verstraete, W., 2008b. Bacterial carbon-
ate precipitation improves the durability of cementitious materials. Cem. Concr.
Res. 38 (7), 10051014.
De Muynck, W., Van Hyfte, E., Verbeken, K., De Belie, N., Verstraete, W., 2008c. In
situ enrichment of carbonate producing bacteria for biodeposition in practice.
In: Proc. of the 1st International Conference on BioGeoCivil Engineering, Delft,
The Netherlands, p. 35.
De Muynck, W., Verbeken, K., De Belie, N., Verstraete, W., 2009. Inuence of the cal-
cium dosage on the effectiveness of bacterially induced carbonate precipitation
on limestone. Ecol. Eng., doi:10.1016/j.ecoleng.2009.03.025, this issue.
Delgado Rodrigues, J., 2001. Consolidation of decayed stones. A delicate problem
with few practical solutions. In: Loureco, P.B., Roca, P. (Eds.), International Sem-
inar on Historical Constructions. Guimares, Portugal.
DeJong, J.T., Fritzges, M.B., Nusslein, K., 2006. Microbially induced cementation to
control sand response to undrained shear. J. Geotech. Geoenviron. 132 (11),
13811392.
Dick, J., De Windt, W., De Graef, B., Saveyn, H., Van der Meeren, P., De Belie, N.,
Verstraete, W., 2006. Bio-deposition of a calcium carbonate layer on degraded
limestone by Bacillus species. Biodegradation 17 (4), 357367.
Douglas, S., Beveridge, T.J., 1998. Mineral formation by bacteria in natural microbial
communities. FEMS Microbiol. Ecol. 26 (2), 7988.
Dreesen, R., Dusar, M., 2004. Historical building stones in the province of Limburg
(NE Belgium): role of petrography in provenance and durability assessment.
Mater. Charact. 53 (24), 273287.
Drew, 1911. The action of some denitrifying bacteria in tropical and temperate seas,
and bacterial precipitation of calcium carbonate in the sea. J. Mar. Biol. Ass. 9,
142155.
Ehrlich, H.L., 1996. How microbes inuence mineral growth and dissolution. Chem.
Geol. 132 (14), 59.
Ehrlich, H.L., 1998. Geomicrobiology: its signicance for geology. Earth Sci. Rev. 45
(12), 4560.
Ercole, C., Cacchio, P., Botta, A.L., Centi, V., Lepidi, A., 2007. Bacterially induced min-
eralization of calcium carbonate: the role of exopolysaccharides and capsular
polysaccharides. Microsc. Microanal. 13 (4250).
Ferris, F.G., Stehmeier, L.G., 1992. Bacteriogenic mineral plugging. USA Patent
US5143155.
Fujita, Y., Ferris, F.G., Lawson, R.D., Colwell, F.S., Smith, R.W., 2000. Calcium carbonate
precipitation by ureolytic subsurface bacteria. Geomicrobiol. J. 17 (4), 305318.
Fujita, Y., Redden, G.D., Ingram, J.C., Cortez, M.M., Ferris, F.G., Smith, R.W., 2004.
Strontium incorporation into calcite generated by bacterial ureolysis. Geochim.
Cosmochim. Acta 68 (15), 32613270.
Gauri, K.L., Parks, L., Jaynes, J., Atlas, R., 1992. Removal of sulphated crust from marble
using sulphate-reducing bacteria. In: Robin, G.M. (Ed.), Stone Cleaning and the
Nature, Soiling and Decay Mechanisms of Stone. Proceedings of the International
conference. 1416 April, Donhead Publishing Ltd., Edinburgh, United Kingdom,
pp. 160165.
Ghosh, P., Mandal, S., Chattopadhyay, B.D., Pal, S., 2005. Use of microorganism to
improve the strength of cement mortar. Cem. Concr. Res. 35 (10), 19801983.
Gollapudi, U.K., Knutson, C.L., Bang, S.S., Islam, M.R., 1995. A new method
for controlling leaching through permeable channels. Chemosphere 30 (4),
695705.
Gonzlez-Munoz, M., Arias, J.M., Montoya, E., Rodriguez-Gallego, M., 1993. Stru-
vite production by Myxococcus coralloides D. Chemosphere 26 (10), 1881
1887.
Gonzlez-Munoz,
M.T., Omar, N.B., Martnez-Canamero, M., Rodrguez-Gallego, M.,
Galindo, A.L., Arias, J., 1996. Struvite and calcite crystallization induced by cel-
lular membranes of Myxococcus xanthus. J. Cryst. Growth 163 (4), 434439.
Gonzlez-Munoz, M.T., Rodriguez-Navarro, C., Jimenez-Lopez, C., Rodriguez-
Gallego, M., 2008. Method and product for protecting and reinforcing
construction and ornamental materials. WO 2008/009771 A1.
135
Gonzlez-Munoz, M.T., 2008. Bacterial biomineralization applied to the protection-
consolidation of ornamental stone: current development and perspectives.
Coalition 15, 1218.
Hammes, F., Boon, N., de Villiers, J., Verstraete, W., Siciliano, S.D., 2003a. Strain-
specic ureolytic microbial calcium carbonate precipitation. Appl. Environ.
Microbiol. 69 (8), 49014909.
Hammes, F., Seka, A., de Knijf, S., Verstraete, W., 2003b. A novel approach to
calcium removal from calcium-rich industrial wastewater. Water Res. 37 (3),
699704.
Hammes, F., Verstraete, W., 2002. Key roles of pH and calcium metabolism in micro-
bial carbonate precipitation. Rev. Environ. Sci. Biotechnol. 1, 37.
Hart, R.T., Fekete, T., Flock, D.L., 1960. The plugging effect of bacteria in sandstone
systems. Can. Min. Metall. Bull. 53, 495501.
Heselmeyer, K., Fisher, U., Krumbein, K.E., Warscheid, T., 1991. Application of Desul-
fovibrio vulgaris for the bioconversion of rock gypsum crusts into calcite.
Bioforum 1/2, 89.
Jack, T.R., Stehmeier, L.G., Ferris, F.G., Islam, M.R., 1991. Microbial selective plugging
to control water channeling. In: Donaldson (Ed.), Microbial Enhancement of Oil
Recovery-Recent Advances. Elsevier Science Publishing Co., New York.
Jimenez-Lopez, C., Jroundi, F., Pascolini, C., Rodriguez-Navarro, C., Pinar-Larrubia,
G., Rodriguez-Gallego, M., Gonzlez-Munoz, M.T., 2008. Consolidation of quarry
calcarenite by calcium carbonate precipitation induced by bacteria activated
among the microbiota inhabiting the stone. Int. Biodeterior. Biodegrad. 62 (4),
352363.
Jimenez-Lopez, C., Rodriguez-Navarro, C., Pinar, G., Carrillo-Rosa, F.J., Rodriguez-
Gallego, M., Gonzlez-Munoz, M.T., 2007. Consolidation of degraded ornamental
porous limestone by calcium carbonate precipitation induced by the microbiota
inhabiting the stone. Chemosphere 68 (10), 19291936.
Jonkers, H., 2007. Self healing concrete: a biological approach. In: van der Zwaag, S.
(Ed.), Self Healing Materials: An alternative Approach to 20 Centuries of Mate-
rials Science. Springer, Netherlands, pp. 195204.
Jonkers, H.M., Schlangen, E., 2007. Crack repair by concrete-immobilized bacteria. In:
Schmets, A.J.M., Van der Zwaag, S. (Eds.), Proc. of First International Conference
on Self Healing Materials. Noordwijk, The Netherlands, p. 7.
Jonkers, H.M., Thijssen, A., Copuroglu, O., Schlangen, E., 2008. Application of bacte-
ria as self-healing agent for the development of sustainable concrete. In: Proc.
of First International Conference on BioGeoCivil Engineering, Delft, The Nether-
lands, p. 25.
Kantzas, A., Ferris, F.G., Jha, K.N., Mourits, F.M., 1992. A novel method of sand consoli-
dation through bacteriogenic mineral plugging. In: Proc. of CIM Annual Technical
Conference, Calgary.
KIK Testrapport, 1997. Test report on the efciency of the FTB consolidant SH
75, an ethylsilicates based consolidant (in Dutch). http://www.ftbremmers.
com/leadmin/dam/Testrapporten/KIK SH75.pdf on November 2008.
Knorre, H., Krumbein, K.E., 2000. Bacterial calcication. In: Riding, E.E., Awramik,
S.M. (Eds.), Microbial Sediments. SpringerVerlag, Berlin, pp. 2531.
Kucharski, E.S., Cord-Ruwisch, R., Whifn, V., Al-Thawadi, S.M.J., 2006. Microbial
biocementation. World intellectual property organization, WO 2006/066326 A1.
Lappin-Scott, H.M., Cusack, F., Costerton, J.W., 1988. Nutrient resuscitation and
growth of starved cells in sandstone cores: a novel approach to enhanced oil
recovery. Appl. Environ. Microbiol. 54 (6), 13731382.
Le Metayer-Levrel, G., Castanier, S., Orial, G., Loubiere, J.F., Perthuisot, J.P., 1999.
Applications of bacterial carbonatogenesis to the protection and regeneration
of limestones in buildings and historic patrimony. Sediment. Geol. 126 (14),
2534.
Lowenstan, H.A., Weiner, S., 1988. On Biomineralization. Oxford University Press,
New York.
Mansch, R., Bock, E., 1998. Biodeterioration of natural stone with special reference
to nitrifying bacteria. Biodegradation 9 (1), 4764.
Mastromei, G., Marvasi, M., Perito, B., 2008. Studies on bacterial carbonate precip-
itation for stone conservation. In: Proc. of BioGeoCivil Engineering Conference,
Delft, Netherlands, pp. 104106.
May, E., 2005. Biobrush research monograph: novel approaches to conserve our
European heritage. EVK4-CT-2001-00055.
McConnaughey, T.A., Whelan, J.F., 1997. Calcication generates protons for nutrient
and bicarbonate uptake. Earth Sci. Rev. 42 (12), 95117.
Mobley, H.L., Hausinger, R.P., 1989. Microbial ureases: signicance, regulation, and
molecular characterization. Microbiol. Mol. Biol. Rev. 53 (1), 85108.
Morita, R., 1980. Calcite precipitation by marine bacteria. Geomicrobiol. J. 2,
6382.
Moropoulou, A., Kouloumbi, N., Haralampopoulos, G., Konstanti, A., Michailidis, P.,
2003. Criteria and methodology for the evaluation of conservation interven-
tions on treated porous stone susceptible to salt decay. Prog. Org. Coat. 48,
259270.
Morse, J.W., 1983. The kinetics of calcium carbonate dissolution and precipitation.
In: Reeder, R.J. (Ed.), Carbonates: Mineralogy and Chemistry, vol. 11. Mineralogic
Society of America, Washington, DC, pp. 227264.
Nemati, M., Greene, E.A., Voordouw, G., 2005. Permeability prole modication
using bacterially formed calcium carbonate: comparison with enzymic option.
Process Biochem. 40 (2), 925933.
Nemati, M., Voordouw, G., 2003. Modication of porous media permeability, using
calcium carbonate produced enzymatically in situ. Enzyme Microb. Technol. 33
(5), 635642.
Nielsen, T.H., Bonde, T.A., Sorensen, J., 1998. Signicance of microbial urea turnover in
N cycling of three Danish agricultural soils. FEMS Microbiol. Ecol. 25 (2), 147157.
136 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136
Orial, G., 2000. La biomineralisation applique la conservation du patrimoine: bilan
de dix ans dexperimentation. Restaurar la memoria, Valladolid, Spain.
Orial, G., Vieweger, T., Loubiere, J.F., 2002. Les mortiers biologiques: une solution
pour la conservation de la sculpture monumentale en pierre. Art Biology and
Conservation, Metropolitan Museum, New York.
Perito, B., Biagiotti, L., Daly, S., Galizzi, A., Tiano, P., Mastromei, G., 2000. Bacterial
genes involved in calcite crystal precipitation. In: Ciferri, O., Tiano, P., Mastromei,
G. (Eds.), Of Microbes and Art: The Role of Microbial Communities in the Degra-
dation and Protection of Cultural Heritage. Plenum Publisher, New York, pp.
219230.
Qian, C., Wang, R., Wang, J., 2007. The method of producing CaCO3 by bacterial
biomineralization. ZL2005100947744.5.
Ramachandran, S.K., Ramakrishnan, V., Bang, S.S., 2001. Remediation of concrete
using micro-organisms. ACI Mater. J. 98, 39.
Ramakrishnan, S.K., Panchalan, R.K., Bang, S.S., 2001. Improvement of concrete dura-
bility by bacterial mineral precipitation. In: 11th International conference on
Fracture, Turin, Italy.
Ranalli, G., Chiavarini, M., Guidetti, V., Marsala, F., Matteini, M., Zanardini, E., Sorlini,
C., 1997. The use of micro-organisms for the removal of sulphates on artistic
stoneworks. Int. Biodeterior. Biodegrad. 40 (24), 255261.
Ranalli, G., Matteini, M., Tosini, I., Zanardini, E., Sorlini, C., 1999. Bioremediation
of cultural heritage: removal of sulphates, nitrates and organic substances. In:
Ciferri, O., Tiano, P., Mastromei, G. (Eds.), Proc. of International Conference on
Microbiology and Conservation Of Microbes and Art: The Role of Microbial
Communities on the Degradation and Protection of Cultural Heritage. Florence,
Italy, pp. 231245.
Rivadeneyra, M.A., Delgado, R., del Moral, A., Ferrer, M.R., Ramos-Cormenzana, A.,
1994. Precipitation of calcium carbonate by Vibrio spp. from an inland saltern.
FEMS Microbiol. Ecol. 13 (3), 197204.
Rivadeneyra, M.A., Parraga, J., Delgado, R., Ramos-Cormenzana, A., Delgado, G., 2004.
Biomineralization of carbonates by Halobacillus trueperi in solid and liquid media
with different salinities. FEMS Microbiol. Ecol. 48, 3946.
Rodriguez-Navarro, C., Jimenez-Lopez, C., Rodriguez-Navarro, A., Gonzlez-Munoz,
M.T., Rodriguez-Gallego, M., 2007. Bacterially mediated mineralization of
vaterite. Geochim. Cosmochim. Acta 71 (5), 11971213.
Rodriguez-Navarro, C., Rodriguez-Gallego, M., Ben Chekroun, K., Gonzlez-Munoz,
M.T., 2003. Conservation of ornamental stone by Myxococcus xanthus-induced
carbonate biomineralization. Appl. Environ. Microbiol. 69 (4), 21822193.
Samonin, V.V., Elikova, E.E., 2004. A study on the absorption of bacterial cells on
porous materials. Microbiology 73 (6), 696701.
Saiz-Jimenez, C., 1997. Biodeterioration vs biodegradation: the role of microorgan-
isms in the removal of pollutants deposited on historic buidlings. Int. Biodeter.
Biodegr. 40 (24), 225232.
Saroj, M., 2006. A process for preparing modied bioconcrete. C04B 28/02 (Indian
patent).
Shen, J., Cheng, X., 2008. Laboratory investigation on restoration of Chinese ancient
masonry buildings using microbial carbonate precipitation. In: 1st BioGeoCivil
Engineering Conference, Delft.
Shinano, H., 1972. Studies of marine microorganisms taking part in the precipitation
of calcium carbonate. Bull. Jpn. Soc. Sci. Fish. 38, 717.
Shirakawa, M., Cincotto, M., Dias, C., Atencio, D., Vanderley, M.J., 2008. Inuence of
carbonation in accelerated chamber previous to biocalcication on ber cement
surface. In: 1st BioGeoCivil Engineering Conference, Delft.
Simon, M.A., Bonner, J.S., Page, C.A., Townsend, R.T., Mueller, D.C., Fuller, C.B., Aut-
enrieth, R.L., 2004. Evaluation of two commercial bioaugmentation products
for enhanced removal of petroleum from a wetland. Ecol. Eng. 22 (45), 263
277.
Stocks-Fischer, S., Galinat, J.K., Bang, S.S., 1999. Microbiological precipitation of
CaCO3 . Soil Biol. Biochem. 31 (11), 15631571.
Stumm, W., Morgan, J.J., 1981. Aquatic Chemistry, 2nd edition. John Wiley, New York.
Sutherland, I.W., Thomson, S., 1975. Comparison of polysaccharides produced by
Myxococcus strains. J. Gen. Microbiol. 89 (JULY), 124132.
Sutton, M., Reis, S., Baker, S., 2008. Atmospheric ammonia: detecting emis-
sion changes and environmental impact. In: Results of an Expert Workshop
Under the Convention on Long-Range Transboundary Air Pollution. Springer,
pp. 490.
Tiano, P., 1995. Stone reinforcement by calcite crystal precipitation induced by
organic matrix macromolecules. Stud. Conserv. 40 (3), 171176.
Tiano, P., Addadi, L., Weiner, S., 1992. Stone reinforcement by induction of cal-
cite crystals using organic matrix macromolecules: feasibility study. In: 7th
International Congress on Deterioration and Conservation of Stone, Lisbon, pp.
13171326.
Tiano, P., Biagiotti, L., Mastromei, G., 1999. Bacterial bio-mediated calcite precipita-
tion for monumental stones conservation: methods of evaluation. J. Microbiol.
Methods 36 (12), 139145.
Tiano, P., Cantisani, E., Sutherland, I., Paget, J.M., 2006. Biomediated reinforcement
of weathered calcareous stones. J. Cult. Herit. 7 (1), 4955.
Urzi, C., Garcia-Valles, M., Vendrell, M., Pernice, A., 1999. Biomineralization processes
on rock and monument surfaces observed in eld and in laboratory conditions.
Geomicrobiol. J. 16, 3954.
Warren, L.A., Maurice, P.A., Parmar, N., Ferris, F.G., 2001. Microbially mediated cal-
cium carbonate precipitation: implications for interpreting calcite precipitation
and for solid-phase capture of inorganic contaminants. Geomicrobiol. J. 18,
93125.
Warscheid, T., Braams, J., 2000. Biodeterioration of stone: a review. Int. Biodeter.
Biodegr. 46 (4), 343368.
Webster, A., May, E., 2006. Bioremediation of weathered-building stone surfaces.
Trends Biotechnol. 24 (6), 255260.
Whifn, V.S., 2004. Microbial CaCO3 precipitation for the production of Bioce-
ment. Ph.D. Thesis, Murdoch University, Australia, pp. 155. http://wwwlib.
murdoch.edu.au/adt/browse/view/adt-MU20041101.142604.
Whifn, V.S., van Paassen, L., Harkes, M.P., 2007. Microbial carbonate precipitation
as a soil improvement technique. Geomicrobiol. J. 24, 417423.
Wooltt, C., Abrey, G., 2008. Poultices: the true or plain poultice and the cleaning
and desalination of historic masonry and sculpture. Retrieved august 2008, from
http://www.buildingconservation.com/articles/poultices/poultice.htm.
Zhong, L., Islam, M.R., 1995. A new microbial plugging process and its impact on frac-
ture remediation. In: Proc. of Society of Petroleum Engineers. Annual Technical
Conference, Dallas, Texas, pp. 703715.