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Ecological Engineering 36 (2010) 118136

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


Microbial carbonate precipitation in construction materials: A review

Willem De Muynck a,b , Nele De Belie a, , Willy Verstraete b,1
Magnel Laboratory for Concrete Research, Dept. of Structural Engineering, Ghent University, Technologiepark Zwijnaarde 904, B-9052 Gent, Belgium
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
attention was paid to the research background that could account for the choice of the microorganism and
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.
2009 Elsevier B.V. All rights reserved.

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

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

0925-8574/$ see front matter 2009 Elsevier B.V. All rights reserved.
W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 119

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

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
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
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, 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
Biobrush (May, 2005) Pseudomonas putida ODAA OAU B4 AW Portland oolitic limestone

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,

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
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

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-

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

situ applications (Orial, 2000).


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


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

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


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


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

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

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

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

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

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


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

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

Added value Ecological, environmentally friendly

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

ODAA: oxidative deamination of amino acids; OAU: organic acid utilization, HU: hydrolysis of urea.
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

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


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)

pH was adjusted with HCl or NaOH.
Dick et al. used 7.5 g L1 CaCl2 2H2 O.
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. 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 125

2NH3 + 2H2 O 2NH4 + + 2OH (9) Finally, calcium chloride was added for a second time in the fourth
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: From the screening procedure described above, 2 strains of B.
sphaericus were selected for further experiments. These strains
CO(NH2 )2 + 2H2 O 2NH4 + + CO3 2 (11)
were shown to decrease the initial water absorption rate by approx-
In the presence of calcium ions, this will result in calcium imately 50%.
carbonate precipitation (Eq. (12)), once a certain level of supersat-
uration is reached: Cementitious materials. As an extension to the above
2 2+ mentioned study, De Muynck et al. (2008a,b) investigated the
CO3 + Ca CaCO3 (12)
biodeposition with B. sphaericus as a surface treatment for cementi-
As calcium ions are bound to the cell wall as a result of the neg- tious materials (Portland cement mortar) with different porosities.
ative charge of the latter, this can result in the formation of crystals In contrast with the treatment procedure described by Dick et al.
on the bacterial cell. In addition, precipitation can also occur in the (2006), all experiments were performed at 28 C under non-sterile
bulk phase of the liquid. A schematic overview, of the ureolytic car- conditions. Due to the alkaline pH of cementitious materials, the
bonate precipitation occurring at the microbial cell wall is given in contamination was expected to be very low. Furthermore, the treat-
Fig. 1. ment procedure was altered: the mortar specimens were immersed
The purpose of the study by Dick et al. (2006) was to identify for 24 h in a 1 day old culture of B. sphaericus containing ca.
the microbial key factors which contribute to the performance 107 cells mL1 , after which they were transferred to fresh medium
of the biodeposition treatment. For the evaluation of the per- containing a calcium source (Table 5). The specimens were removed
formance, the authors investigated the water absorption rate of from the solution after 3 days.
treated and untreated Euville limestone. The key factors had to The authors demonstrated that the biodeposition treatment
be easy in use and applicable for quick screening. The following resulted in an increased resistance of mortar specimens towards
parameters have been examined: calcite deposition on limestone carbonation, chloride penetration and freezing and thawing, espe-
cubes, pH increase, urea degrading capacity, EPS production, cially for more porous mortars with higher water to cement ratios
biolm formation, -potential and deposition of dense crystal (w/c). Moreover, the biodeposition treatment showed a similar pro-
layers. Of these parameters, the -potential proved to be the factor tection towards degradation processes as some of the conventional
with the greatest predictive power to screen microorganisms for surface treatments under investigation (silanes, siloxanes, silicates
good limestone restoration, reecting the effect on the initial and acrylates).
water absorption. The -potential is a measure of the potential of According to the authors, the biodeposition treatment on
the electric layer at the surface of the cells, and is, therefore, an cementitious materials should be regarded as a coating system. This
important parameter in the adhesion and surface colonization by could be attributed to the fact that the carbonate precipitation was
bacteria. Due to the positive -potential of the limestone, bacteria mainly a surface phenomenon due to the limited penetration of
with a highly negative -potential will be more easily retained. The the bacteria in the porous matrix. From thin section analyses, the
second important key factor was the specic urea degradation rate. authors observed that the majority of the surface was covered with
Bacteria with a high initial specic urea degradation rate show a a layer of crystals with thicknesses within the range of 1040 m,
high afnity for urea. This allows for a high substrate turnover for in which often larger crystals (up to 110 m) could be found. The
a limited amount of cells. morphology of the crystals was observed to be highly dependent
The bacteria which had been selected for screening were iso- on the medium composition. Much research on bacterial induced
lated from an ureolytic calcication reactor. This type of reactor precipitation has been conducted with calcium chloride as the cal-
had been previously developed by the same research group for the cium source (Adolphe et al., 1990; Ferris and Stehmeier, 1992; Bang
removal of calcium from calcium-rich wastewater (Hammes et al., et al., 2001). As chloride ions are detrimental to the reinforcement
2003). Calcifying sludge was obtained through the stimulation of in concrete structures, the use of calcium acetate as an alternative
autochthonous ureolytic organisms, by means of repeated addi- calcium source was investigated. In the event calcium chloride was
tions of urea. From this sludge, bacteria were isolated and screened used as the calcium source, rhombohedral carbonate crystals were
for their ability to precipitate calcite on agar plates containing obtained. In the presence of calcium acetate, spherulitic crystals
urea and calcium chloride. Although urease activity is widespread were observed (Fig. 2). However, no differences in the protective
among different groups of microorganisms, it was mainly microor- effect were observed between biodeposition treatments with a dif-
ganisms closely related to the Bacillus sphaericus group which ferent calcium source. Therefore the authors concluded that from
were shown to proliferate and express the urease gene under the these two salts, calcium acetate should be used for biodeposition
given cultivation conditions (Hammes et al., 2003). The ability of on cementitious materials.
B. sphaericus to precipitate calcium carbonate had been previously
described by Cacchio et al. (2003). Among several strains isolated 3.1.5. Procedure according to the Biobrush consortium (United
from a limestone cave, a B. sphaericus strain was shown to rapidly Kingdom)
precipitate CaCO3 in B4 medium (Table 5), even at low tempera- The basic aim of the Biobrush (BIOremediation for Building
tures such as 4 C. Furthermore, calcifying bacteria were found not Restoration of the Urban Stone Heritage) project was to integrate
to solubilise carbonates. the existing knowledge on the application of microorganisms for
For the deposition of a layer of carbonate on the surface, Dick et the remediation of damaged stone into a conservation practice. Fur-
al. (2006) rst proposed the establishment of a biolm. For that pur- thermore, the goal was to sequentially link the processes of salt
pose, limestone cubes were immersed for 2 weeks in liquid medium removal to the processes of consolidation (May, 2005).
inoculated with 1% of the different strains. The surface was rewet- The use of microorganisms has been investigated for the removal
ted each 2 h for 5 min by shaking. After the 2 weeks ended, calcium of nitrates, sulphates and organic matter present on the surface
chloride was added to the medium in order to precipitate calcium of artworks (Gauri et al., 1992; Ranalli et al., 1999). In addition
carbonate. In the third week, the specimens were suspended in to the elimination of black crusts, microbial sulphate removal also
fresh medium in order to have a second phase of biolm growth. 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. 3.2. Application of organic matrix molecules; procedure
Heselmeyer et al. (1991) obtained the complete removal of gyp- according to the Bioreinforce consortium (Italy)
sum crusts from marble samples in laboratory conditions using
a strain of Desulfovibrio vulgaris. The procedure was further opti- Tiano et al. (1999) commented on the use of viable cells for the
mized by Ranalli et al. (1997), who used sepiolite as a carrier formation of new minerals inside the stone. This was the result
material for D. vulgaris and Desulfovibrio desulfuricans. The use of of their experiments with Micrococcus spp. and Bacillus subtilis
sepiolite not only provided anaerobic conditions and humidity, but strains on Pietra di Lecce bioclastic limestone. In these experi-
also enabled the authors to shorten the treatment time. Additional ments, bacteria were applied by brushing sterilized specimens that
improvements were made by Cappitelli et al. (2006) who reported were soaked with distilled water, reaching a nal concentration of
on the superiority of Carbogel as a delivery system for the bacte- 106 cells cm2 . Subsequently, the bacteria were fed daily by wetting
ria. The use of Carbogel allowed for a higher retention of viable with a small amount of B4 medium (Table 5) for a period of 15 days.
bacteria and signicantly decreased the time needed for entrap- Experiments were performed at 28 C under non-sterile conditions.
ment of the microorganisms as compared to the use of sepiolite. According to the authors, the decrease in water absorption after
In addition, methods were presented to avoid the precipitation a biodeposition treatment is for a large part attributable to the
of black iron sulde. The optimized methodology appeared to be physical obstruction of pores, rather than to the stable presence of
superior to chemical treatments involving the use of ethylenedi- newly precipitated calcite. Furthermore, the authors commented
aminetetraacetic acid (EDTA), since no sodium sulphate was formed on some possible negative consequences, such as (1) the pres-
(Cappitelli et al., 2007). ence of products of new formation, due to the chemical reactions
As Cappitelli et al. (2006, 2007) were among the members of between the stone minerals and some by-products originating from
the Biobrush consortium, the use of Carbogel was subsequently the metabolism of viable heterotrophic bacteria and (2) the forma-
introduced into the eld of biodeposition. According to the consor- tion of stained patches, due to the growth of air-borne micro-fungi
tium, these delivery systems could be used to control the possible related to the presence of organic nutrients necessary for bacterial
harmful side effects of bacteria to stone. In addition, it was noticed development.
that the application of calcinogenic bacteria by spraying alone only To avoid some of these problems, the authors proposed the use
resulted in a limited change of the capillary water uptake of Port- of natural and synthetic polypeptides to control the growth of cal-
land stone. According to the consortium, the latter is attributable cite crystals in the pores. The rst suggestions in this direction
to the limited colonization of the stone as a result of drying already date from the time at which the Calcite Bioconcept treat-
out. ment was developed. Tiano et al. (1992) and Tiano (1995) proposed
Within the framework of the Biobrush project regarding biode- the use of organic matrix macromolecules (OMM) extracted from
position, bacteria isolated from a stream in Somerset (UK), and Mytilus californianus shells to induce the precipitation of calcium
bacteria from culture collections that had been reported to have carbonate within the pores of the stone. The organic matrix was
calcifying activity, were screened for their ability to deposit calcite shown to produce a more relevant and durable carbonate precipi-
in solid and liquid modied B4 media (Table 5). From the 10 isolates tation compared to the single use of calcium chloride or hydroxide.
that were retained and assessed for their ability to deposit calcite This precipitation resulted in a slight decrease in porosity and water
on stone surfaces, Pseudomonas putida was chosen for further study absorption by capillarity (Tiano, 1995).
in eld trials. The latter has a low risk to humans and is sensitive However, the practical application was hindered by the com-
to most of the tested antibiotics and precipitated calcite in a wide plexity of the extraction procedure and the very low yield of usable
temperature range (May, 2005). product (Tiano et al., 1999). Given this, the authors searched for
In these eld trials, bacteria were applied to the stone by alternative starting materials by changing the nature of the organic
brushing. Subsequently, the bacteria were covered with moistened macromolecules involved. As these bio inducing macromolecules
Japanese paper, above which a 11.5 cm thick layer of Carbogel pre- (BIM) are usually rich in aspartic acid groups, Tiano et al. (2006)
pared with modied B4 was applied. TrisHCl buffer was added to proposed the alternative use of acid functionalized proteins such
the Carbogel to adjust the low pH of this carrier. Finally, the gel as polyaspartic acid. Calcium and carbonate ions for crystal growth
was covered with a polyethylene sheet. As a result of this treat- were supplied by means of an ammonium carbonate and calcium
ment a decrease of the water absorption and open porosity by 1% chloride solution or a saturated solution of bicarbonate, and were
and 5%, respectively, was obtained. In order for this treatment to be supplemented in some cases by calcite nanoparticles, in order
effective as a consolidant, a 2 weeks treatment was observed to be to maintain a saturated carbonate solution in the pore over a
necessary. prolonged period. Proteins, calcium ions and nanoparticles were
W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 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.,
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
Fig. 2. Scanning electron micrographs of untreated (A) and biodeposition treated
bacteria from the microbial community of the stone, as a more
(B and C) CEM I mortar specimens. Notice the differences in crystal morphology user friendly method for the in situ consolidation of ornamental
obtained with different calcium sources: predominantly rhombohedral crystals in stone. In addition to their work on decayed limestone fragments
the case of calcium chloride (A) and spherulitic crystals with calcium acetate (B). (Jimenez-Lopez et al., 2007), this technique was recently proposed
for the treatment of new stones used for replacement purposes
introduced in the stone by means of spraying. According to the (Jimenez-Lopez et al., 2008).
authors, the method is most suitable for the use on marble statues Upon comparison of the microbial community identied in non-
and objects of high aesthetic value where conservation is required treated quarry stone and that identied in the non-treated decayed
with the minimum change in the chemistry of the object. Field test stone, the authors observed for the latter the presence of microor-
results, however, indicate that the effects of the BIM treatment were ganisms related to the quarry from which the stone was extracted
rather small. The consolidating effect and the decrease in water and microorganisms related to the environment and contamination
uptake were very low compared to the use of ethylsilicates, i.e. to which the stone was exposed.
15% over 12 mm depth compared to 30% up to 10 mm depth (as Some of the identied bacteria, Pseudomonas and Bacillus, had
measured with the drilling resistance measuring system) and 17% already been reported to produce calcium carbonate both in
compared to 60%, respectively (Tiano et al., 2006). laboratory conditions and in nature. These chemoorganotrophic
Elucidation of the genetic background of crystal formation in organisms are able to grow in culture media containing amino acids
bacteria has been proposed as an alternative way for the produc- such as nutrient agar and tryptic soy agar (Jimenez-Lopez et al.,
tion of inducing macromolecules. This was one of the objectives 2007).
of the European Bioreinforce (BIOmediated calcite precipitation From these ndings, the authors proposed the use of bacto-
for monumental stones REINFORCEment) project. According to 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 in the eld of geotechnical engineering, i.e. plugging, strengthen-
and nitrogen, which favours alkalinisation due to the oxidative ing and improvement of soils (Ferris and Stehmeier, 1992; Zhong
deamination of amino acids. Furthermore, as no carbohydrates and Islam, 1995; Nemati and Voordouw, 2003; Whifn et al., 2007).
were supplied, the probability of acid production, which is detri- Recent advances, however, indicate the potential use of this tech-
mental to the stone, was believed to be minimal. According to the nique for the remediation of cracks in building materials, strength
authors, this procedure is much easier than the use of bacterially improvement and self-healing of cementitious materials.
inoculated media, since difculties linked to the need of specialized
persons and equipment to work with microorganisms or tech- 4.1. Biological mortar (France)
nical requirements to ensure optimal growth conditions would
be avoided (Jimenez-Lopez et al., 2007). However, the fact that The knowledge and experiences obtained with the Calcite Bio-
microorganisms do not need to be introduced not only leads to a concept treatment for limestone, have resulted in the development
decrease of the overall cost, but also ensures that this method is not of a biological mortar for the remediation of small cavities on
covered by the claims of the patent by Adolphe et al. (1990). Conse- limestone surfaces. The aim of the biological mortar was to avoid
quently, Gonzlez-Munoz et al. (2008) applied for a new patent for some of the problems related to chemical and physical incom-
the protection and reinforcement of construction and ornamental patibilities of commonly used repair mortars with the underlying
materials by means of the application of an activator medium able material, especially in the case of brittle materials (Castanier,
to induce the formation of calcium carbonate. 1995; Le Metayer-Levrel et al., 1999; Orial et al., 2002, Personal
Sonication test results demonstrated that the new cement cre- communication by Loubire (Chief of Calcite Bioconcept), 2008;
ated by the microbial community and/or the combined action of http://www.calcitebioconcept.com/).
the microbial community and M. xanthus was more resistant than In general, a mortar refers to a workable paste consisting of a
that created by the sole action of either M. xanthus or the culture binder, aggregates and water to bind building materials together
media. Furthermore, the authors did not observe any changes in the and to ll the gaps between them. In particular, a biological mortar
porosity of the stone. In addition, limited exopolysaccharide pro- refers to a mixture of bacteria, nely ground limestone and a nutri-
duction was observed (Jimenez-Lopez et al., 2007). The latter could tional medium containing a calcium salt. The term biological refers
be somewhat expected as Rodriguez-Navarro et al. (2003) noted to the microbial origin of the binder, i.e. microbiologically produced
that organic lms are unable to attach to the stones under shak- calcium carbonate. Similar to lime mortars, the produced calcium
ing conditions, as was the case in the work by Jimenez-Lopez et al. carbonate cements the aggregates together. Cementation occurs as
(2007, 2008). a result of the nucleation and growth of carbonate crystals at the
Due to the time required for the activation of the microbial com- surface of the aggregates, especially at the contact areas between
munity, Jimenez-Lopez et al. (2007) proposed the additional use of them.
M. xanthus for those restoration interventions in which time is an The optimization of the mortar composition encompassed the
issue and fast formation of calcium carbonate is required. dosage and composition of the three main components, i.e. lime-
Very recently, the application of an activator medium has been stone powder, nutrients and bacterial paste. The mortars were
successfully applied in situ on calcarenite stone (Monasterio de San evaluated based upon their appearance (cohesion and colour),
Jeronimo and Hospital Real, Granada). Preliminary results show the the presence of micro-cracks and the resistance towards fractur-
effectiveness of the treatment in terms of colour changes (negligi- ing. Concerning the medium composition, some adjustments were
ble) and surface resistance by means of a peeling test (Personal made to the initial method, as was used for biodeposition purposes.
communication by Rodriguez-Navarro, 2008). The amount of nutrient solution introduced during the fabrication
Although the formation of endospores was previously consid- of the mortar was sufcient to support bacterial activity. Repeated
ered a potential drawback for the use of Bacillus in stone conserva- external applications of the nutrient solution were unable to com-
tion (Rodriguez-Navarro et al., 2003), spore forming bacteria, able pletely wet the mortar. Furthermore, they resulted in discolorations
to germinate upon the application of the culture media, contribute at the surface and were, therefore, rapidly omitted. Additionally, the
in large extent to the precipitation of carbonate by the method biological mortars necessitated the use of larger amounts of bacte-
described by Jimenez-Lopez et al. (2007). Drawbacks to the use of ria and as a result the composition of the nutrient medium had to
spore forming bacteria were related to the possible uncontrolled be altered.
growth of bacteria upon germination. However, Le Metayer-Levrel Based on the different evaluation parameters, best results
et al. (1999) found that no increases in the microbial activity or were obtained with one part of bacterial paste (containing
changes in the autochthonous microbiota were observed immedi- 109 cells mL1 ), one part of nutritional medium and two parts
ately or 4 years after the application of calcinogenic bacteria. of limestone powder. Limestone powder with a granulometry
The long activation times of this technique inspired De Muynck between 40 and 160 m was observed to be the most suited. The
et al. (2008c) to develop an in situ enrichment of carbonate pro- technique has already been successfully tested on a small scale on
ducing bacteria. For that purpose, different media were developed sculptures of the Amiens Cathedral and on a portal of the church
which allowed a rapid growth of carbonate producing strains upon of Argenton-Chteau (France). Visual observations 2 years after
exposure to the surrounding air. Among the different metabolic the treatment indicated a satisfactory appearance of the repaired
pathways under investigation, conditions optimal for carbonate zones. (Le Metayer-Levrel et al., 1999; Orial et al., 2002).
precipitation were most rapidly obtained upon the hydrolysis of
urea. Nevertheless, the rate of urea hydrolysis and biodeposition 4.2. Remediation of cracks in concrete (USA, Belgium)
remained low compared to pure cultures of B. sphaericus.
In the recovery of heavy oil from oil elds, where water is more
readily removed than the viscous oil, the ability to selectively plug
4. Biocementation porous rock to focus pumping energy in oil rich zones is highly
desirable (Hart et al., 1960; Lappin-Scott et al., 1988). Because of
Besides the deposition of a layer of carbonate on the surface of the cost and unsatisfactory performance of some of the chemi-
building materials, MICP has also been used for the generation of cally cross-linked polymers, many workers suggested that insoluble
binder-based materials. Initial developments were mainly situated biopolymers and biomass generated by injection of indigenous
W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 129

microorganisms can be used to selectively plug off zones of high was shown to protect the enzyme from environmental changes,
water permeability (Lappin-Scott et al., 1988; Jack et al., 1991; as the immobilized urease retained higher enzymatic activities at
Gollapudi et al., 1995). In addition, the use of a microbial mineral high temperatures and in the presence of high concentrations of
plugging system based on the precipitation of carbonates was sug- pronase. While the rate of calcite precipitation of the immobilized
gested (Ferris and Stehmeier, 1992; Zhong and Islam, 1995). While enzyme was slower compared to that of the free enzyme, lower
initial research on MICP in sand columns was mainly focused on concentrations of the former where needed to obtain the theoreti-
the decrease of porosity and permeability as a result of the physi- cal maximum precipitation in a period of 24 h. Although the authors
cal presence of the newly formed carbonates (Ferris and Stehmeier, mentioned ongoing research on the use of immobilized urease in
1992), recent investigations focus on the improvement of strength the remediation of surface cracks in concrete, to our knowledge no
as a result of the cementation of sand particles (Whifn, 2004; published results are available at the moment.
Kucharski et al., 2006). The latter is due to the particle binding As an extension to their research on biodeposition on cementi-
properties of the microbially produced carbonates. tious materials, De Belie and De Muynck (2008) further investigated
The hydrolysis of urea was selected as a very suitable pathway the use of microbially induced carbonate precipitation for the repair
for the production of carbonate ions due to its ability to alkalinize of cracks in concrete. For the protection of B. sphaericus from the
the environment. Furthermore, urea is an important organic nitro- alkaline pH conditions, bacteria were immobilized in a silica sol.
gen carrier in natural environments and is commonly used as an Upon the addition of a salt, a bioceramic material (biocer) was
agricultural fertilizer (Nielsen et al., 1998). Moreover, the ability to formed, which was able to bridge the crack. Subsequent addition
hydrolyze urea is widely distributed among indigenous bacteria in of a urea and calcium chloride solution resulted in the formation of
soils and groundwater systems (Mobley and Hausinger, 1989; Fujita carbonate crystals inside the pores of the biocer and concomitantly
et al., 2000). Urea-utilizing bacteria such as Sporosarcina pasteurii sealing of the crack. As a result, a decrease of the water permeabil-
and Sporosarcina ureae are commonly isolated from soil, water, ity, similar to that obtained with traditional epoxy injections, was
sewage and incrustations on urinals. observed.
The participation of S. pasteurii in sand consolidation has been
demonstrated by Kantzas et al. (1992). Gollapudi et al. (1995) fur- 4.3. Bacterial concrete (USA, India)
ther investigated the use of S. pasteurii for the plugging of sand
columns. Although the bacteria were mixed with the sand slurry, Besides external application of bacteria in the case of reme-
consolidation mainly occurred near the surface. Stocks-Fischer et diation of cracks, microorganisms have also been applied in the
al. (1999) showed that microorganisms directly participated in the concrete mixture. Until now, research has mainly focused on the
calcite precipitation by providing a nucleation site and by creating consequences of this addition on the material properties of con-
an alkaline environment which favoured the precipitation of calcite. crete, i.e. strength and durability. Both properties depend on the
Zhong and Islam (1995) used the consolidation of sand mixtures for microstructure of the concrete. However, the effects of the presence
the remediation of cracks in granite. Cracks in granite were packed of the microorganisms and/or the microbially induced carbonates
with a mixture of bacteria, nutrients and a ller material. Among on the microstructure still need to be elucidated, especially the
the different materials that were mixed with S. pasteurii, the silica interaction between the biomass and the cement matrix.
fume (10%) and sand (90%) mixture lead to the highest compressive Ramachandran et al. (2001) investigated the use of microbio-
strength and lowest permeability. logically induced mineral precipitation for the improvement of the
As a further extension to this research, Ramachandran et al. compressive strength of Portland cement mortar cubes. This study
(2001) investigated the microbiological remediation of cracks in identied the effect of the buffer solution and type and amount of
concrete. The authors proposed MICP as an effective way to seal microorganisms, i.e. S. pasteurii and P. aeruginosa, used. Further-
cracks. The appearance of cracks and ssures is an inevitable phe- more, in order to study the effect of the biomass, the inuence
nomenon during the ageing process of concrete structures upon of both living and dead cells was investigated. Before addition to
exposure to weather changes. If left untreated, cracks tend to the mortar mixture, bacteria were centrifuged and washed twice.
expand further and eventually lead to costly repair. Specimens with The nal pellets were then suspended in either saline or phos-
cracks lled with bacteria, nutrients and sand demonstrated a sig- phate buffer, which was subsequently added to the mixture. After
nicant increase in compressive strength and stiffness values when demolding, the mortar specimens were stored in a solution con-
compared with those without cells. The presence of calcite was, taining urea and calcium chloride for 7 days. Subsequently, the
however, limited to the surface areas of the crack. The authors specimens were cured in air until the measurement of the com-
attributed this to the fact that S. pasteurii grows more actively in pressive strength.
the presence of oxygen. Still, the highly alkaline pH (1213) of con- At lower concentrations, the presence of S. pasteurii was shown
crete was a major hindering factor to the growth of the moderate to increase the compressive strength of mortar cubes. While the 28-
alkaliphile S. pasteurii, whose growth optimum is around a pH of day compressive strength of the control cubes amounted to about
9. In order to protect the cells from the high pH, Day et al. (2003) 55 1 MPa, specimens treated with 103 cells cm3 had a compres-
investigated the effect of different ller materials on the effective- sive strength of about 65 1 MPa. The contribution of P. aeruginosa
ness of the crack remediation. Beams treated with bacteria and to the strength was found to be insignicant. From the X-ray diffrac-
polyurethane showed a higher improvement in stiffness compared tion (XRD) analysis, no signicant increased amounts of calcite
to ller materials such as lime, silica, y ash and sand. According could be found in mortar specimens treated with bacteria. This
to the authors, the porous nature of the polyurethane minimizes could be attributed to the inhibition of the microorganisms by the
transfer limitations to substrates and supports the growth of bac- high pH and the lack of oxygen inside the mortar mixture. The over-
teria more efciently than other lling materials, enabling an all increase of strength, therefore, resulted from the presence of an
accumulation of calcite in deeper areas of the crack. No differ- adequate amount of organic substances in the matrix due to the
ences could be observed between the overall performances of free microbial biomass. However, an increase of the biomass, as dead
or polyurethane immobilized cells in the precipitation of carbon- cells in particular, resulted in a decreased strength. According to the
ate (Bang et al., 2001). In addition to this research, Bachmeier et al. authors, this could be attributed to the disintegration of the organic
(2002) investigated the precipitation of calcium carbonate with the matter with time, making the matrix more porous (Ramachandran
urease enzyme immobilized on polyurethane. The immobilization 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

Table 6
Approximate costs of surface treatments.

Treatment Price D /unit Dosage unit/m2 Product D /m2 No. of applic. Prod. + applic. D /m2

Calcite bioconcept 2328a


Growth medium 1 D g1 23 g 23 1
Nutrical 0.2 D g1 816 g(5) 24(5) 5
Water repellents 2.54 D L1 0.51 L 1.254 1 1525
Consolidants 1015 D L1 >1 L >10 1 >30
Unaltered stone.
Sculptured and degraded stone.

Ramakrishnan et al. (2001) investigated the effect of this tech- ponents, microorganisms and nutrients, in the matrix to ensure
nique on the durability of concrete. The presence of bacteria was minimal externally needed triggers. Therefore, the authors inves-
observed to increase the resistance of concrete towards alkali, sul- tigated the compatibility of different organic compounds with the
fate, freeze thaw attack and drying shrinkage; the effect being more cement matrix. Moreover, suitable bacteria should be able to sur-
pronounced with increasing concentrations of bacterial cells. The vive concrete incorporation for prolonged periods of time. For that
authors attributed this to the presence of a calcite layer on the sur- purpose, alkali-resistant spore forming bacteria related to the genus
face, as conrmed by XRD analysis, lowering the permeability of Bacillus, Bacillus pseudormus DSM 8715 and Bacillus cohnii DSM
the specimens. The best results were obtained with the phosphate 6307, were selected. In addition, the bacteria were added as spores,
buffer. as these are known for their ability to endure extreme mechanical
Ghosh et al. (2005) demonstrated the positive effect of the and chemical stress. On top of this, the authors decided to choose
addition of Shewanella on the compressive strength of mortar spec- a pathway different from the hydrolysis of urea for the production
imens. Contrary to the aforementioned research, these authors did of carbonate ions. In this way, possible negative effects of the pro-
not intend mineral precipitation, as these specimens were cured in duced ammonia on the reinforcement corrosion and degradation
air and not in a nutrient containing medium. An increase of 25% of the concrete matrix (when further oxidized by bacteria to yield
of the 28 days compressive strength was obtained for a cell con- nitric acid) could be avoided. Among the components selected, cal-
centration of about 105 cells mL1 and a water to cement ratio of cium lactate did not substantially affect the compressive strength
0.4. For these samples, the presence of a brous material inside the values. Furthermore, the addition of a high number of bacterial
pores could be noticed. As a result, a modication of the pore size spores (108 cm3 ) resulted in a decrease of strength of less than 10%.
distribution was observed. The positive effect of the addition of She- For the evaluation of the mineral producing capacity, healing
wanella improved with increasing curing times. For a concentration agent-incorporated specimens and control specimens were broken
of 105 cells mL1 , an increase of the compressive strength of 17% and to pieces after 7 or 28 days curing, immersed in tap water for 8 days
25% was observed after 7 and 28 days, respectively. However, no and subsequently analyzed by ESEM. While a massive production of
increase of the compressive strength was observed with additions larger-sized CaCO3 precipitates was observed for the 7 days cured
of Escherichia coli to the mortar mixture. This led the authors to sug- specimens, no differences could be observed between the healing
gest that the choice of the microorganism plays an important role agent incorporated specimens and control specimens after 28 days.
in the improvement of the compressive strength. More specically, The authors related this to a decrease of the viability of the spores
the production of EPS by the bacteria seemed to be of importance. 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).
4.4. Self-healing concrete (the Netherlands)
5. Cost evaluation
As an extension to the aforementioned research, Jonkers (2007)
and Jonkers and Schlangen (2007) investigated the use of bac- 5.1. Biodeposition
teria as self-healing agents for the autonomous remediation of
cracks in concrete (Fig. 3). In contrast with previous studies, such Table 6 gives an overview of the costs related to the application
an approach necessitated the presence of all the reaction com- of surface treatments to building materials (Personal communi-
W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 131

cation by an employee of the Belgian company FTB remmers, of the Notre Dame de Paris. The treatment has also been applied on
2008; http://www.ftbremmers.com/). The costs of the biodeposi- the facade of warehouses (ex. Galeries Lafayette, Paris), hotels and
tion treatment are attributable both to the price of the product apartments in and around Paris. As a consequence, the company
and the number of applications required. The theoretic price of the Calcite Bioconcept has an estimated annual turnover of the order of
product depends on the price of the microorganisms and the price 100,000150,000 D (Personal communication by Loubire, 2008).
of the nutrients. One kilogram of lyophilized bacteria (1011 CFU g1 )
costs around 1100 D kg1 . As bacteria are applied in a concentra-
5.2. Biocementation
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
In contrast with the biodeposition treatment, the added value
on the porosity of the stone, the dosage ranges between 0.04 and
of the biocementation treatment in building materials is less pro-
0.08 kg m2 , bringing the cost of the nutrients to 715 D m2 . This
nounced. As a consequence, more difculties in competing with
brings the total product cost around 1017 D m2 . In addition, the
traditional treatments can be expected.
total price of the treatment amounts to about 2328 D m2 . The
In the case of the biological mortar, a similar performance could
latter includes the costs of application and the added value of the
be obtained with traditional lime mortars, also being compatible
product. In case heavily degraded surfaces need to be treated, the
with limestone. As mortars consist of a mixture of sand, water and
cost of the treatment will be between 35 and 40 D m2 (Personal
a binder, it is the latter which will make up the cost when com-
communication by Loubire, 2008).
paring the two kinds of mortars. For non-hydraulic lime mortars,
In case a carrier material is applied, such as proposed by
the cost of the binder amounts to about 0.6 D kg1 (Carmeuse, Bel-
the Biobrush consortium (May, 2005), the biodeposition treat-
gium, 2008). For biological mortars, however, the binder consists
ment becomes even more costly. The price of high density
of a mixture of nutrients (about 180 D kg1 ) and a bacterial paste
polyethylene sheets, as used on external wall assemblies, amounts
(1100 D kg1 ) (Personal communication by Loubire, 2008).
to about 2.3 D m2 (http://order.americanmicroinc.com/cgi-
Especially in the case of crack repair of cementitious materials,
bin/americanmicroinc/VB10X25X6.html). For the formation of
at the moment, little or no added value is obtained. Because of the
a gel, 10 g L1 of Carbogel (27.6 D kg1 , Carbogel, Brazil, 2008;
fact that (organic) carrier materials are needed to protect the bac-
http://www.carbogel.com.br/) is required. Considering a proposed
teria from the alkaline environment, the ecological aspect of the
thickness of the gel of about 1 cm, this will bring the cost per m2
treatment has been largely reduced. Furthermore, the method cur-
to about 2.8 D m2 . The Japanese paper brings about an extra cost
rently seems unfeasible to be readily applicable in practice due to
of about 12.3 D m2 (http://japanesepaperplace.com/retail/retail-
the large amount of specialist work needed.
products/conservation-papers.htm). As a result of the carrier
Only in the case of self-healing building materials can a sig-
material, however, less applications of moisture (and hence nutri-
nicant added value be expected. The latter is, however, largely
ents) will be necessary. The resulting decrease in cost will be
attributable to the concept of self-healing materials, decreasing the
marginal compared to the costs of the carrier. Additionally, the
needs for manual inspection and repair. The research is still in its
disposal of the carrier material will also present an extra cost.
infancy, and it will be largely questionable whether bacteria will be
As mentioned before, the method proposed by Jimenez-Lopez
able to remain viable for a prolonged time and upon activation be
et al. (2007) could offer an economical advantage over the Cal-
able to seal the cracks.
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- 6. Considerations
ber of applications of nutrients, and hence, loss of the economical
advantage. In our opinion, the feasibility of the biodeposition treatment in
Due to the price of its constituents, the biodeposition treatment practice largely depends on the time required for carbonate produc-
will never be able to compete with some of the traditional surface tion, and hence, precipitation to occur. The latter has also important
treatments on a pure economical basis. The focus of this kind of consequences on the economical aspect of the treatment.
treatment should, therefore, be on the added value compared to In the case of the application of calcinogenic bacteria, longer
other treatments. times required for precipitation to occur necessitate longer periods
The biodeposition treatment presents an ecological, envi- during which the building material has to remain wet. This is due to
ronmentally friendly alternative over the other treatments. the fact that microorganisms require a minimum amount of water
Furthermore, the application of a layer of calcium carbonate to to remain active. With increasing times for precipitation, increasing
limestone ts in the current restoration concept of compatibility. amounts of EPS production, biolm formation and hence plugging
Some of the traditional surface treatments, typically organic resins, can be expected. In order to ensure the presence of a sufcient
have shown long-term incompatibilities with the stone. The latter amount of water, multiple applications of nutrients over several
resulted in a much more intense damage to the stone than would days (Le Metayer-Levrel et al., 1999) or the application of a carrier
have occurred without restoration, necessitating replacement and material (May, 2005) have been proposed. However, both measures
costly repair. have a signicant inuence on the total cost of the treatment, as
In addition, the biological deposited crystals show some unique can be seen from the previous paragraphs. Increasing the number
properties. Due to their growth on pre-existing calcite crystals and of applications of nutrients increases the cost of the treatment due
the incorporation of organic molecules, these crystals are strongly to the extra man hours needed, while the use of a carrier material
attached to the surface, exerting a consolidating effect (Rodriguez- has a major inuence on the product cost.
Navarro et al., 2003). Furthermore, by adding pigments to the From the different microbial metabolic pathways proposed for
medium, it is also possible to create a surcial patina, allowing the biodeposition, the hydrolysis of urea results, without any doubt in
concealment of new replacement stone (Le Metayer-Levrel et al., the fastest production of carbonate ions and hence precipitation
1999). of calcium carbonate. This is due to the fact that the hydrolysis
Until now, practical applications of the biodeposition treatment of urea is a very rapid process and depends on only one enzyme.
have been mainly limited to France. The treatment has been applied As a consequence, no additional nutrients are necessary for the
on several historic monuments across the country, including a part 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 carbonate, resulting in a decrease of the pH, counteracts the pH
could be drastically decreased. Consequently, the outgrowth of increase as a result of the release of ammonia.
other microorganisms (e.g. fungi and heterotrophic bacteria) will Nonetheless, even in the case of the ureolytic biodeposition
be highly unlikely. This makes the hydrolysis of urea a very feasible treatment the production of ammonium will be rather low com-
pathway for applications in situ. From the above, it is clear that the pared to conventional sources of nitrogen pollution, i.e. agriculture
hydrolysis of urea could present some economical advantages over and domestic waste water. The treatment of 1 m2 of building mate-
the other pathways. rial with 1 L of a biodeposition medium containing 10 g L1 urea,
In addition to the feasibility, which is governed by the time results in the production of 4.7 g N. For comparison, from waste
required for production of carbonates to occur (see previous para- water treatment plants it can be calculated that one person pro-
graphs), the efciency of the biodeposition treatment has been duces between 6 and 16 g of N per day (DeCuyper and Loutz, 1992).
observed to depend on the speed of precipitation. The presence of ammonium might also present some risks to
Rodriguez-Navarro et al. (2003) reported on the importance the stone itself. First of all, the presence of an ammonium salt might
of the type and structure of the precipitated CaCO3 polymorphs present some risks related to salt damage. Depending on the type
(vaterite or calcite) on the efciency of the biodeposition treatment. of calcium salt used, ammonium acetate or ammonium chloride
The presence of well developed rhombohedral calcite crystals will be present in the stone after treatment. To our knowledge, no
resulted in a more pronounced consolidating effect compared to reports are available on the effect of these salts on stone. Therefore,
the presence of tiny acicular vaterite crystals. future investigations should investigate the retention of these salts
Differences in size and morphology of the crystals can be in the stone.
attributed to differences in the saturation state of a system pre- Secondly, ammonium can be converted to nitric acid by the
ceding nucleation, with large rhombohedral calcite crystals being activity of nitrifying bacteria, resulting in damage to the stone.
formed at relatively low supersaturation and vaterite crystals being However, Mansch and Bock (1998) observed that the initial col-
produced under highly supersaturated conditions. From the above, onization of natural stone by nitrifying bacteria takes several years.
the authors concluded that fast precipitation could result in a lower In addition, the extent of colonization is mainly governed by the pH
efciency of the biodeposition treatment. of the pore solution, with a pH between 7 and 9 being optimal for
According to Rodriguez-Navarro et al. (2003), the presence of growth. As the initial pH of the biodeposition liquid is around 9.3,
a phosphate buffer could explain for the occurrence of rhombo- the activity of the nitrifying bacteria will be suppressed. Moreover,
hedral crystals. They attributed this to the buffering effect of the the applied chemoorganotrophic carbonate producing bacteria will
phosphate, preventing rapid local pH variations, and hence, rapid outcompete the nitrifying bacteria for oxygen during the precipita-
changes in the saturation state of the system. However, from the tion process. As a result of the precipitation, however, the pH will
papers by Jimenez-Lopez et al. (2007, 2008), it appears that the drop to a value of about seven. Therefore, in order to avoid nitrica-
phosphate buffer is not the only compositional difference between tion in the long-term, the presence of large amounts of ammonium
the M-3 and M-3P media (composition see Table 5). From the salts should be avoided. From long-term observations on the ef-
graphs indicating the removal of calcium ions from solution, it can ciency of the Calcite Bioconcept treatment, however, no damages
be clearly observed that the initial concentration of calcium ions to the stone have been reported.
was much higher in the M-3 medium (50 mM Ca2+ ) compared If higher concentrations of ammonium should be produced, as
to that of the M-3P medium (35 mM Ca2+ ). The latter could be might be the case for the hydrolysis of urea, the use of a paste
attributed to the precipitation of calcium phosphate which was might offer an attractive solution. The latter is one of the most
removed before the start of the experiment (Personal communi- commonly applied methods for the removal of salts from build-
cation by Jimenez-Lopez et al., 2008). As a result, the M-3 medium ing materials (Wooltt and Abrey, 2008; Carretero et al., 2006).
showed initially a higher saturation state compared to the M-3P Upon wet application, the paste facilitates the dissolution of salts
medium. within stones and migration of ions to the outside, where they
In spite of the high speed of carbonate formation and calcium recrystallize and are retained. Once dry, the paste can be easily
dosages used, De Muynck et al. (2009) obtained an excellent water- removed. Different types of pastes or combinations thereof have
proong and consolidating effect with an ureolytic biodeposition been applied for such purposes: paper pulp, clay materials (sepio-
treatment on Euville limestone. From SEM examinations, the pres- lite, bentonite) and cellulose derivatives. As a result of their unique
ence of rhombohedral crystals could be clearly observed. properties, many of these materials have also been used for the
Besides the hydrolysis of urea, most MICP treatments rely on immobilization of microorganisms in a variety of elds. A com-
the production of ammonia for the alkalinization of the culture bination of these two applications has already been applied for
medium. Because of the fact that atmospheric ammonia is being the removal of black crusts on stone artworks (Ranalli et al., 1997;
recognized as a pollutant, the in situ use of such treatments might Cappitelli et al., 2006). While Carbogel was observed to remove
raise some issues of environmental concern. Atmospheric ammo- about 42% of the calcium ions from a black crust, the combina-
nia is known to contribute to several environmental problems, tion of the former with sulphate reducing bacteria led to a total
including direct toxic effects on vegetation, atmospheric nitro- removal efciency of about 95%. Besides removing the produced
gen deposition, leading to the eutrophication and acidication of ammonium, the use of a paste will also protect the bacteria from
sensitive ecosystems, and to the formation of secondary partic- drying out, enhancing the overall biodeposition treatment, as was
ulate matter in the atmosphere, with effects on human health, observed by the Biobrush consortium (May, 2005). As seen before,
atmospheric visibility and global radiative balance (Sutton et al., however, the use of a paste results in a higher cost for the treat-
2008). ment.
However, when the concentration of ammonia generating com- In their search for alternative approaches towards the Calcite
pounds does not exceed the concentration of the calcium salt, it is Bioconcept method, most researchers have focused on the use of
possible to decrease the emission of ammonia to a great extent. De different organisms or metabolic pathways. Little attention, how-
Muynck et al. (2009) observed in their biodeposition experiments ever, has been paid to the inuence of the dosage (g m2 ) or
that the pH of the solution remained about 7. At these pH values, concentration (g L1 ) of the calcium salt and the nutrients (i.e.
ammonium will be the predominating compound. The neutral pH carbonate precursor components such as urea or amino acids)
could be attributed to the fact that the precipitation of calcium on the global effectiveness of the treatment. In many cases, the
W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 133

authors just applied the medium which had been used to illustrate tribution could be considered as one of the most determining
the carbonate precipitation potential of the strain. An overview of factors. Samonin and Elikova (2004) reported that for a maxi-
the different concentrations of calcium salts used can be seen in mum adsorption of microbial cells, the adsorbent pores must be
Table 5. 25 times larger than the cells. Therefore, the amount of bacte-
The importance of the calcium dosage on the overall effective- ria retained in high macroporosity stones will be higher than in
ness of the biodeposition treatment can be easily demonstrated high microporosity stones. As a consequence, carbonate precip-
by the following example. In the Calcite Bioconcept method, the itation can occur at higher depths in macroporous stone. From
total calcium dosage amounts to about 5.5 g m2 calcium chlo- SEM analyses, precipitation has been observed at depths of about
ride (MW 147 g mol1 ) or 1.5 g m2 calcium. Theoretically, this will 100 m for the Calcite Bioconcept treatment (Personal communi-
result in an overall precipitation of about 3.74 g calcium carbonate cation by Loubire, 2008). As mentioned earlier, Rodriguez-Navarro
(MW 100 g mol1 ) per square meter of stone surface. Assuming a et al. (2003) observed precipitation at depths greater than 500 m
density of calcium carbonate of 2.71 g cm3 and a homogenous pre- in a bioclastic calcarenite. De Muynck et al. (2008b) observed an
cipitation over 1 m2 of a non-porous stone, this corresponds with increased amount of biomass adsorption in mortar specimens with
a layer of calcium carbonate of about 1.38 m in thickness. In the increasing water to cement ratio (w/c). The authors attributed this
case of a porous stone, a smaller thickness is expected due to the to the increasing amount of pores with a diameter larger than 1 m
high surface area of the pores. In practice, however, bacteria will in specimens with increasing w/c. Since the amount of capillary
be mainly retained in the pores of the stone, especially those pores pores between 2 and 10 m is rather limited in cementitious mate-
with a diameter larger than 1 m. Consequently, precipitation will rials, the authors concluded that for these types of materials, the
mainly occur around large pores, and layer thicknesses greater than biodeposition treatment is mainly a surface phenomenon. This was
1 m can be observed. This is in agreement with the ndings of Le also observed from thin sections, where a layer of crystals within
Metayer-Levrel et al. (1999) and Orial (2000), who observed layer the range of 1040 m on the surface was found, correspond-
thicknesses of about 23 m and 45 m, respectively. ing with the theoretical thickness calculated from the calcium
Let us now consider the methodology as proposed by Rodriguez- dosage.
Navarro et al. (2003). In one of their experiments, the authors From Table 1 it is clear that the differences between the various
submerged limestone prisms of 2.5 by 4.5 by 0.5 cm in an Erlen- methodologies are not limited to the mediator used for precipita-
meyer ask containing 100 mL of M-3 solution. As such, the tion. In addition, different research groups used different dosages
theoretical calcium dosage amounts to about 339 g m2 calcium of calcium salts and different application procedures on different
acetate (MW 230 g mol1 ) or 59 g m2 calcium, corresponding with types of stone. Besides the different metabolic pathways and bacte-
an overall precipitation of about 147 g calcium carbonate per square ria proposed, the difference in inoculum size could also account for
meter. This will in theory result in a layer of calcium carbonate with the differences in time required for precipitation to occur. Further-
a thickness of about 54 m on the surface of the limestone prisms. more, many experiments were performed under sterile conditions.
Although the authors did not report on the thickness of the carbon- However, for applications in situ growth and activity are required
ate layer, cementation was found up to depths larger than 500 m under non-sterile conditions. This could potentially inuence on
(Rodriguez-Navarro et al., 2003). the microbial activity. From the above mentioned it should be clear
As a result of the more pronounced precipitation of calcium that this will hamper any quantitative comparison between the dif-
carbonate in the case of the methodology proposed by Rodriguez- ferent treatments. Additionally, such a comparison is even more
Navarro et al. (2003), a larger consolidating effect can be observed difcult due to the fact that different authors used different eval-
compared to the Calcite Bioconcept treatment. In their work on con- uation parameters and procedures (Table 1). Some authors mainly
solidation of sand columns by means of biocementation, Whifn focused on the waterproong effect (Dick et al., 2006), while others
et al. (2007) observed that a minimum amount of carbonate mainly investigated the strengthening effect (Rodriguez-Navarro
precipitation per m3 of sand was required in order to obtain a sig- et al., 2003; Jimenez-Lopez et al., 2007). In addition to these two
nicant consolidating effect. DeJong et al. (2006) observed that effects, Tiano et al. (1999) further proposed the evaluation of the
the cementing effect occurred as a result of the precipitated cal- visual aspect before and after treatment by means of colorimetric
cite forming bonds at the particle-particle contacts of sand grains. analysis.
With increasing concentrations of precipitated carbonate, increas- Therefore, the next step in research regarding the application of
ing bond formation and hence consolidation can be obtained. calcinogenic bacteria should be a qualitative and quantitative eval-
Therefore, increasing amounts of carbonate precipitates could uation of the different methodologies under identical conditions.
result in an increased protective effect of the biodeposition treat- Besides the evaluation of the protective performance (strength-
ment. This was indeed observed by De Muynck et al. (2009), who ening and waterproong (incl. porosity)), the inuence of the
noticed an increased waterproong with increasing numbers of treatment on the visual aspect should be investigated. From this,
treatments or increasing the concentration of the crystal precur- the exact role of the microorganism and the metabolic pathway can
sors in one treatment. The latter is also known to play a role in the be distinguished among the other parameters contributing to the
speed, and hence, the type of crystals that are formed, affecting the overall effectiveness. An expanded knowledge on these factors will
global effectiveness of a treatment (Whifn et al., 2007). no doubt contribute to the added value of the biodeposition treat-
An increase in the dosage of the calcium salt could, however, lead ment, which is an ecological, compatible surface treatment with a
to an accumulation of salts in the stone, which could depending on high protective effect.
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- 7. Future perspectives
eral tests did not reveal any problems related to salt damage. The
chlorides are rapidly washed away as a result of raining (Personal In 2010, the patent of Adolphe et al. (1990) will expire.
communication, Loubire, 2008). This will certainly lead to further explorations of the biodepo-
Besides the dosage of the calcium salt used, the type of stone sition technique by the different research groups. As a result,
will also have a major impact on the global performance of the reports on experiences from life size experiments can be soon
treatment. The porosity, and more specically the pore size dis- expected.
134 W. De Muynck et al. / Ecological Engineering 36 (2010) 118136

In addition, promising results on the use of microorganisms for been hampered as a result of the differences in experimental pro-
the improvement of the durability of building materials have drawn cedures between the different research groups. In our opinion, too
the attention of research groups all over the world. Until now, work little attention has been paid to the calcium dosage and the type
on biodeposition was mainly concentrated in Europe, while much of stone, which could largely attribute for the differences observed
of the work on remediation of cracks in concrete has been done between the various treatments. In this review, some recommen-
in the USA. Recently, results from preliminary studies on bacteri- dations have been made to improve the in situ feasibility of this
ally induced carbonate precipitation from other research groups type of treatment, both from an economical and practical point of
have come to our knowledge. In China, researchers are investigat- view. In addition to the biodeposition treatment, the use of bacte-
ing the use of microbially induced carbonate precipitation for the rially induced carbonates as a binder, i.e. biocementation, has been
restoration of ancient masonry buildings (Shen and Cheng, 2008) addressed. An overview has been given of the different elds of
and the protection of concrete surfaces (Chunxiang et al., 2009). applications and their future prospects.
Furthermore, the method of producing CaCO3 by bacterial biomin-
eralization has been patented in China by Qian et al. (2007). The Acknowledgements
latter was possible, as the patent by Adolphe et al. (1990) was
limited to European countries. In Brazil, Shirakawa et al. (2008) This research was funded by a BOF grant from Ghent Univer-
are working on biodeposition on ber cement roof tiles. In India, sity. The authors would like to thank Nico Boon, Bart De Gusseme,
researchers have applied for a patent in which the use of Shewanella Melissa Dunkle and Siegfried E. Vlaeminck for critically reading the
for the improvement of the strength of concrete was described manuscript.
(Saroj, 2006).
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