Ecological Engineering 36 (2010) 118136

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

Review

Microbial carbonate precipitation in construction materials: A review

Willem De Muynck a,b , Nele De Belie a, , Willy Verstraete b,1
a b

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
Evidence of microbial involvement in carbonate precipitation has led to the exploration of this process in the eld of construction materials. One of the rst patented applications concerned the protection of ornamental stone by means of a microbially deposited carbonate layer, i.e. biodeposition. The promising 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 performing. 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 interpretation of the differences observed in effectiveness. Furthermore, recommendations to improve the in situ feasibility of the biodeposition method are postulated. In the second part of this paper, the use of microbially induced carbonates as a binder material, i.e. biocementation, is discussed. Bacteria have been added to concrete for the improvement of compressive strength and the remediation of cracks. Current studies are evaluating the potential of bacteria as self-healing agents for the autonomous decrease of permeability of concrete upon crack formation. 2009 Elsevier B.V. All rights reserved.

Article history: Received 12 August 2008 Received in revised form 11 February 2009 Accepted 13 February 2009

Keywords: Bacteria Stone Biomineralization Biodeposition Biomortar Biocement Bioconcrete Calcite Conservation MICP

1. Introduction Construction materials such as stone and concrete are subjected to the weathering action of several physical, chemical and biological factors (Saiz-Jimenez, 1997; Le Metayer-Levrel et al., 1999; Warscheid and Braams, 2000). Because of their composition and textural characteristics, carbonate stones (limestones, dolostones and marbles) are particularly susceptible to weathering. Progressive dissolution of the mineral matrix as a consequence of weathering leads to an increase of the porosity, and as a result, a decrease of the mechanical features (Tiano et al., 1999). In order to decrease the susceptibility to decay, many conservation treatments have been applied with the aim of modifying some of the stone characteristics. Water repellents have been applied to protect stone from the ingress of water and other weathering agents. The use of stone consolidants aims at re-establishing the cohesion between grains of deteriorated stone. However, both conservation treatments are subject to frequent controversy due to their nonreversible action and their limited long-term performance. Because

Corresponding author. Tel.: +32 092645522; fax: +32 092645845. E-mail addresses: Willem.DeMuynck@UGent.be (W. De Muynck), Nele.DeBelie@UGent.be (N. De Belie), Willy.Verstraete@UGent.be (W. Verstraete). 1 Tel.: +32 092645976; fax: +32 092646248. 0925-8574/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2009.02.006

of problems related to incompatibility with the stone, both water repellents and consolidants have often been reported to accelerate stone decay. (Clifton and Frohnsdorff, 1982; Delgado Rodrigues, 2001; Moropoulou et al., 2003). Organic treatments commonly result in the formation of incompatible and often harmful surface lms. Additionally, because large quantities of organic solvents are used, they contribute to pollution (Camaiti et al., 1988; Rodriguez-Navarro et al., 2003). Inorganic consolidation may be preferable since stone materials and protective or consolidating materials share some physico-chemical afnity (Rodriguez-Navarro et al., 2003). Some researchers have tried to develop methods based on the reintroduction of calcite into the pores of limestone. The lime-water technique, i.e. application of a saturated solution of calcium hydroxide, has been proposed and experimented both for wall painting mortars and for some deteriorated calcareous stones, in order to impart a slight water repellent and consolidating effect (Tiano et al., 1999). As of yet, little success has been achieved in consolidating stone with inorganic materials. Some of the reasons for the poor performance of inorganic consolidants are their tendencies to produce shallow and hard crusts because of their poor penetration abilities, the formation of soluble salts as reaction by-products, growth of precipitated crystals and the questionable ability of some of them to bind stone particles together (Clifton and Frohnsdorff, 1982). In the case of the calcite reintroduction methods, the latter is attributable to the production

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119

of many small crystallites, which are not chemically bound to the internal surface of the pore and which are not able to bridge the pores (Tiano et al., 2006). Recently, bacterially induced carbonate precipitation has been proposed as an environmentally friendly method to protect decayed ornamental stone. The method relies on the bacterially induced formation of a compatible carbonate precipitate on limestone, and unlike the lime-water treatment, the carbonate cement appears to be highly coherent (Le Metayer-Levrel et al., 1999). In addition, this technique has been explored for the improvement of the durability of cementitious materials (Ramachandran et al., 2001; Ramakrishnan et al., 2001; De Muynck et al., 2008a,b). 2. Microbially induced carbonate precipitation (MICP) Like other biomineralization processes, calcium carbonate (CaCO3 ) precipitation can occur by two different mechanisms: biologically controlled or induced (Lowenstan and Weiner, 1988). In biologically controlled mineralization, the organism controls the process, i.e. nucleation and growth of the mineral particles, to a high degree. The organism synthesizes minerals in a form that is unique to that species, independently of environmental conditions. Examples of controlled mineralization are magnetite formation in magnetotactic bacteria (Bazylinski et al., 2007) and silica deposition in the unicellular algae coccolithophores and diatoms, respectively (Barabesi et al., 2007). However, calcium carbonate production by bacteria is generally regarded as induced, as the type of mineral produced is largely dependent on the environmental conditions (Rivadeneyra et al., 1994) and no specialized structures or specic molecular mechanism are thought to be involved (Barabesi et al., 2007). Different types of bacteria, as well as abiotic factors (salinity and composition of the medium) seem to contribute in a variety of ways to calcium carbonate precipitation in a wide range of different environments (Knorre and Krumbein, 2000; Rivadeneyra et al., 2004). Calcium carbonate precipitation is a rather straightforward chemical process governed mainly by four key factors: (1) the calcium concentration, (2) the concentration of dissolved inorganic carbon (DIC), (3) the pH and (4) the availability of nucleation sites (Hammes and Verstraete, 2002). CaCO3 precipitation requires sufcient calcium and carbonate ions so that the ion activity product (IAP) exceeds the solubility constant (Kso ) (Eqs. (1) and (2)). From the comparison of the IAP with the Kso the saturation state () of the system can be dened; if > 1 the system is oversaturated and precipitation is likely (Morse, 1983): Ca2+ + CO3 2 CaCO3 = a(Ca
2+

(1) with K so calcite, 25 = 4.8 10

9

)a(CO3

)/K so

(2)

The concentration of carbonate ions is related to the concentration of DIC and the pH of a given aquatic system. In addition, the concentration of DIC depends on several environmental parameters such as temperature and the partial pressure of carbon dioxide (for systems exposed to the atmosphere). The equilibrium reactions and constants governing the dissolution of CO2 in aqueous media (25 C and 1 atm) are given in Eqs. (3)(6) (Stumm and Morgan, 1981): CO2(g) CO2(aq.)
+

(pK H = 1.468)

(3) (4) (5) (6)

CO2(aq.) + H2 O H2 CO3 H2 CO3 H + HCO3 HCO3 CO3

2 +

(pK = 2.84) (pK 1 = 6.352) (pK 2 = 10.329)

+H

With H2 CO3 = CO2(aq.) + H2 CO3 .

Microorganisms can inuence precipitation by altering almost any of the precipitation parameters described above, either separately or in various combinations with one another (Hammes and Verstraete, 2002). However, the primary role has been ascribed to their ability to create an alkaline environment through various physiological activities. Both autotrophic and heterotrophic pathways are involved in the creation of such an alkaline environment (for an extensive review, see Castanier et al., 1999). While the environmental conditions of heterotrophic pathways are diverse (aerobiosis, anaerobiosis and microaerophily), carbonate precipitation always appears to be a response of the heterotrophic bacterial communities to an enrichment of the environment in organic matter (Castanier et al., 1999). A rst heterotrophic pathway involves the sulphur cycle, in particular the dissimilatory sulphate reduction, which is carried out by sulphate reducing bacteria under anoxic conditions. A second heterotrophic pathway involves the nitrogen cycle, and more specically, (1) the oxidative deamination of amino acids in aerobiosis, (2) the dissimilatory reduction of nitrate in anaerobiosis or microaerophily and (3) the degradation of urea or uric acid in aerobiosis. Another microbial process that leads to an increase of both the pH and the concentration of dissolved inorganic carbon is the utilization of organic acids (Braissant et al., 2002), a process which has been commonly used in microbial carbonate precipitation experiments. The precipitation pathways described above are general in nature, which accounts for the common occurrence of microbial carbonate precipitation (MCP) and validates the statement by Boquet et al. (1973) that under suitable conditions, most bacteria are capable of inducing carbonate precipitation. In addition, carbonate particles can also be produced by ion exchange through the cell membrane (Rivadeneyra et al., 1994; Castanier et al., 1999). Besides changes induced in the macro-environment, bacteria have also been reported to inuence calcium carbonate precipitation by acting as sites of nucleation or calcium enrichment (Morita, 1980). Due to the presence of several negatively charged groups on the cell wall, at a neutral pH, positively charged metal ions can be bound on bacterial surfaces (Douglas and Beveridge, 1998; Ehrlich, 1998). Such bound metal ions (e.g. calcium) may subsequently react with anions (e.g. carbonate) to form an insoluble salt (e.g. calcium carbonate). In the case of a sufcient excess of the required cations and anions, the metal salt on the cell surface initiates mineral formation by acting as a nucleation site. The anion (e.g. carbonate) in this reaction may be a product of the bacterial metabolism, or it may have an abiotic origin (Ehrlich, 1998). Furthermore, it has been demonstrated that specic bacterial outer structures (glycocalyx and parietal polymers) consisting of exopolysaccharides and amino acids play an essential role in the morphology and mineralogy of bacterially induced carbonate precipitation (Braissant et al., 2003; Ercole et al., 2007). The actual role of the bacterial precipitation remains, however, a matter of debate. Some authors believe this precipitation to be an unwanted and accidental by-product of the metabolism (Knorre and Krumbein, 2000) while others think that it is a specic process with ecological benets for the precipitating organisms (Ehrlich, 1996; McConnaughey and Whelan, 1997). The evidence of microbial involvement in carbonate precipitation has subsequently led to the exploration of this process in a variety of elds. A rst series of applications is situated in the eld of bioremediation. In addition to conventional bioremediation strategies which rely on the biodegradation of organic pollutants (Chaturvedi et al., 2006; Simon et al., 2004), the use of MICP has been proposed for the removal of metal ions. Applications include the treatment of groundwater contaminated with heavy metals (Warren et al., 2001) and radionucleotides (Fujita et al., 2004), the removal of calcium from wastewater (Hammes et al., 2003).

120 Table 1 Characteristics of the various biodeposition processes. Characteristic Denition Type Goals Where/when Mediator How Research group Added value (Current) Limitations Status of use Process Biodeposition

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

Biologically induced deposition of a carbonate layer on the surface of building materials Surface treatment Improvement of the durability, consolidation and decrease of water absorption Applied on the surface of building materials such as stone, bricks and concrete Microorganisms Organic matrix molecules (OMM) Spraying of bacteria and nutrients Spraying of OMM and carbonate rich solution Application of poultice Calcite Bioconcept, Granada University, Ghent Bioreinforce consortium University Biobrush consortium Ecological, environmentally friendly, compatibility Possibility of including pigments Costs of bacteria and nutrients Costs of chemicals Rather limited efcacy The Calcite Bioconcept: laboratory and in situ Laboratory and in situ experiments experiments Several research groups: laboratory experiments Restoration sector Limestone, concrete Marble statues

Activator medium Application of nutrient media Granada University

Long activation period required Laboratory and in situ experiments

Market Niche

Limestone

Another series of applications aims at modifying the properties of soil, i.e. for the enhancement of oil recovery from oil reservoirs (Nemati and Voordouw, 2003; Nemati et al., 2005), plugging (Ferris and Stehmeier, 1992) and strengthening of sand columns (DeJong et al., 2006; Whifn et al., 2007). Moreover, microbially induced precipitation has been investigated for its potential to improve the durability of construction materials such as limestone and cementitious materials. The latter is dealt with in this review paper and can be divided into processes for the deposition of a protective surface layer with consolidating and/or waterproong properties, i.e. biodeposition (Tables 1 and 2), and processes for the generation of a biologically induced binder, i.e. biocementation (Tables 3 and 4). 3. Biodeposition Adolphe et al. (1990) were among the rst to consider the use of microbially induced carbonate precipitation (MICP) for the protection of ornamental stone. They applied for a patent regarding the use of calcinogenic bacteria on stone surfaces, as is discussed in Section 3.1.2. The promising results of this so-called Calcite Bioconcept technique encouraged different research groups to evaluate alternative approaches for the biomediated carbonate precipitation on limestone. These approaches can be mainly divided into those falling within and those falling outside the specications of the patent by Adolphe et al. (1990), i.e. the application of calcinogenic bacteria to a stone surface. The rst series of approaches (Sections 3.1.33.1.6), those falling within the patent specications, are characterized by the use of different microorganisms, metabolic pathways or delivery systems to overcome some of the potential limitations of the Calcite Bioconcept technique. The selection of a microorganism by the different research groups was often based on their experiences from previous studies on microbially induced mineral precipitation. In the second series of approaches, no microorganisms are applied to the surface. These approaches can be divided into studies where inducing macromolecules are supplied to the stone together with a supersaturated solution of calcium carbonate (Section 3.2) and studies which obtain carbonate precipitation by the microbiota inhabiting the stone (Section 3.3). In the latter, only nutrients are added to the stone. 3.1. Application of calcinogenic bacteria Before going into detail on the different methodologies proposed, a short chronological overview is given on the work

preceding the application of microbially induced carbonate production to building materials. 3.1.1. From carbonate precipitation in natural environments and laboratory conditions to applications in situ When exposed to atmospheric conditions, soft limestone quickly acquires a protective skin (calcin) through dissolution of carbonates within the pore water, evaporation and precipitation of calcite at or near the exposed surface (Dreesen and Dusar, 2004). This layer demonstrates a higher hardness and density compared to the underlying layers. As a result of atmospheric pollutants, however, this layer slowly degrades, losing its protective role. The discovery that bacteria contribute to the formation of limestone has led to the suggestion to use bacteria for the re-establishment of this calcin. Boquet et al. (1973) were among the rst to demonstrate the ability of soil bacteria to precipitate calcium carbonate under laboratory conditions. While previous research only concerned marine bacteria in liquid media (Drew, 1911; Shinano, 1972), the authors investigated crystal formation by soil bacteria on solid media. The authors obtained the best results with B4 medium (Table 5). Among the organisms tested, several Bacillus strains (incl. Bacillus cereus) and Pseudomonas aeruginosa were observed to form crystals. The authors concluded that crystal formation is a function of the medium, and that under suitable conditions most bacteria can form crystals. In parallel with the work done by this Spanish research group, Adolphe and Billy (1974) succeeded in the formation of calcite in the laboratory by bacteria isolated from tuff and travertine. Between 1983 and 1987, Castanier et al. (1999) investigated the different mechanisms responsible for the microbial formation of calcium carbonate, evidencing the microbial origin of limestone. Adolphe et al. (1989) further demonstrated the bacterial origin of the calcite crusts in extreme climates, such as Greenland and the Sahara desert. In addition, the team observed the great resistance of these layers towards erosion. From the above ndings, Adolphe et al. (1990) applied for a patent for the treatment of articial surfaces by virtue of a surface coating produced by microorganisms. In addition, a company, Calcite Bioconcept, was created. 3.1.2. Procedure according to Calcite Bioconcept (France) Although the ability of bacteria to precipitate calcium carbonate 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 Limestone Mediator Microorganisms Authors Calcite Bioconcept (Le Metayer-Levrel et al., 1999) Tiano et al. (1999) Rodriguez-Navarro et al. (2003) Dick et al. (2006) Biobrush (May, 2005) Organic matrix molecules Tiano (1995) and Tiano et al. (2006) Organism/molecule Bacillus cereus Micrococcus sp. Bacillus subtilis Myxococcus xanthus Bacillus sphaericus Pseudomonas putida Mytilus californianus shell extracts Metabolisma ODAA ODAA OAU ODAA OAU HU ODAA OAU n.a. Solutionb Growth medium (CB) and Nutrical B4 M-3, M-3P SF B4 AW Ammonium carbonate method or supersaturated bicarbonate solution Stone type (porosity) Tuffeau (40%), Saint Maximin (30%) Pietra di Lecce, bioclastic limestone (40%) Bioclastic calcarenite (2432%) Euville, crinoidal limestone (16%) Portland oolitic limestone (20%) Sound and articially aged marble (Gioia calcitic marble, 13.5%), limestone (Pietra di Lecce, 40%) and dolostone (Pietra dAngera, bioclastic, 20%)

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

Aspartic acid Bacillus cell fragments Activator medium Jimenez-Lopez et al. (2007) Microbiota inhabiting the stone ODAA OAU M-3, M-3P, CC Decayed limestone and quarry calcarenite stone from La Escribania (2432%) Concrete/mortar with CEM I 52.5 N (1520%); w/c 0.5, 0.6 and 0.7 Evaluation procedures Application procedure Bacteria Calcite bioconcept Culture in exponential phase: 107 to 109 cells mL1 Overnight culture: 106 cells cm2 Spraying Nutrients/chemicals Spraying (5 times) Septicity conditions Stone NS N/C NS Appl. NS Water absorption (Karsten pipe), SEM analysis, surface roughness (imprint moulding), colorimetry and Plate count Water absorption (contact sponge), colorimetric measurements, stone cohesion(drilling resistance measuring system) Stone cohesion (sonicator bath), weight increase, XRD and SEM analysis, porosimetry analysis Water absorption (immersion test) SEM analysis Water absorption and drying due to evaporation Water absorption (contact sponge), colorimetric measurements, stone cohesion (drilling resistance measuring system, peeling tape test), staining of newly formed calcite with Alizarin Red S and Calcein

Cementitious materials

Microorganisms

De Muynck et al. (2008a,b)

Bacillus sphaericus

HU

SF

Authors

Experimental methods Inoculum

Tiano et al.

Brushing on water saturated specimens

Wetting every day for 15 days

NS

Rodriguez-Navarro et al.

2% inoculum

Immersion in growing bacterial culture (shaking or stationary conditions) for 30 days

Dick et al. Biobrush Tiano et al.

1% inoculum 108 cells mL1 n.a.

Immersion in growing bacterial culture (intermediate wetting) for 28 days Spraying In Carbogel n.a. Immersion in test solution or spraying (in situ tests)

S NS NS

S NS NS

S NS NS

121

122

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

to investigate the viability and performance of these bacteria in situ. The technique was further optimized and industrialized as a result of the collaboration between the University of Nantes, the Laboratory for the research of historic monuments (LRMH) and the company Calcite Bioconcept (Le Metayer-Levrel et al., 1999). The rst step comprised the search for suitable microorganisms. Bacteria were isolated from natural carbonate producing environments 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 economical 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 operational conditions, i.e. in aerobiosis and microaerophily. More specically, the media contain a source of proteins for the oxidative deamination of amino acids in aerobiosis and a source of 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 a suitable bacterial suspension culture (Tables 1 and 2). Subsequently, 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 feeding 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., 1999). The rst application in situ was carried out in 1993 in Thouars on 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, evaluation of the roughness and colorimetry and SEM examination. SEM images indicated the abundant development of calcinogenic bacterial populations, illustrating the viability of B. cereus on stone surfaces. The presence of the biocalcin decreased the water absorption rate to a signicant extent (5 times lower) while retaining the permeability for gas. Furthermore, no inuence on the aesthetic appearance could be observed (Le Metayer-Levrel et al., 1999). Long-term evaluation of the biocalcin layer has shown differences in the durability behaviour related to the orientation of the fac ade 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 restore the protective effect of the biocalcin (Orial, 2000). Similar experiments were applied on limestone statuaries which had been placed in different climatic environments. Experiments were performed on two types of limestone, Tuffeau and Saint-Maximim. The former is a ne-grained limestone characterized 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

Appl.

N/C

Septicity conditions

S and NS

Stone

Nutrients/chemicals

Immersion in a growing bacterial culture for 30 days

Immersion for 4 days

NS

NS

NS

Stone cohesion (sonicator bath), weight increase, XRD and SEM analysis, porosimetry analysis Weight increase, Water absorption gas, permeability, chloride migration, Carbonation, Freezing and thawing, Thin sections, SEM, XRD analysis n.a.: not applicable, N/C: nutrients/chemicals, Appl.: application, NS: non-sterile and S: sterile a ODAA: oxidative deamination of amino acids; OAU: organic acid utilization, HU: hydrolysis of urea. b Composition see Table 5. Application procedure Jimenez-Lopez et al. Microbiota inhabiting the stone De Muynck et al. Overnight culture: 107 to 109 cells mL1 Inoculum Immersion for 1 day Bacteria

Table 2 (Continued)

Authors

Experimental methods

Evaluation procedures

W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 Table 3 Characteristics of the various biocementation processes. Characteristic Process Biocementation Remediation of cracks Denition Type Goals Bacterial concrete Self-healing concrete Admixture Self-healing of cracks Biomortar Binder

123

Generation of a biologically induced binder Surface treatment Admixture Improvement of the durability External crack repair Strength improvement

Strength development formation of a carbonate-binder-based mortar

Where/when

Applied in cracks on the surface of concrete and mortar Application of bacteria with a carrier/binder material immersion in nutrient solution Ecological, environmentally friendly

Applied in the concrete mixture Application of bacteria in the mixture, lower amount of bacteria (1 wt% admixture) Changes in microstructure Laboratory experiments

How

Application of mortar mixture to repair broken limestone fragments or to ll cavities in stone Application of bacteria and nutrients in the mixture, lower amount of bacteria (1 wt% admixture) Self-healing capacity Laboratory experiments

Application of bacteria and nutrients in the mixture, higher amount of bacteria (25 vol.% binder) Compatibility Small scale applications in practice Restoration sector eco-friendly reuse of brick

Added value

Status of use (current) Limitations

Laboratory experiments

Market niche

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 Eco-friendly repair of cracks Repair of difcult to reach concrete

and maritime environment appeared to be very aggressive, with almost complete loss of the biocalcin after 4 years of exposure. However, in the urban environment, the biocalcin retained its protective effect, with treated statues showing little damage compared to untreated ones. Furthermore, after 4 years of exposure to urban conditions, no undesired biological colonisation could be observed (Orial, 2000). By adding natural pigments into the nutritional medium, it is also possible to create a surcial patina with the biodeposition treatment. The pigments are integrated into the biocalcin and thus give a persistent light colouring to the stone. This technique has been proposed to conceal some newly replaced stones on a monument fac ade (Le Metayer-Levrel et al., 1999). 3.1.3. Procedure according to the University of Granada (Spain) Rodriguez-Navarro et al. (2003) addressed two important limitations of the calcite method. As the thickness of the bioconsolidating cement was limited to only a few microns, this method seemed to be ineffective for in-depth consolidation. Moreover, the formation of a supercial lm consisting of a mixture of biological remains, plugged stone pores and provided no consolidation (Rodriguez-Navarro et al., 2003). Besides, the authors commented on the potential drawback of the use of Bacillus in stone conser-

vation. According to these authors, the formation of endospores may lead to germination and uncontrolled biolm growth under appropriate conditions (i.e. temperature, humidity and nutrient availability). The authors, therefore proposed the use of Myxococcus xanthus for the creation of a consolidating carbonate matrix in the porous system of limestone. Their research group previously demonstrated the ability of this species to induce the precipitation of carbonates, phosphates and sulfates in a wide range of solid and liquid media (Gonzlez-Munoz et al., 1993, 1996; Ben Omar et al., 1995, 1998; Ben Chekroun et al., 2004; Rodriguez-Navarro et al., 2007), being the rst to describe struvite ((NH4 )MgPO4 6H2 O) formation by myxobacteria (Ben Omar et al., 1994). Furthermore, they were able to obtain the crystallization of struvite and calcite by dead cells et al., 1996). and cellular fractions of M. xanthus (Gonzlez-Munoz The latter is an abundant Gram-negative, non-pathogenic aerobic soil bacterium which belongs to a peculiar microbial group whose complex life cycle involves a remarkable process of morphogenesis and differentiation. In the tested culture media, no formation of a dormant stage was observed. Additionally, when applied on stone specimens, no fruiting bodies were observed upon drying. As a result of this cell death, no uncontrolled bacterial growth was observed.

Table 4 Overview of the different applications in which biocementation has been used in building materials. Application Biological mortar Remediation of cracks in concrete Bacterial concrete Self-healing Author Calcite bioconcept Ramachandran et al. De Belie et al. Ramachandran et al. Ghosh et al. Jonkers et al. Organism Bacillus cereus Bacillus pasteurii Bacillus sphaericus Bacillus pasteurii Shewanella Bacillus pseudormus Bacillus cohnii Metabolisma ODAA HU HU HU OAU Solutionb Nutrical SF Growth and biocementation medium (DB) SF Calcium lactate

a b

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 Nutrients Growth medium (CB) Peptone Yeast extract KNO3 NaCl Growth medium + CaCl2 2H2 O Actical Natamycine Calcium acetate Yeast extract Glucose BactoCasitone Ca(CH2 COO)2 4H2 O K2 CO3 (1/2)H2 O M-3 + phosphate buffer BactoCasitone Ca(CH2 COO)2 4H2 O CaCl2 2H2 O NaHCO3 Yeast extract Nutrient broth Urea CaCl2 2H2 O NH4 Cl NaHCO3 Yeast extract Urea Urea CaCl2 2H2 O Conc. Orial (2000) pHa References

Nutrical

Orial (2000)

B4

0.25 wt% 0.4 wt% 1 wt% 1 wt% 1 wt% 0.2 wt% 10 mM 0.3 wt% 0.4 wt% 0.1 wt% 0.3 wt% 0.1 wt% 3 g L1 20 g L1 1.45.6 g L1 10 g L1 2.12 g L1 20 g L1 20 g L1 20 g L1 50 g L1

Boquet et al.

M-3

Rodriguez-Navarro et al. (2003) Rodriguez-Navarro et al. (2003) Jimenez-Lopez et al. (2008)

M-3P CC

8 8

SFb , c

Stocks-Fischer et al. (1999)

Growth medium (DB) Biodeposition (DB)

7 7

Whifn (2004) De Belie and De Muynck (2008)

a b c

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 medium containing a pancreatic digest of casein as the nitrogen source. Also, the effect of a phosphate buffer on the carbonate production was investigated. Biodeposition experiments were performed both under static and non-static conditions. Sterilized calcarenite samples were submerged into a certain volume of M-3 or M-3P (Table 5) which was subsequently inoculated (1%, v/v) with M. xanthus. All experiments were performed at 28 C under sterile conditions. The phosphate buffer had a profound effect on the bacterial cell yield and the carbonate productivity, as well as on the supersaturation preceding the nucleation of carbonate crystals. A greater bacterial production also led to a higher yield in calcite crystals. Furthermore, the buffering effect of the phosphate prevented rapid local pH variations and, concomitantly the occurrence of a high supersaturation. As a result, the deposited carbonate crystals were shown to be strongly adhered to the surface of the pores, since the newly formed carbonates were more resistant to mechanical stress in the form of sonication than the calcite crystals in the stone. The authors attributed this to their epitaxial growth on pre-existing calcite crystals and to the incorporation of organic molecules. Apparently, the presence of organic molecules causes a misalignment of different domains within a single crystal. The authors observed carbonate cementation to a depth of several hundred micrometers (>500 m) without the occurrence of any plugging or blocking of the pores. Plugging is mainly a consequence of extracellular polymeric substance (EPS) lm formation (Tiano et al., 1999). In accordance with this, only limited EPS

production was observed in stones submerged in M-3 and M-3P media under static conditions. These ndings are in sharp contrast with the abundant production of EPS by M. xanthus described by Sutherland and Thomson (1975). The latter could be attributed to differences in culture medium composition and culture conditions, as was suggested by the authors. 3.1.4. Procedure according to the University of Ghent (Belgium) 3.1.4.1. Euville limestone. Dick et al. (2006), a Ghent University research team, proposed the microbial hydrolysis of urea as a strategy to obtain a restoring and protective calcite layer on degraded limestone. The hydrolysis of urea (Eqs. (1)(5)) presents several advantages over the other carbonate generating pathways, as it can be easily controlled and it has the potential to produce high amounts of carbonate within a short period of time. The hydrolysis of urea is catalyzed by means of urease. As a consequence, urea is degraded to carbonate and ammonium, resulting in an increase of the pH and carbonate concentration in the bacterial environment (Stocks-Fischer et al., 1999). One mole of urea is hydrolyzed intracellularly to one mole of ammonia and one mole of carbamate (Eq. (7)), which spontaneously hydrolyzes to one mole of ammonia and carbonic acid (Eq. (8)). These products subsequently equilibrate in water to form bicarbonate and two moles of ammonium and hydroxide ions (Eqs. (9) and (10)): CO(NH2 )2 + H2 O H2 COOH + NH3 NH2 COOH + H2 O NH3 + H2 CO3 (7) (8)

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2NH3 + 2H2 O 2NH4 + + 2OH 2OH + H2 CO3 CO3

2

(9) (10)

+ 2H2 O

The global reaction can be written as follows: CO(NH2 )2 + 2H2 O 2NH4 + + CO3 2 (11)

In the presence of calcium ions, this will result in calcium carbonate precipitation (Eq. (12)), once a certain level of supersaturation is reached: CO3
2

Finally, calcium chloride was added for a second time in the fourth week. All experiments were performed at 28 C under sterile conditions. From the screening procedure described above, 2 strains of B. sphaericus were selected for further experiments. These strains were shown to decrease the initial water absorption rate by approximately 50%. 3.1.4.2. Cementitious materials. As an extension to the above mentioned study, De Muynck et al. (2008a,b) investigated the biodeposition with B. sphaericus as a surface treatment for cementitious materials (Portland cement mortar) with different porosities. In contrast with the treatment procedure described by Dick et al. (2006), all experiments were performed at 28 C under non-sterile conditions. Due to the alkaline pH of cementitious materials, the contamination was expected to be very low. Furthermore, the treatment procedure was altered: the mortar specimens were immersed for 24 h in a 1 day old culture of B. sphaericus containing ca. 107 cells mL1 , after which they were transferred to fresh medium containing a calcium source (Table 5). The specimens were removed from the solution after 3 days. The authors demonstrated that the biodeposition treatment resulted in an increased resistance of mortar specimens towards carbonation, chloride penetration and freezing and thawing, especially for more porous mortars with higher water to cement ratios (w/c). Moreover, the biodeposition treatment showed a similar protection towards degradation processes as some of the conventional surface treatments under investigation (silanes, siloxanes, silicates and acrylates). According to the authors, the biodeposition treatment on cementitious materials should be regarded as a coating system. This could be attributed to the fact that the carbonate precipitation was mainly a surface phenomenon due to the limited penetration of the bacteria in the porous matrix. From thin section analyses, the authors observed that the majority of the surface was covered with a layer of crystals with thicknesses within the range of 1040 m, in which often larger crystals (up to 110 m) could be found. The morphology of the crystals was observed to be highly dependent on the medium composition. Much research on bacterial induced precipitation has been conducted with calcium chloride as the calcium source (Adolphe et al., 1990; Ferris and Stehmeier, 1992; Bang et al., 2001). As chloride ions are detrimental to the reinforcement in concrete structures, the use of calcium acetate as an alternative calcium source was investigated. In the event calcium chloride was used as the calcium source, rhombohedral carbonate crystals were obtained. In the presence of calcium acetate, spherulitic crystals were observed (Fig. 2). However, no differences in the protective effect were observed between biodeposition treatments with a different calcium source. Therefore the authors concluded that from these two salts, calcium acetate should be used for biodeposition on cementitious materials. 3.1.5. Procedure according to the Biobrush consortium (United Kingdom) The basic aim of the Biobrush (BIOremediation for Building Restoration of the Urban Stone Heritage) project was to integrate the existing knowledge on the application of microorganisms for the remediation of damaged stone into a conservation practice. Furthermore, the goal was to sequentially link the processes of salt removal to the processes of consolidation (May, 2005). The use of microorganisms has been investigated for the removal of nitrates, sulphates and organic matter present on the surface of artworks (Gauri et al., 1992; Ranalli et al., 1999). In addition to the elimination of black crusts, microbial sulphate removal also results in the conversion of gypsum to calcite. As such, this method

+ Ca

2+

CaCO3

(12)

As calcium ions are bound to the cell wall as a result of the negative charge of the latter, this can result in the formation of crystals on the bacterial cell. In addition, precipitation can also occur in the bulk phase of the liquid. A schematic overview, of the ureolytic carbonate precipitation occurring at the microbial cell wall is given in Fig. 1. The purpose of the study by Dick et al. (2006) was to identify the microbial key factors which contribute to the performance of the biodeposition treatment. For the evaluation of the performance, the authors investigated the water absorption rate of treated and untreated Euville limestone. The key factors had to be easy in use and applicable for quick screening. The following parameters have been examined: calcite deposition on limestone cubes, pH increase, urea degrading capacity, EPS production, biolm formation, -potential and deposition of dense crystal layers. Of these parameters, the -potential proved to be the factor with the greatest predictive power to screen microorganisms for good limestone restoration, reecting the effect on the initial water absorption. The -potential is a measure of the potential of the electric layer at the surface of the cells, and is, therefore, an important parameter in the adhesion and surface colonization by bacteria. Due to the positive -potential of the limestone, bacteria with a highly negative -potential will be more easily retained. The second important key factor was the specic urea degradation rate. Bacteria with a high initial specic urea degradation rate show a high afnity for urea. This allows for a high substrate turnover for a limited amount of cells. The bacteria which had been selected for screening were isolated from an ureolytic calcication reactor. This type of reactor had been previously developed by the same research group for the removal of calcium from calcium-rich wastewater (Hammes et al., 2003). Calcifying sludge was obtained through the stimulation of autochthonous ureolytic organisms, by means of repeated additions of urea. From this sludge, bacteria were isolated and screened for their ability to precipitate calcite on agar plates containing urea and calcium chloride. Although urease activity is widespread among different groups of microorganisms, it was mainly microorganisms closely related to the Bacillus sphaericus group which were shown to proliferate and express the urease gene under the given cultivation conditions (Hammes et al., 2003). The ability of B. sphaericus to precipitate calcium carbonate had been previously described by Cacchio et al. (2003). Among several strains isolated from a limestone cave, a B. sphaericus strain was shown to rapidly precipitate CaCO3 in B4 medium (Table 5), even at low temperatures such as 4 C. Furthermore, calcifying bacteria were found not to solubilise carbonates. For the deposition of a layer of carbonate on the surface, Dick et al. (2006) rst proposed the establishment of a biolm. For that purpose, limestone cubes were immersed for 2 weeks in liquid medium inoculated with 1% of the different strains. The surface was rewetted each 2 h for 5 min by shaking. After the 2 weeks ended, calcium chloride was added to the medium in order to precipitate calcium carbonate. In the third week, the specimens were suspended in fresh medium in order to have a second phase of biolm growth.

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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 microenvironment of the bacteria (A). In the presence of calcium ions, this can result in a local supersaturation and hence heterogeneous precipitation of calcium carbonate on the bacterial cell wall (B). After a while, the whole cell becomes encapsulated (C), limiting nutrient transfer, resulting in cell death. Image (D) shows the imprints of bacterial cells involved in carbonate precipitation. A more in-depth representation can be found in Hammes and Verstraete (2002).

could be considered as a special kind of biodeposition treatment. Heselmeyer et al. (1991) obtained the complete removal of gypsum crusts from marble samples in laboratory conditions using a strain of Desulfovibrio vulgaris. The procedure was further optimized by Ranalli et al. (1997), who used sepiolite as a carrier material for D. vulgaris and Desulfovibrio desulfuricans. The use of sepiolite not only provided anaerobic conditions and humidity, but also enabled the authors to shorten the treatment time. Additional improvements were made by Cappitelli et al. (2006) who reported on the superiority of Carbogel as a delivery system for the bacteria. The use of Carbogel allowed for a higher retention of viable bacteria and signicantly decreased the time needed for entrapment of the microorganisms as compared to the use of sepiolite. In addition, methods were presented to avoid the precipitation of black iron sulde. The optimized methodology appeared to be superior to chemical treatments involving the use of ethylenediaminetetraacetic acid (EDTA), since no sodium sulphate was formed (Cappitelli et al., 2007). As Cappitelli et al. (2006, 2007) were among the members of the Biobrush consortium, the use of Carbogel was subsequently introduced into the eld of biodeposition. According to the consortium, these delivery systems could be used to control the possible harmful side effects of bacteria to stone. In addition, it was noticed that the application of calcinogenic bacteria by spraying alone only resulted in a limited change of the capillary water uptake of Portland stone. According to the consortium, the latter is attributable to the limited colonization of the stone as a result of drying out. Within the framework of the Biobrush project regarding biodeposition, bacteria isolated from a stream in Somerset (UK), and bacteria from culture collections that had been reported to have calcifying activity, were screened for their ability to deposit calcite in solid and liquid modied B4 media (Table 5). From the 10 isolates that were retained and assessed for their ability to deposit calcite on stone surfaces, Pseudomonas putida was chosen for further study in eld trials. The latter has a low risk to humans and is sensitive to most of the tested antibiotics and precipitated calcite in a wide temperature range (May, 2005). In these eld trials, bacteria were applied to the stone by brushing. Subsequently, the bacteria were covered with moistened Japanese paper, above which a 11.5 cm thick layer of Carbogel prepared with modied B4 was applied. TrisHCl buffer was added to the Carbogel to adjust the low pH of this carrier. Finally, the gel was covered with a polyethylene sheet. As a result of this treatment a decrease of the water absorption and open porosity by 1% and 5%, respectively, was obtained. In order for this treatment to be effective as a consolidant, a 2 weeks treatment was observed to be necessary.

3.2. Application of organic matrix molecules; procedure according to the Bioreinforce consortium (Italy) Tiano et al. (1999) commented on the use of viable cells for the formation of new minerals inside the stone. This was the result of their experiments with Micrococcus spp. and Bacillus subtilis strains on Pietra di Lecce bioclastic limestone. In these experiments, bacteria were applied by brushing sterilized specimens that were soaked with distilled water, reaching a nal concentration of 106 cells cm2 . Subsequently, the bacteria were fed daily by wetting with a small amount of B4 medium (Table 5) for a period of 15 days. Experiments were performed at 28 C under non-sterile conditions. According to the authors, the decrease in water absorption after a biodeposition treatment is for a large part attributable to the physical obstruction of pores, rather than to the stable presence of newly precipitated calcite. Furthermore, the authors commented on some possible negative consequences, such as (1) the presence of products of new formation, due to the chemical reactions between the stone minerals and some by-products originating from the metabolism of viable heterotrophic bacteria and (2) the formation of stained patches, due to the growth of air-borne micro-fungi related to the presence of organic nutrients necessary for bacterial development. To avoid some of these problems, the authors proposed the use of natural and synthetic polypeptides to control the growth of calcite crystals in the pores. The rst suggestions in this direction already date from the time at which the Calcite Bioconcept treatment was developed. Tiano et al. (1992) and Tiano (1995) proposed the use of organic matrix macromolecules (OMM) extracted from Mytilus californianus shells to induce the precipitation of calcium carbonate within the pores of the stone. The organic matrix was shown to produce a more relevant and durable carbonate precipitation compared to the single use of calcium chloride or hydroxide. This precipitation resulted in a slight decrease in porosity and water absorption by capillarity (Tiano, 1995). However, the practical application was hindered by the complexity of the extraction procedure and the very low yield of usable product (Tiano et al., 1999). Given this, the authors searched for alternative starting materials by changing the nature of the organic macromolecules involved. As these bio inducing macromolecules (BIM) are usually rich in aspartic acid groups, Tiano et al. (2006) proposed the alternative use of acid functionalized proteins such as polyaspartic acid. Calcium and carbonate ions for crystal growth were supplied by means of an ammonium carbonate and calcium chloride solution or a saturated solution of bicarbonate, and were supplemented in some cases by calcite nanoparticles, in order to maintain a saturated carbonate solution in the pore over a prolonged period. Proteins, calcium ions and nanoparticles were

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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 formation (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 conclude that calcinogenic strains might have a subcellular structure, resistant to the methods used to kill cells (sonication, autoclaving), 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 fac ade (Mastromei et al., 2008). In addition, Barabesi et al. (2007) reported on a gene cluster of B. subtilis involved in calcium carbonate precipitation. From UV mutagenesis experiments, six mutants impaired in calcite crystal formation were isolated. Sequence analysis of the mutated genes revealed that in many cases their putative function was linked to the fatty acid metabolism (Perito et al., 2000; Barabesi et al., 2007). Further experiments are ongoing to investigate the link between this kind of metabolism and calcium precipitation. 3.3. Application of an activator medium (Spain) Concerning possible changes of the activity and composition of the autochthonous microbiota upon addition of an inoculated culture media to ornamental stone, Rodriguez-Navarro et al. (2003) pointed out the possibility of a synergetic contribution of the former to the overall biodeposition process. In fact, Urzi et al. (1999) previously demonstrated that the majority of bacteria isolated from building materials are able to induce carbonate precipitation under laboratory conditions. From the above, Jimenez-Lopez et al. (2007) proposed the application of a culture medium, able to activate the calcinogenic bacteria from the microbial community of the stone, as a more user friendly method for the in situ consolidation of ornamental stone. In addition to their work on decayed limestone fragments (Jimenez-Lopez et al., 2007), this technique was recently proposed for the treatment of new stones used for replacement purposes (Jimenez-Lopez et al., 2008). Upon comparison of the microbial community identied in nontreated quarry stone and that identied in the non-treated decayed stone, the authors observed for the latter the presence of microorganisms related to the quarry from which the stone was extracted and microorganisms related to the environment and contamination to which the stone was exposed. Some of the identied bacteria, Pseudomonas and Bacillus, had already been reported to produce calcium carbonate both in laboratory conditions and in nature. These chemoorganotrophic organisms are able to grow in culture media containing amino acids such as nutrient agar and tryptic soy agar (Jimenez-Lopez et al., 2007). From these ndings, the authors proposed the use of bactocasitone as a way to activate the calcinogenic bacteria from the

Fig. 2. Scanning electron micrographs of untreated (A) and biodeposition treated (B and C) CEM I mortar specimens. Notice the differences in crystal morphology obtained with different calcium sources: predominantly rhombohedral crystals in the case of calcium chloride (A) and spherulitic crystals with calcium acetate (B).

introduced in the stone by means of spraying. According to the authors, the method is most suitable for the use on marble statues and objects of high aesthetic value where conservation is required with the minimum change in the chemistry of the object. Field test results, however, indicate that the effects of the BIM treatment were rather small. The consolidating effect and the decrease in water uptake were very low compared to the use of ethylsilicates, i.e. 15% over 12 mm depth compared to 30% up to 10 mm depth (as measured with the drilling resistance measuring system) and 17% compared to 60%, respectively (Tiano et al., 2006). Elucidation of the genetic background of crystal formation in bacteria has been proposed as an alternative way for the production of inducing macromolecules. This was one of the objectives of the European Bioreinforce (BIOmediated calcite precipitation for monumental stones REINFORCEment) project. According to

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stone microbial community. Bacto-casitone is a source of carbon and nitrogen, which favours alkalinisation due to the oxidative deamination of amino acids. Furthermore, as no carbohydrates were supplied, the probability of acid production, which is detrimental to the stone, was believed to be minimal. According to the authors, this procedure is much easier than the use of bacterially inoculated media, since difculties linked to the need of specialized persons and equipment to work with microorganisms or technical requirements to ensure optimal growth conditions would be avoided (Jimenez-Lopez et al., 2007). However, the fact that microorganisms do not need to be introduced not only leads to a decrease of the overall cost, but also ensures that this method is not covered by the claims of the patent by Adolphe et al. (1990). Conse et al. (2008) applied for a new patent for quently, Gonzlez-Munoz the protection and reinforcement of construction and ornamental materials by means of the application of an activator medium able to induce the formation of calcium carbonate. Sonication test results demonstrated that the new cement created by the microbial community and/or the combined action of the microbial community and M. xanthus was more resistant than that created by the sole action of either M. xanthus or the culture media. Furthermore, the authors did not observe any changes in the porosity of the stone. In addition, limited exopolysaccharide production was observed (Jimenez-Lopez et al., 2007). The latter could be somewhat expected as Rodriguez-Navarro et al. (2003) noted that organic lms are unable to attach to the stones under shaking conditions, as was the case in the work by Jimenez-Lopez et al. (2007, 2008). Due to the time required for the activation of the microbial community, Jimenez-Lopez et al. (2007) proposed the additional use of M. xanthus for those restoration interventions in which time is an issue and fast formation of calcium carbonate is required. Very recently, the application of an activator medium has been successfully applied in situ on calcarenite stone (Monasterio de San Jeronimo and Hospital Real, Granada). Preliminary results show the effectiveness of the treatment in terms of colour changes (negligible) and surface resistance by means of a peeling test (Personal communication by Rodriguez-Navarro, 2008). Although the formation of endospores was previously considered a potential drawback for the use of Bacillus in stone conservation (Rodriguez-Navarro et al., 2003), spore forming bacteria, able to germinate upon the application of the culture media, contribute in large extent to the precipitation of carbonate by the method described by Jimenez-Lopez et al. (2007). Drawbacks to the use of spore forming bacteria were related to the possible uncontrolled growth of bacteria upon germination. However, Le Metayer-Levrel et al. (1999) found that no increases in the microbial activity or changes in the autochthonous microbiota were observed immediately or 4 years after the application of calcinogenic bacteria. The long activation times of this technique inspired De Muynck et al. (2008c) to develop an in situ enrichment of carbonate producing bacteria. For that purpose, different media were developed which allowed a rapid growth of carbonate producing strains upon exposure to the surrounding air. Among the different metabolic pathways under investigation, conditions optimal for carbonate precipitation were most rapidly obtained upon the hydrolysis of urea. Nevertheless, the rate of urea hydrolysis and biodeposition remained low compared to pure cultures of B. sphaericus.

in the eld of geotechnical engineering, i.e. plugging, strengthening and improvement of soils (Ferris and Stehmeier, 1992; Zhong and Islam, 1995; Nemati and Voordouw, 2003; Whifn et al., 2007). Recent advances, however, indicate the potential use of this technique for the remediation of cracks in building materials, strength improvement and self-healing of cementitious materials. 4.1. Biological mortar (France) The knowledge and experiences obtained with the Calcite Bioconcept treatment for limestone, have resulted in the development of a biological mortar for the remediation of small cavities on limestone surfaces. The aim of the biological mortar was to avoid some of the problems related to chemical and physical incompatibilities of commonly used repair mortars with the underlying material, especially in the case of brittle materials (Castanier, 1995; Le Metayer-Levrel et al., 1999; Orial et al., 2002, Personal communication by Loubire (Chief of Calcite Bioconcept), 2008; http://www.calcitebioconcept.com/). In general, a mortar refers to a workable paste consisting of a binder, aggregates and water to bind building materials together and to ll the gaps between them. In particular, a biological mortar refers to a mixture of bacteria, nely ground limestone and a nutritional medium containing a calcium salt. The term biological refers to the microbial origin of the binder, i.e. microbiologically produced calcium carbonate. Similar to lime mortars, the produced calcium carbonate cements the aggregates together. Cementation occurs as a result of the nucleation and growth of carbonate crystals at the surface of the aggregates, especially at the contact areas between them. The optimization of the mortar composition encompassed the dosage and composition of the three main components, i.e. limestone powder, nutrients and bacterial paste. The mortars were evaluated based upon their appearance (cohesion and colour), the presence of micro-cracks and the resistance towards fracturing. Concerning the medium composition, some adjustments were made to the initial method, as was used for biodeposition purposes. The amount of nutrient solution introduced during the fabrication of the mortar was sufcient to support bacterial activity. Repeated external applications of the nutrient solution were unable to completely wet the mortar. Furthermore, they resulted in discolorations at the surface and were, therefore, rapidly omitted. Additionally, the biological mortars necessitated the use of larger amounts of bacteria and as a result the composition of the nutrient medium had to be altered. Based on the different evaluation parameters, best results were obtained with one part of bacterial paste (containing 109 cells mL1 ), one part of nutritional medium and two parts of limestone powder. Limestone powder with a granulometry between 40 and 160 m was observed to be the most suited. The technique has already been successfully tested on a small scale on sculptures of the Amiens Cathedral and on a portal of the church of Argenton-Chteau (France). Visual observations 2 years after the treatment indicated a satisfactory appearance of the repaired zones. (Le Metayer-Levrel et al., 1999; Orial et al., 2002). 4.2. Remediation of cracks in concrete (USA, Belgium) In the recovery of heavy oil from oil elds, where water is more readily removed than the viscous oil, the ability to selectively plug porous rock to focus pumping energy in oil rich zones is highly desirable (Hart et al., 1960; Lappin-Scott et al., 1988). Because of the cost and unsatisfactory performance of some of the chemically cross-linked polymers, many workers suggested that insoluble biopolymers and biomass generated by injection of indigenous

4. Biocementation Besides the deposition of a layer of carbonate on the surface of building materials, MICP has also been used for the generation of binder-based materials. Initial developments were mainly situated

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microorganisms can be used to selectively plug off zones of high water permeability (Lappin-Scott et al., 1988; Jack et al., 1991; Gollapudi et al., 1995). In addition, the use of a microbial mineral plugging system based on the precipitation of carbonates was suggested (Ferris and Stehmeier, 1992; Zhong and Islam, 1995). While initial research on MICP in sand columns was mainly focused on the decrease of porosity and permeability as a result of the physical presence of the newly formed carbonates (Ferris and Stehmeier, 1992), recent investigations focus on the improvement of strength as a result of the cementation of sand particles (Whifn, 2004; Kucharski et al., 2006). The latter is due to the particle binding properties of the microbially produced carbonates. The hydrolysis of urea was selected as a very suitable pathway for the production of carbonate ions due to its ability to alkalinize the environment. Furthermore, urea is an important organic nitrogen carrier in natural environments and is commonly used as an agricultural fertilizer (Nielsen et al., 1998). Moreover, the ability to hydrolyze urea is widely distributed among indigenous bacteria in soils and groundwater systems (Mobley and Hausinger, 1989; Fujita et al., 2000). Urea-utilizing bacteria such as Sporosarcina pasteurii and Sporosarcina ureae are commonly isolated from soil, water, sewage and incrustations on urinals. The participation of S. pasteurii in sand consolidation has been demonstrated by Kantzas et al. (1992). Gollapudi et al. (1995) further investigated the use of S. pasteurii for the plugging of sand columns. Although the bacteria were mixed with the sand slurry, consolidation mainly occurred near the surface. Stocks-Fischer et al. (1999) showed that microorganisms directly participated in the calcite precipitation by providing a nucleation site and by creating an alkaline environment which favoured the precipitation of calcite. Zhong and Islam (1995) used the consolidation of sand mixtures for the remediation of cracks in granite. Cracks in granite were packed with a mixture of bacteria, nutrients and a ller material. Among the different materials that were mixed with S. pasteurii, the silica fume (10%) and sand (90%) mixture lead to the highest compressive strength and lowest permeability. As a further extension to this research, Ramachandran et al. (2001) investigated the microbiological remediation of cracks in concrete. The authors proposed MICP as an effective way to seal cracks. The appearance of cracks and ssures is an inevitable phenomenon during the ageing process of concrete structures upon exposure to weather changes. If left untreated, cracks tend to expand further and eventually lead to costly repair. Specimens with cracks lled with bacteria, nutrients and sand demonstrated a signicant increase in compressive strength and stiffness values when compared with those without cells. The presence of calcite was, however, limited to the surface areas of the crack. The authors attributed this to the fact that S. pasteurii grows more actively in the presence of oxygen. Still, the highly alkaline pH (1213) of concrete was a major hindering factor to the growth of the moderate alkaliphile S. pasteurii, whose growth optimum is around a pH of 9. In order to protect the cells from the high pH, Day et al. (2003) investigated the effect of different ller materials on the effectiveness of the crack remediation. Beams treated with bacteria and polyurethane showed a higher improvement in stiffness compared to ller materials such as lime, silica, y ash and sand. According to the authors, the porous nature of the polyurethane minimizes transfer limitations to substrates and supports the growth of bacteria more efciently than other lling materials, enabling an accumulation of calcite in deeper areas of the crack. No differences could be observed between the overall performances of free or polyurethane immobilized cells in the precipitation of carbonate (Bang et al., 2001). In addition to this research, Bachmeier et al. (2002) investigated the precipitation of calcium carbonate with the urease enzyme immobilized on polyurethane. The immobilization

was shown to protect the enzyme from environmental changes, as the immobilized urease retained higher enzymatic activities at high temperatures and in the presence of high concentrations of pronase. While the rate of calcite precipitation of the immobilized enzyme was slower compared to that of the free enzyme, lower concentrations of the former where needed to obtain the theoretical maximum precipitation in a period of 24 h. Although the authors mentioned ongoing research on the use of immobilized urease in the remediation of surface cracks in concrete, to our knowledge no published results are available at the moment. As an extension to their research on biodeposition on cementitious materials, De Belie and De Muynck (2008) further investigated the use of microbially induced carbonate precipitation for the repair of cracks in concrete. For the protection of B. sphaericus from the alkaline pH conditions, bacteria were immobilized in a silica sol. Upon the addition of a salt, a bioceramic material (biocer) was formed, which was able to bridge the crack. Subsequent addition of a urea and calcium chloride solution resulted in the formation of carbonate crystals inside the pores of the biocer and concomitantly sealing of the crack. As a result, a decrease of the water permeability, similar to that obtained with traditional epoxy injections, was observed. 4.3. Bacterial concrete (USA, India) Besides external application of bacteria in the case of remediation of cracks, microorganisms have also been applied in the concrete mixture. Until now, research has mainly focused on the consequences of this addition on the material properties of concrete, i.e. strength and durability. Both properties depend on the microstructure of the concrete. However, the effects of the presence of the microorganisms and/or the microbially induced carbonates on the microstructure still need to be elucidated, especially the interaction between the biomass and the cement matrix. Ramachandran et al. (2001) investigated the use of microbiologically induced mineral precipitation for the improvement of the compressive strength of Portland cement mortar cubes. This study identied the effect of the buffer solution and type and amount of microorganisms, i.e. S. pasteurii and P. aeruginosa, used. Furthermore, in order to study the effect of the biomass, the inuence of both living and dead cells was investigated. Before addition to the mortar mixture, bacteria were centrifuged and washed twice. The nal pellets were then suspended in either saline or phosphate buffer, which was subsequently added to the mixture. After demolding, the mortar specimens were stored in a solution containing urea and calcium chloride for 7 days. Subsequently, the specimens were cured in air until the measurement of the compressive strength. At lower concentrations, the presence of S. pasteurii was shown to increase the compressive strength of mortar cubes. While the 28day compressive strength of the control cubes amounted to about 55 1 MPa, specimens treated with 103 cells cm3 had a compressive strength of about 65 1 MPa. The contribution of P. aeruginosa to the strength was found to be insignicant. From the X-ray diffraction (XRD) analysis, no signicant increased amounts of calcite could be found in mortar specimens treated with bacteria. This could be attributed to the inhibition of the microorganisms by the high pH and the lack of oxygen inside the mortar mixture. The overall increase of strength, therefore, resulted from the presence of an adequate amount of organic substances in the matrix due to the microbial biomass. However, an increase of the biomass, as dead cells in particular, resulted in a decreased strength. According to the authors, this could be attributed to the disintegration of the organic matter with time, making the matrix more porous (Ramachandran et al., 2001).

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Fig. 3. Schematic drawing of conventional concrete (AC) versus bacteria-based self-healing concrete (DF). Crack ingress chemicals degrade the material matrix and accelerate corrosion of the reinforcement (AC). Incorporated bacteria-based healing agent activated by ingress water seals and prevents further cracking (DF) (courtesy of Jonkers). Table 6 Approximate costs of surface treatments. Treatment Calcite bioconcept Growth medium Nutrical Water repellents Consolidants
a b

Price D /unit

Dosage unit/m2

Product D /m2

No. of applic.

Prod. + applic. D /m2 2328a 3540b

1 D g1 0.2 D g1 2.54 D L1 1015 D L1

23 g 816 g(5) 0.51 L >1 L

23 24(5) 1.254 >10

1 5 1 1

1525 >30

Unaltered stone. Sculptured and degraded stone.

Ramakrishnan et al. (2001) investigated the effect of this technique on the durability of concrete. The presence of bacteria was observed to increase the resistance of concrete towards alkali, sulfate, freeze thaw attack and drying shrinkage; the effect being more pronounced with increasing concentrations of bacterial cells. The authors attributed this to the presence of a calcite layer on the surface, as conrmed by XRD analysis, lowering the permeability of the specimens. The best results were obtained with the phosphate buffer. Ghosh et al. (2005) demonstrated the positive effect of the addition of Shewanella on the compressive strength of mortar specimens. Contrary to the aforementioned research, these authors did not intend mineral precipitation, as these specimens were cured in air and not in a nutrient containing medium. An increase of 25% of the 28 days compressive strength was obtained for a cell concentration of about 105 cells mL1 and a water to cement ratio of 0.4. For these samples, the presence of a brous material inside the pores could be noticed. As a result, a modication of the pore size distribution was observed. The positive effect of the addition of Shewanella improved with increasing curing times. For a concentration of 105 cells mL1 , an increase of the compressive strength of 17% and 25% was observed after 7 and 28 days, respectively. However, no increase of the compressive strength was observed with additions of Escherichia coli to the mortar mixture. This led the authors to suggest that the choice of the microorganism plays an important role in the improvement of the compressive strength. More specically, the production of EPS by the bacteria seemed to be of importance. 4.4. Self-healing concrete (the Netherlands) As an extension to the aforementioned research, Jonkers (2007) and Jonkers and Schlangen (2007) investigated the use of bacteria as self-healing agents for the autonomous remediation of cracks in concrete (Fig. 3). In contrast with previous studies, such an approach necessitated the presence of all the reaction com-

ponents, microorganisms and nutrients, in the matrix to ensure minimal externally needed triggers. Therefore, the authors investigated the compatibility of different organic compounds with the cement matrix. Moreover, suitable bacteria should be able to survive concrete incorporation for prolonged periods of time. For that purpose, alkali-resistant spore forming bacteria related to the genus Bacillus, Bacillus pseudormus DSM 8715 and Bacillus cohnii DSM 6307, were selected. In addition, the bacteria were added as spores, as these are known for their ability to endure extreme mechanical and chemical stress. On top of this, the authors decided to choose a pathway different from the hydrolysis of urea for the production of carbonate ions. In this way, possible negative effects of the produced ammonia on the reinforcement corrosion and degradation of the concrete matrix (when further oxidized by bacteria to yield nitric acid) could be avoided. Among the components selected, calcium lactate did not substantially affect the compressive strength values. Furthermore, the addition of a high number of bacterial spores (108 cm3 ) resulted in a decrease of strength of less than 10%. For the evaluation of the mineral producing capacity, healing agent-incorporated specimens and control specimens were broken to pieces after 7 or 28 days curing, immersed in tap water for 8 days and subsequently analyzed by ESEM. While a massive production of larger-sized CaCO3 precipitates was observed for the 7 days cured specimens, no differences could be observed between the healing agent incorporated specimens and control specimens after 28 days. The authors related this to a decrease of the viability of the spores upon incorporation in the cement matrix. The decrease in viability appears to be linked with a decrease of the matrix pores size diameter (Jonkers and Schlangen, 2007; Jonkers et al., 2008). 5. Cost evaluation 5.1. Biodeposition Table 6 gives an overview of the costs related to the application of surface treatments to building materials (Personal communi-

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cation by an employee of the Belgian company FTB remmers, 2008; http://www.ftbremmers.com/). The costs of the biodeposition treatment are attributable both to the price of the product and the number of applications required. The theoretic price of the product depends on the price of the microorganisms and the price of the nutrients. One kilogram of lyophilized bacteria (1011 CFU g1 ) costs around 1100 D kg1 . As bacteria are applied in a concentration of 23 g m2 this results in a cost of 2.23.3 D m2 . The costs of the nutrients are estimated to be about 180 D kg1 . Depending on the porosity of the stone, the dosage ranges between 0.04 and 0.08 kg m2 , bringing the cost of the nutrients to 715 D m2 . This brings the total product cost around 1017 D m2 . In addition, the total price of the treatment amounts to about 2328 D m2 . The latter includes the costs of application and the added value of the product. In case heavily degraded surfaces need to be treated, the cost of the treatment will be between 35 and 40 D m2 (Personal communication by Loubire, 2008). In case a carrier material is applied, such as proposed by the Biobrush consortium (May, 2005), the biodeposition treatment becomes even more costly. The price of high density polyethylene sheets, as used on external wall assemblies, amounts to about 2.3 D m2 (http://order.americanmicroinc.com/cgibin/americanmicroinc/VB10X25X6.html). For the formation of a gel, 10 g L1 of Carbogel (27.6 D kg1 , Carbogel, Brazil, 2008; http://www.carbogel.com.br/) is required. Considering a proposed thickness of the gel of about 1 cm, this will bring the cost per m2 to about 2.8 D m2 . The Japanese paper brings about an extra cost of about 12.3 D m2 (http://japanesepaperplace.com/retail/retailproducts/conservation-papers.htm). As a result of the carrier material, however, less applications of moisture (and hence nutrients) will be necessary. The resulting decrease in cost will be marginal compared to the costs of the carrier. Additionally, the disposal of the carrier material will also present an extra cost. As mentioned before, the method proposed by Jimenez-Lopez et al. (2007) could offer an economical advantage over the Calcite 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 number of applications of nutrients, and hence, loss of the economical advantage. Due to the price of its constituents, the biodeposition treatment will never be able to compete with some of the traditional surface treatments on a pure economical basis. The focus of this kind of treatment should, therefore, be on the added value compared to other treatments. The biodeposition treatment presents an ecological, environmentally friendly alternative over the other treatments. Furthermore, the application of a layer of calcium carbonate to limestone ts in the current restoration concept of compatibility. Some of the traditional surface treatments, typically organic resins, have shown long-term incompatibilities with the stone. The latter resulted in a much more intense damage to the stone than would have occurred without restoration, necessitating replacement and costly repair. In addition, the biological deposited crystals show some unique properties. Due to their growth on pre-existing calcite crystals and the incorporation of organic molecules, these crystals are strongly attached to the surface, exerting a consolidating effect (RodriguezNavarro et al., 2003). Furthermore, by adding pigments to the medium, it is also possible to create a surcial patina, allowing the concealment of new replacement stone (Le Metayer-Levrel et al., 1999). Until now, practical applications of the biodeposition treatment have been mainly limited to France. The treatment has been applied on several historic monuments across the country, including a part

of the Notre Dame de Paris. The treatment has also been applied on the fac ade of warehouses (ex. Galeries Lafayette, Paris), hotels and apartments in and around Paris. As a consequence, the company Calcite Bioconcept has an estimated annual turnover of the order of 100,000150,000 D (Personal communication by Loubire, 2008). 5.2. Biocementation In contrast with the biodeposition treatment, the added value of the biocementation treatment in building materials is less pronounced. As a consequence, more difculties in competing with traditional treatments can be expected. In the case of the biological mortar, a similar performance could be obtained with traditional lime mortars, also being compatible with limestone. As mortars consist of a mixture of sand, water and a binder, it is the latter which will make up the cost when comparing the two kinds of mortars. For non-hydraulic lime mortars, the cost of the binder amounts to about 0.6 D kg1 (Carmeuse, Belgium, 2008). For biological mortars, however, the binder consists of a mixture of nutrients (about 180 D kg1 ) and a bacterial paste (1100 D kg1 ) (Personal communication by Loubire, 2008). Especially in the case of crack repair of cementitious materials, at the moment, little or no added value is obtained. Because of the fact that (organic) carrier materials are needed to protect the bacteria from the alkaline environment, the ecological aspect of the treatment has been largely reduced. Furthermore, the method currently seems unfeasible to be readily applicable in practice due to the large amount of specialist work needed. Only in the case of self-healing building materials can a signicant added value be expected. The latter is, however, largely attributable to the concept of self-healing materials, decreasing the needs for manual inspection and repair. The research is still in its infancy, and it will be largely questionable whether bacteria will be able to remain viable for a prolonged time and upon activation be able to seal the cracks. 6. Considerations In our opinion, the feasibility of the biodeposition treatment in practice largely depends on the time required for carbonate production, and hence, precipitation to occur. The latter has also important consequences on the economical aspect of the treatment. In the case of the application of calcinogenic bacteria, longer times required for precipitation to occur necessitate longer periods during which the building material has to remain wet. This is due to the fact that microorganisms require a minimum amount of water to remain active. With increasing times for precipitation, increasing amounts of EPS production, biolm formation and hence plugging can be expected. In order to ensure the presence of a sufcient amount of water, multiple applications of nutrients over several days (Le Metayer-Levrel et al., 1999) or the application of a carrier material (May, 2005) have been proposed. However, both measures have a signicant inuence on the total cost of the treatment, as can be seen from the previous paragraphs. Increasing the number of applications of nutrients increases the cost of the treatment due to the extra man hours needed, while the use of a carrier material has a major inuence on the product cost. From the different microbial metabolic pathways proposed for biodeposition, the hydrolysis of urea results, without any doubt in the fastest production of carbonate ions and hence precipitation of calcium carbonate. This is due to the fact that the hydrolysis of urea is a very rapid process and depends on only one enzyme. As a consequence, no additional nutrients are necessary for the long-term maintenance of the bacterial activity. And additionally,

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the time of wetting and thus the number of nutrient applications could be drastically decreased. Consequently, the outgrowth of other microorganisms (e.g. fungi and heterotrophic bacteria) will be highly unlikely. This makes the hydrolysis of urea a very feasible pathway for applications in situ. From the above, it is clear that the hydrolysis of urea could present some economical advantages over the other pathways. In addition to the feasibility, which is governed by the time required for production of carbonates to occur (see previous paragraphs), the efciency of the biodeposition treatment has been observed to depend on the speed of precipitation. Rodriguez-Navarro et al. (2003) reported on the importance of the type and structure of the precipitated CaCO3 polymorphs (vaterite or calcite) on the efciency of the biodeposition treatment. The presence of well developed rhombohedral calcite crystals resulted in a more pronounced consolidating effect compared to the presence of tiny acicular vaterite crystals. Differences in size and morphology of the crystals can be attributed to differences in the saturation state of a system preceding nucleation, with large rhombohedral calcite crystals being formed at relatively low supersaturation and vaterite crystals being produced under highly supersaturated conditions. From the above, the authors concluded that fast precipitation could result in a lower efciency of the biodeposition treatment. According to Rodriguez-Navarro et al. (2003), the presence of a phosphate buffer could explain for the occurrence of rhombohedral crystals. They attributed this to the buffering effect of the phosphate, preventing rapid local pH variations, and hence, rapid changes in the saturation state of the system. However, from the papers by Jimenez-Lopez et al. (2007, 2008), it appears that the phosphate buffer is not the only compositional difference between the M-3 and M-3P media (composition see Table 5). From the graphs indicating the removal of calcium ions from solution, it can be clearly observed that the initial concentration of calcium ions was much higher in the M-3 medium (50 mM Ca2+ ) compared to that of the M-3P medium (35 mM Ca2+ ). The latter could be attributed to the precipitation of calcium phosphate which was removed before the start of the experiment (Personal communication by Jimenez-Lopez et al., 2008). As a result, the M-3 medium showed initially a higher saturation state compared to the M-3P medium. In spite of the high speed of carbonate formation and calcium dosages used, De Muynck et al. (2009) obtained an excellent waterproong and consolidating effect with an ureolytic biodeposition treatment on Euville limestone. From SEM examinations, the presence of rhombohedral crystals could be clearly observed. Besides the hydrolysis of urea, most MICP treatments rely on the production of ammonia for the alkalinization of the culture medium. Because of the fact that atmospheric ammonia is being recognized as a pollutant, the in situ use of such treatments might raise some issues of environmental concern. Atmospheric ammonia is known to contribute to several environmental problems, including direct toxic effects on vegetation, atmospheric nitrogen deposition, leading to the eutrophication and acidication of sensitive ecosystems, and to the formation of secondary particulate matter in the atmosphere, with effects on human health, atmospheric visibility and global radiative balance (Sutton et al., 2008). However, when the concentration of ammonia generating compounds does not exceed the concentration of the calcium salt, it is possible to decrease the emission of ammonia to a great extent. De Muynck et al. (2009) observed in their biodeposition experiments that the pH of the solution remained about 7. At these pH values, ammonium will be the predominating compound. The neutral pH could be attributed to the fact that the precipitation of calcium

carbonate, resulting in a decrease of the pH, counteracts the pH increase as a result of the release of ammonia. Nonetheless, even in the case of the ureolytic biodeposition treatment the production of ammonium will be rather low compared to conventional sources of nitrogen pollution, i.e. agriculture and domestic waste water. The treatment of 1 m2 of building material with 1 L of a biodeposition medium containing 10 g L1 urea, results in the production of 4.7 g N. For comparison, from waste water treatment plants it can be calculated that one person produces between 6 and 16 g of N per day (DeCuyper and Loutz, 1992). The presence of ammonium might also present some risks to the stone itself. First of all, the presence of an ammonium salt might present some risks related to salt damage. Depending on the type of calcium salt used, ammonium acetate or ammonium chloride will be present in the stone after treatment. To our knowledge, no reports are available on the effect of these salts on stone. Therefore, future investigations should investigate the retention of these salts in the stone. Secondly, ammonium can be converted to nitric acid by the activity of nitrifying bacteria, resulting in damage to the stone. However, Mansch and Bock (1998) observed that the initial colonization of natural stone by nitrifying bacteria takes several years. In addition, the extent of colonization is mainly governed by the pH of the pore solution, with a pH between 7 and 9 being optimal for growth. As the initial pH of the biodeposition liquid is around 9.3, the activity of the nitrifying bacteria will be suppressed. Moreover, the applied chemoorganotrophic carbonate producing bacteria will outcompete the nitrifying bacteria for oxygen during the precipitation process. As a result of the precipitation, however, the pH will drop to a value of about seven. Therefore, in order to avoid nitrication in the long-term, the presence of large amounts of ammonium salts should be avoided. From long-term observations on the efciency of the Calcite Bioconcept treatment, however, no damages to the stone have been reported. If higher concentrations of ammonium should be produced, as might be the case for the hydrolysis of urea, the use of a paste might offer an attractive solution. The latter is one of the most commonly applied methods for the removal of salts from building materials (Wooltt and Abrey, 2008; Carretero et al., 2006). Upon wet application, the paste facilitates the dissolution of salts within stones and migration of ions to the outside, where they recrystallize and are retained. Once dry, the paste can be easily removed. Different types of pastes or combinations thereof have been applied for such purposes: paper pulp, clay materials (sepiolite, bentonite) and cellulose derivatives. As a result of their unique properties, many of these materials have also been used for the immobilization of microorganisms in a variety of elds. A combination of these two applications has already been applied for the removal of black crusts on stone artworks (Ranalli et al., 1997; Cappitelli et al., 2006). While Carbogel was observed to remove about 42% of the calcium ions from a black crust, the combination of the former with sulphate reducing bacteria led to a total removal efciency of about 95%. Besides removing the produced ammonium, the use of a paste will also protect the bacteria from drying out, enhancing the overall biodeposition treatment, as was observed by the Biobrush consortium (May, 2005). As seen before, however, the use of a paste results in a higher cost for the treatment. In their search for alternative approaches towards the Calcite Bioconcept method, most researchers have focused on the use of different organisms or metabolic pathways. Little attention, however, has been paid to the inuence of the dosage (g m2 ) or concentration (g L1 ) of the calcium salt and the nutrients (i.e. carbonate precursor components such as urea or amino acids) on the global effectiveness of the treatment. In many cases, the

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authors just applied the medium which had been used to illustrate the carbonate precipitation potential of the strain. An overview of the different concentrations of calcium salts used can be seen in Table 5. The importance of the calcium dosage on the overall effectiveness of the biodeposition treatment can be easily demonstrated by the following example. In the Calcite Bioconcept method, the total calcium dosage amounts to about 5.5 g m2 calcium chloride (MW 147 g mol1 ) or 1.5 g m2 calcium. Theoretically, this will result in an overall precipitation of about 3.74 g calcium carbonate (MW 100 g mol1 ) per square meter of stone surface. Assuming a density of calcium carbonate of 2.71 g cm3 and a homogenous precipitation over 1 m2 of a non-porous stone, this corresponds with a layer of calcium carbonate of about 1.38 m in thickness. In the case of a porous stone, a smaller thickness is expected due to the high surface area of the pores. In practice, however, bacteria will be mainly retained in the pores of the stone, especially those pores with a diameter larger than 1 m. Consequently, precipitation will mainly occur around large pores, and layer thicknesses greater than 1 m can be observed. This is in agreement with the ndings of Le Metayer-Levrel et al. (1999) and Orial (2000), who observed layer thicknesses of about 23 m and 45 m, respectively. Let us now consider the methodology as proposed by RodriguezNavarro et al. (2003). In one of their experiments, the authors submerged limestone prisms of 2.5 by 4.5 by 0.5 cm in an Erlenmeyer ask containing 100 mL of M-3 solution. As such, the theoretical calcium dosage amounts to about 339 g m2 calcium acetate (MW 230 g mol1 ) or 59 g m2 calcium, corresponding with an overall precipitation of about 147 g calcium carbonate per square meter. This will in theory result in a layer of calcium carbonate with a thickness of about 54 m on the surface of the limestone prisms. Although the authors did not report on the thickness of the carbonate layer, cementation was found up to depths larger than 500 m (Rodriguez-Navarro et al., 2003). As a result of the more pronounced precipitation of calcium carbonate in the case of the methodology proposed by RodriguezNavarro et al. (2003), a larger consolidating effect can be observed compared to the Calcite Bioconcept treatment. In their work on consolidation of sand columns by means of biocementation, Whifn et al. (2007) observed that a minimum amount of carbonate precipitation per m3 of sand was required in order to obtain a signicant consolidating effect. DeJong et al. (2006) observed that the cementing effect occurred as a result of the precipitated calcite forming bonds at the particle-particle contacts of sand grains. With increasing concentrations of precipitated carbonate, increasing bond formation and hence consolidation can be obtained. Therefore, increasing amounts of carbonate precipitates could result in an increased protective effect of the biodeposition treatment. This was indeed observed by De Muynck et al. (2009), who noticed an increased waterproong with increasing numbers of treatments or increasing the concentration of the crystal precursors in one treatment. The latter is also known to play a role in the speed, and hence, the type of crystals that are formed, affecting the global effectiveness of a treatment (Whifn et al., 2007). An increase in the dosage of the calcium salt could, however, lead to an accumulation of salts in the stone, which could depending on the anion give rise to eforescence or damage related to crystallization. As mentioned earlier, the use of a paste could prevent this from happening Regarding the Calcite Bioconcept treatment, several tests did not reveal any problems related to salt damage. The chlorides are rapidly washed away as a result of raining (Personal communication, Loubire, 2008). Besides the dosage of the calcium salt used, the type of stone will also have a major impact on the global performance of the treatment. The porosity, and more specically the pore size dis-

tribution could be considered as one of the most determining factors. Samonin and Elikova (2004) reported that for a maximum adsorption of microbial cells, the adsorbent pores must be 25 times larger than the cells. Therefore, the amount of bacteria retained in high macroporosity stones will be higher than in high microporosity stones. As a consequence, carbonate precipitation can occur at higher depths in macroporous stone. From SEM analyses, precipitation has been observed at depths of about 100 m for the Calcite Bioconcept treatment (Personal communication by Loubire, 2008). As mentioned earlier, Rodriguez-Navarro et al. (2003) observed precipitation at depths greater than 500 m in a bioclastic calcarenite. De Muynck et al. (2008b) observed an increased amount of biomass adsorption in mortar specimens with increasing water to cement ratio (w/c). The authors attributed this to the increasing amount of pores with a diameter larger than 1 m in specimens with increasing w/c. Since the amount of capillary pores between 2 and 10 m is rather limited in cementitious materials, the authors concluded that for these types of materials, the biodeposition treatment is mainly a surface phenomenon. This was also observed from thin sections, where a layer of crystals within the range of 1040 m on the surface was found, corresponding with the theoretical thickness calculated from the calcium dosage. From Table 1 it is clear that the differences between the various methodologies are not limited to the mediator used for precipitation. In addition, different research groups used different dosages of calcium salts and different application procedures on different types of stone. Besides the different metabolic pathways and bacteria proposed, the difference in inoculum size could also account for the differences in time required for precipitation to occur. Furthermore, many experiments were performed under sterile conditions. However, for applications in situ growth and activity are required under non-sterile conditions. This could potentially inuence on the microbial activity. From the above mentioned it should be clear that this will hamper any quantitative comparison between the different treatments. Additionally, such a comparison is even more difcult due to the fact that different authors used different evaluation parameters and procedures (Table 1). Some authors mainly focused on the waterproong effect (Dick et al., 2006), while others mainly investigated the strengthening effect (Rodriguez-Navarro et al., 2003; Jimenez-Lopez et al., 2007). In addition to these two effects, Tiano et al. (1999) further proposed the evaluation of the visual aspect before and after treatment by means of colorimetric analysis. Therefore, the next step in research regarding the application of calcinogenic bacteria should be a qualitative and quantitative evaluation of the different methodologies under identical conditions. Besides the evaluation of the protective performance (strengthening and waterproong (incl. porosity)), the inuence of the treatment on the visual aspect should be investigated. From this, the exact role of the microorganism and the metabolic pathway can be distinguished among the other parameters contributing to the overall effectiveness. An expanded knowledge on these factors will no doubt contribute to the added value of the biodeposition treatment, which is an ecological, compatible surface treatment with a high protective effect.

7. Future perspectives In 2010, the patent of Adolphe et al. (1990) will expire. This will certainly lead to further explorations of the biodeposition technique by the different research groups. As a result, reports on experiences from life size experiments can be soon expected.

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In addition, promising results on the use of microorganisms for the improvement of the durability of building materials have drawn the attention of research groups all over the world. Until now, work on biodeposition was mainly concentrated in Europe, while much of the work on remediation of cracks in concrete has been done in the USA. Recently, results from preliminary studies on bacterially induced carbonate precipitation from other research groups have come to our knowledge. In China, researchers are investigating the use of microbially induced carbonate precipitation for the restoration of ancient masonry buildings (Shen and Cheng, 2008) and the protection of concrete surfaces (Chunxiang et al., 2009). Furthermore, the method of producing CaCO3 by bacterial biomineralization has been patented in China by Qian et al. (2007). The latter was possible, as the patent by Adolphe et al. (1990) was limited to European countries. In Brazil, Shirakawa et al. (2008) are working on biodeposition on ber cement roof tiles. In India, researchers have applied for a patent in which the use of Shewanella for the improvement of the strength of concrete was described (Saroj, 2006). It is clear that the work done by several research groups, focusing on different materials, can only improve our understanding on the possibilities and limitations of biotechnological applications on building materials. However, as was already indicated by Webster and May (2006), the challenge for the immediate future is to translate some of the promising results obtained in the eld of bioremediation of building materials into practical applications. In accordance with the authors, we agree that the acceptance and satisfactory use of biotechnological applications by conservators requires knowledge on the risk factors, in particular the longterm effects of the applied bacteria and their nutrient media. In order to avoid any undesirable secondary effects, Gonzlez-Munoz (2008) already stressed the importance of investigations on the effects of the medium composition on the growth of autochthonous bacteria. By excluding the use of carbohydrates in the medium, Jimenez-Lopez et al. (2007) already indicated that the growth of acid producing bacteria could be avoided. Future investigations should also focus on the retention of nutrients and metabolic products in the stone, as they have an inuence on the survival, growth and biolm formation of the microorganisms inside the stone. Finally, research is needed on biodeposition on heavily degraded stone. Experiments have shown that ethylsilicates are unable to consolidate large grains (1.53 mm) of Euville limestone (KIK Testrapport, 1997). Such particles can be often found as a result of natural weathering. Biodeposition or more specically the concept of biological mortars could be used for the cementation of such coarse grains.

been hampered as a result of the differences in experimental procedures between the different research groups. In our opinion, too little attention has been paid to the calcium dosage and the type of stone, which could largely attribute for the differences observed between the various treatments. In this review, some recommendations have been made to improve the in situ feasibility of this type of treatment, both from an economical and practical point of view. In addition to the biodeposition treatment, the use of bacterially induced carbonates as a binder, i.e. biocementation, has been addressed. An overview has been given of the different elds of applications and their future prospects. Acknowledgements This research was funded by a BOF grant from Ghent University. The authors would like to thank Nico Boon, Bart De Gusseme, Melissa Dunkle and Siegfried E. Vlaeminck for critically reading the manuscript. References
Adolphe, J.P., Billy, C., 1974. Biosynthse de calcite par une association bactrienne arobie. C. R. Acad. Sci. Paris 278, 28732875. Adolphe, J.P., Hourimche, A., Loubire, J.F., Paradas, J., Soleilhavoup, F., 1989. Les formations carbonates dorigine bactrienne. Formations continentales dAfrique du Nord. Bull. Soc. Geol. Fr. 8 (5), 5562. Adolphe, J.P., Loubire, J.F., Paradas, J., Soleilhavoup, F., 1990. Procd de traitement biologique dune surface articielle. European patent 90400G97.0. (after French patent 8903517, 1989). Bachmeier, K.L., Williams, A.E., Warmington, J.R., Bang, S.S., 2002. Urease activity in microbiologically-induced calcite precipitation. J. Biotechnol. 93 (2), 171 181. Bang, S.S., Galinat, J.K., Ramakrishnan, V., 2001. Calcite precipitation induced by polyurethane-immobilized Sporosarcina pasteurii. Enzyme Microb. Technol. 28 (45), 404409. Barabesi, C., Galizzi, A., Mastromei, G., Rossi, M., Tamburini, E., Perito, B., 2007. Bacillus subtilis gene cluster involved in calcium carbonate biomineralization. J. Bacteriol. 189 (1), 228235. Bazylinski, D.A., Frankel, R.B., Konhauser, K.O., 2007. Modes of biomineralization of magnetite by microbes. Geomicrobiol. J. 24, 465475. Ben Chekroun, K., Rodriguez-Navarro, C., Gonzlez-Munoz, M.T., Arias, J.M., Cultrone, G., Rodriguez-Gallego, M., 2004. Precipitation and growth morphology of calcium carbonate induced by Myxococcus xanthus: implications for recognition of bacterial carbonates. J. Sediment. Res. 74 (6), 868876. Ben Omar, N., Entrena, M., Gonzlez-Munoz, M.T., Arias, J.M., 1994. Effects of Ph and phosphate on the production of struvite by Myxococcus xanthus. Geomicrobiol. J. 12 (2), 8190. Ben Omar, N., Gonzlez-Munoz, M.T., Penalver, J.M.A., 1998. Struvite crystallization on Myxococcus cells. Chemosphere 36 (3), 475481. Ben Omar, N., Martnez-Canamero, M., Gonzlez-Munoz, M.T., Maria Arias, J., Huertas, F., 1995. Myxococcus xanthus killed cells as inducers of struvite crystallization. Its possible role in the biomineralization processes. Chemosphere 30 (12), 23872396. Boquet, E., Boronat, A., Ramos-Cormenzana, A., 1973. Production of calcite (calcium carbonate) crystals by soil bacteria is a general phenomenon. Nature 246 (5434), 527529. Braissant, O., Cailleau, G., Dupraz, C., Verrecchia, E., 2003. Bacterially induced mineralization of calcium carbonate in terrestrial environments: the role of exopolysaccharides and amino acids. J. Sediment. Res. 73 (3), 485 490. Braissant, O., Verrecchia, E., Aragno, M., 2002. Is the contribution of bacteria to terrestrial carbon budget greatly underestimated? Naturwissenschaften 89 (8), 366370. Cacchio, P., Ercole, C., Cappuccio, G., Lepidi, A., 2003. Calcium carbonate precipitation by bacterial strains isolated from a limestone cave and from a loamy soil. Geomicrobiol. J. 20 (2), 8598. Camaiti, M., Borselli, G., Matteol, U., 1988. Prodotti consalidanti impiegati nelle operazioni di restauro. Edilizia 10, 125134. Cappitelli, F., Toniolo, L., Sansonetti, A., Gulotta, D., Ranalli, G., Zanardini, E., Sorlini, C., 2007. Advantages of using microbial technology over traditional chemical technology in removal of black crusts from stone surfaces of historical monuments. Appl. Environ. Microbiol. 73 (17), 56715675. Cappitelli, F., Zanardini, E., Ranalli, G., Mello, E., Daffonchio, D., Sorlini, C., 2006. Improved methodology for bioremoval of black crusts on historical stone artworks by use of sulfate-reducing bacteria. Appl. Environ. Microbiol. 72 (5), 37333737. Carretero, M.I., Bernab, J.M., Galan, E., 2006. Application of sepiolite-cellulose pastes for the removal of salts from building stones. Appl. Clay Sci. 33, 4351.

8. Summary The knowledge about the microbial origin of limestone has resulted in research concerning MICP for the protection of ornamental stone. The rst patent in which this method has been described already dates from almost two decades ago. Since then, different research groups have searched for alternative approaches to obtain a protective layer of calcium carbonate on the surface of building materials (biodeposition). Some authors suggested the use of alternative microorganisms or metabolic pathways, while others obtained precipitation without the application of calcinogenic bacteria, consequently falling outside the scope of the claims of the Calcite Bioconcept Patent. The goal of this review is to provide an in-depth overview of the different methodologies, allowing for a qualitative comparison of their performance. A quantitative evaluation however, has

W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 Castanier, S. 1995. Nouvelles compositions pour mortier biologique, procd de recourvrement dun surface ou de comblement dune cavit laide des compositions. French patent. No. 95 05861. Castanier, S., Le Metayer-Levrel, G., Perthuisot, J.-P., 1999. Ca-carbonates precipitation and limestone genesisthe microbiogeologist point of view. Sediment. Geol. 126 (14), 923. Chaturvedi, S., Chandra, R., Rai, V., 2006. Isolation and characterization of Phragmites australis (L.) rhizosphere bacteria from contaminated site for bioremediation of colored distillery efuent. Ecol. Eng. 27 (3), 202207. Chunxiang, Q., Jianyun, W., Ruixing, W., Liang, C., 2009. Corrosion protection of cement-based building materials by surface deposition of CaCO3 by Bacillus pasteurii. Mater. Sci. Eng. 29 (4), 12731280. Clifton, J.R., Frohnsdorff, G.J.C., 1982. Stone consolidating materials: a status report. In: Conservation of Historic Stone Buildings and Monuments. National Academy Press, Washington, DC, pp. 287311. Day, J.L., Ramakrishnan, V., Bang, S.S., 2003. Microbiologically induced sealant for concrete crack remediation. In: Proc. of 16th Engineering Mechanics Conference, Seattle. DeCuyper, K., Loutz, S., 1992. Les caractristiques des eaux uses domestiques. Tribune Cebedeau 45 (560), 719. De Belie, N., De Muynck, W., 2008. Crack repair in concrete using biodeposition. In: Proc. of ICCRR Cape Town, South Africa. De Muynck, W., Cox, K., De Belie, N., Verstraete, W., 2008a. Bacterial carbonate precipitation as an alternative surface treatment for concrete. Constr. Build. Mater. 22 (5), 875885. De Muynck, W., Debrouwer, D., De Belie, N., Verstraete, W., 2008b. Bacterial carbonate precipitation improves the durability of cementitious materials. Cem. Concr. Res. 38 (7), 10051014. De Muynck, W., Van Hyfte, E., Verbeken, K., De Belie, N., Verstraete, W., 2008c. In situ enrichment of carbonate producing bacteria for biodeposition in practice. In: Proc. of the 1st International Conference on BioGeoCivil Engineering, Delft, The Netherlands, p. 35. De Muynck, W., Verbeken, K., De Belie, N., Verstraete, W., 2009. Inuence of the calcium dosage on the effectiveness of bacterially induced carbonate precipitation on limestone. Ecol. Eng., doi:10.1016/j.ecoleng.2009.03.025, this issue. Delgado Rodrigues, J., 2001. Consolidation of decayed stones. A delicate problem with few practical solutions. In: Lourec o, P.B., Roca, P. (Eds.), International Seminar on Historical Constructions. Guimares, Portugal. DeJong, J.T., Fritzges, M.B., Nusslein, K., 2006. Microbially induced cementation to control sand response to undrained shear. J. Geotech. Geoenviron. 132 (11), 13811392. Dick, J., De Windt, W., De Graef, B., Saveyn, H., Van der Meeren, P., De Belie, N., Verstraete, W., 2006. Bio-deposition of a calcium carbonate layer on degraded limestone by Bacillus species. Biodegradation 17 (4), 357367. Douglas, S., Beveridge, T.J., 1998. Mineral formation by bacteria in natural microbial communities. FEMS Microbiol. Ecol. 26 (2), 7988. Dreesen, R., Dusar, M., 2004. Historical building stones in the province of Limburg (NE Belgium): role of petrography in provenance and durability assessment. Mater. Charact. 53 (24), 273287. Drew, 1911. The action of some denitrifying bacteria in tropical and temperate seas, and bacterial precipitation of calcium carbonate in the sea. J. Mar. Biol. Ass. 9, 142155. Ehrlich, H.L., 1996. How microbes inuence mineral growth and dissolution. Chem. Geol. 132 (14), 59. Ehrlich, H.L., 1998. Geomicrobiology: its signicance for geology. Earth Sci. Rev. 45 (12), 4560. Ercole, C., Cacchio, P., Botta, A.L., Centi, V., Lepidi, A., 2007. Bacterially induced mineralization of calcium carbonate: the role of exopolysaccharides and capsular polysaccharides. Microsc. Microanal. 13 (4250). Ferris, F.G., Stehmeier, L.G., 1992. Bacteriogenic mineral plugging. USA Patent US5143155. Fujita, Y., Ferris, F.G., Lawson, R.D., Colwell, F.S., Smith, R.W., 2000. Calcium carbonate precipitation by ureolytic subsurface bacteria. Geomicrobiol. J. 17 (4), 305318. Fujita, Y., Redden, G.D., Ingram, J.C., Cortez, M.M., Ferris, F.G., Smith, R.W., 2004. Strontium incorporation into calcite generated by bacterial ureolysis. Geochim. Cosmochim. Acta 68 (15), 32613270. Gauri, K.L., Parks, L., Jaynes, J., Atlas, R., 1992. Removal of sulphated crust from marble using sulphate-reducing bacteria. In: Robin, G.M. (Ed.), Stone Cleaning and the Nature, Soiling and Decay Mechanisms of Stone. Proceedings of the International conference. 1416 April, Donhead Publishing Ltd., Edinburgh, United Kingdom, pp. 160165. Ghosh, P., Mandal, S., Chattopadhyay, B.D., Pal, S., 2005. Use of microorganism to improve the strength of cement mortar. Cem. Concr. Res. 35 (10), 19801983. Gollapudi, U.K., Knutson, C.L., Bang, S.S., Islam, M.R., 1995. A new method for controlling leaching through permeable channels. Chemosphere 30 (4), 695705. Gonzlez-Munoz, M., Arias, J.M., Montoya, E., Rodriguez-Gallego, M., 1993. Struvite production by Myxococcus coralloides D. Chemosphere 26 (10), 1881 1887. Gonzlez-Munoz, M.T., Omar, N.B., Martnez-Canamero, M., Rodrguez-Gallego, M., Galindo, A.L., Arias, J., 1996. Struvite and calcite crystallization induced by cellular membranes of Myxococcus xanthus. J. Cryst. Growth 163 (4), 434439. Gonzlez-Munoz, M.T., Rodriguez-Navarro, C., Jimenez-Lopez, C., RodriguezGallego, M., 2008. Method and product for protecting and reinforcing construction and ornamental materials. WO 2008/009771 A1.

135

Gonzlez-Munoz, M.T., 2008. Bacterial biomineralization applied to the protectionconsolidation of ornamental stone: current development and perspectives. Coalition 15, 1218. Hammes, F., Boon, N., de Villiers, J., Verstraete, W., Siciliano, S.D., 2003a. Strainspecic ureolytic microbial calcium carbonate precipitation. Appl. Environ. Microbiol. 69 (8), 49014909. Hammes, F., Seka, A., de Knijf, S., Verstraete, W., 2003b. A novel approach to calcium removal from calcium-rich industrial wastewater. Water Res. 37 (3), 699704. Hammes, F., Verstraete, W., 2002. Key roles of pH and calcium metabolism in microbial carbonate precipitation. Rev. Environ. Sci. Biotechnol. 1, 37. Hart, R.T., Fekete, T., Flock, D.L., 1960. The plugging effect of bacteria in sandstone systems. Can. Min. Metall. Bull. 53, 495501. Heselmeyer, K., Fisher, U., Krumbein, K.E., Warscheid, T., 1991. Application of Desulfovibrio vulgaris for the bioconversion of rock gypsum crusts into calcite. Bioforum 1/2, 89. Jack, T.R., Stehmeier, L.G., Ferris, F.G., Islam, M.R., 1991. Microbial selective plugging to control water channeling. In: Donaldson (Ed.), Microbial Enhancement of Oil Recovery-Recent Advances. Elsevier Science Publishing Co., New York. Jimenez-Lopez, C., Jroundi, F., Pascolini, C., Rodriguez-Navarro, C., Pinar-Larrubia, G., Rodriguez-Gallego, M., Gonzlez-Munoz, M.T., 2008. Consolidation of quarry calcarenite by calcium carbonate precipitation induced by bacteria activated among the microbiota inhabiting the stone. Int. Biodeterior. Biodegrad. 62 (4), 352363. Jimenez-Lopez, C., Rodriguez-Navarro, C., Pinar, G., Carrillo-Rosa, F.J., Rodriguez Gallego, M., Gonzlez-Munoz, M.T., 2007. Consolidation of degraded ornamental porous limestone by calcium carbonate precipitation induced by the microbiota inhabiting the stone. Chemosphere 68 (10), 19291936. Jonkers, H., 2007. Self healing concrete: a biological approach. In: van der Zwaag, S. (Ed.), Self Healing Materials: An alternative Approach to 20 Centuries of Materials Science. Springer, Netherlands, pp. 195204. Jonkers, H.M., Schlangen, E., 2007. Crack repair by concrete-immobilized bacteria. In: Schmets, A.J.M., Van der Zwaag, S. (Eds.), Proc. of First International Conference on Self Healing Materials. Noordwijk, The Netherlands, p. 7. Jonkers, H.M., Thijssen, A., Copuroglu, O., Schlangen, E., 2008. Application of bacteria as self-healing agent for the development of sustainable concrete. In: Proc. of First International Conference on BioGeoCivil Engineering, Delft, The Netherlands, p. 25. Kantzas, A., Ferris, F.G., Jha, K.N., Mourits, F.M., 1992. A novel method of sand consolidation through bacteriogenic mineral plugging. In: Proc. of CIM Annual Technical Conference, Calgary. KIK Testrapport, 1997. Test report on the efciency of the FTB consolidant SH 75, an ethylsilicates based consolidant (in Dutch). http://www.ftbremmers. com/leadmin/dam/Testrapporten/KIK SH75.pdf on November 2008. Knorre, H., Krumbein, K.E., 2000. Bacterial calcication. In: Riding, E.E., Awramik, S.M. (Eds.), Microbial Sediments. SpringerVerlag, Berlin, pp. 2531. Kucharski, E.S., Cord-Ruwisch, R., Whifn, V., Al-Thawadi, S.M.J., 2006. Microbial biocementation. World intellectual property organization, WO 2006/066326 A1. Lappin-Scott, H.M., Cusack, F., Costerton, J.W., 1988. Nutrient resuscitation and growth of starved cells in sandstone cores: a novel approach to enhanced oil recovery. Appl. Environ. Microbiol. 54 (6), 13731382. Le Metayer-Levrel, G., Castanier, S., Orial, G., Loubiere, J.F., Perthuisot, J.P., 1999. Applications of bacterial carbonatogenesis to the protection and regeneration of limestones in buildings and historic patrimony. Sediment. Geol. 126 (14), 2534. Lowenstan, H.A., Weiner, S., 1988. On Biomineralization. Oxford University Press, New York. Mansch, R., Bock, E., 1998. Biodeterioration of natural stone with special reference to nitrifying bacteria. Biodegradation 9 (1), 4764. Mastromei, G., Marvasi, M., Perito, B., 2008. Studies on bacterial carbonate precipitation for stone conservation. In: Proc. of BioGeoCivil Engineering Conference, Delft, Netherlands, pp. 104106. May, E., 2005. Biobrush research monograph: novel approaches to conserve our European heritage. EVK4-CT-2001-00055. McConnaughey, T.A., Whelan, J.F., 1997. Calcication generates protons for nutrient and bicarbonate uptake. Earth Sci. Rev. 42 (12), 95117. Mobley, H.L., Hausinger, R.P., 1989. Microbial ureases: signicance, regulation, and molecular characterization. Microbiol. Mol. Biol. Rev. 53 (1), 85108. Morita, R., 1980. Calcite precipitation by marine bacteria. Geomicrobiol. J. 2, 6382. Moropoulou, A., Kouloumbi, N., Haralampopoulos, G., Konstanti, A., Michailidis, P., 2003. Criteria and methodology for the evaluation of conservation interventions on treated porous stone susceptible to salt decay. Prog. Org. Coat. 48, 259270. Morse, J.W., 1983. The kinetics of calcium carbonate dissolution and precipitation. In: Reeder, R.J. (Ed.), Carbonates: Mineralogy and Chemistry, vol. 11. Mineralogic Society of America, Washington, DC, pp. 227264. Nemati, M., Greene, E.A., Voordouw, G., 2005. Permeability prole modication using bacterially formed calcium carbonate: comparison with enzymic option. Process Biochem. 40 (2), 925933. Nemati, M., Voordouw, G., 2003. Modication of porous media permeability, using calcium carbonate produced enzymatically in situ. Enzyme Microb. Technol. 33 (5), 635642. Nielsen, T.H., Bonde, T.A., Sorensen, J., 1998. Signicance of microbial urea turnover in N cycling of three Danish agricultural soils. FEMS Microbiol. Ecol. 25 (2), 147157.

136

W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 Shirakawa, M., Cincotto, M., Dias, C., Atencio, D., Vanderley, M.J., 2008. Inuence of carbonation in accelerated chamber previous to biocalcication on ber cement surface. In: 1st BioGeoCivil Engineering Conference, Delft. Simon, M.A., Bonner, J.S., Page, C.A., Townsend, R.T., Mueller, D.C., Fuller, C.B., Autenrieth, R.L., 2004. Evaluation of two commercial bioaugmentation products for enhanced removal of petroleum from a wetland. Ecol. Eng. 22 (45), 263 277. Stocks-Fischer, S., Galinat, J.K., Bang, S.S., 1999. Microbiological precipitation of CaCO3 . Soil Biol. Biochem. 31 (11), 15631571. Stumm, W., Morgan, J.J., 1981. Aquatic Chemistry, 2nd edition. John Wiley, New York. Sutherland, I.W., Thomson, S., 1975. Comparison of polysaccharides produced by Myxococcus strains. J. Gen. Microbiol. 89 (JULY), 124132. Sutton, M., Reis, S., Baker, S., 2008. Atmospheric ammonia: detecting emission changes and environmental impact. In: Results of an Expert Workshop Under the Convention on Long-Range Transboundary Air Pollution. Springer, pp. 490. Tiano, P., 1995. Stone reinforcement by calcite crystal precipitation induced by organic matrix macromolecules. Stud. Conserv. 40 (3), 171176. Tiano, P., Addadi, L., Weiner, S., 1992. Stone reinforcement by induction of calcite crystals using organic matrix macromolecules: feasibility study. In: 7th International Congress on Deterioration and Conservation of Stone, Lisbon, pp. 13171326. Tiano, P., Biagiotti, L., Mastromei, G., 1999. Bacterial bio-mediated calcite precipitation for monumental stones conservation: methods of evaluation. J. Microbiol. Methods 36 (12), 139145. Tiano, P., Cantisani, E., Sutherland, I., Paget, J.M., 2006. Biomediated reinforcement of weathered calcareous stones. J. Cult. Herit. 7 (1), 4955. Urzi, C., Garcia-Valles, M., Vendrell, M., Pernice, A., 1999. Biomineralization processes on rock and monument surfaces observed in eld and in laboratory conditions. Geomicrobiol. J. 16, 3954. Warren, L.A., Maurice, P.A., Parmar, N., Ferris, F.G., 2001. Microbially mediated calcium carbonate precipitation: implications for interpreting calcite precipitation and for solid-phase capture of inorganic contaminants. Geomicrobiol. J. 18, 93125. Warscheid, T., Braams, J., 2000. Biodeterioration of stone: a review. Int. Biodeter. Biodegr. 46 (4), 343368. Webster, A., May, E., 2006. Bioremediation of weathered-building stone surfaces. Trends Biotechnol. 24 (6), 255260. Whifn, V.S., 2004. Microbial CaCO3 precipitation for the production of Biocement. Ph.D. Thesis, Murdoch University, Australia, pp. 155. http://wwwlib. murdoch.edu.au/adt/browse/view/adt-MU20041101.142604. Whifn, V.S., van Paassen, L., Harkes, M.P., 2007. Microbial carbonate precipitation as a soil improvement technique. Geomicrobiol. J. 24, 417423. Wooltt, C., Abrey, G., 2008. Poultices: the true or plain poultice and the cleaning and desalination of historic masonry and sculpture. Retrieved august 2008, from http://www.buildingconservation.com/articles/poultices/poultice.htm. Zhong, L., Islam, M.R., 1995. A new microbial plugging process and its impact on fracture remediation. In: Proc. of Society of Petroleum Engineers. Annual Technical Conference, Dallas, Texas, pp. 703715.

Orial, G., 2000. La biomineralisation applique la conservation du patrimoine: bilan de dix ans dexperimentation. Restaurar la memoria, Valladolid, Spain. Orial, G., Vieweger, T., Loubiere, J.F., 2002. Les mortiers biologiques: une solution pour la conservation de la sculpture monumentale en pierre. Art Biology and Conservation, Metropolitan Museum, New York. Perito, B., Biagiotti, L., Daly, S., Galizzi, A., Tiano, P., Mastromei, G., 2000. Bacterial genes involved in calcite crystal precipitation. In: Ciferri, O., Tiano, P., Mastromei, G. (Eds.), Of Microbes and Art: The Role of Microbial Communities in the Degradation and Protection of Cultural Heritage. Plenum Publisher, New York, pp. 219230. Qian, C., Wang, R., Wang, J., 2007. The method of producing CaCO3 by bacterial biomineralization. ZL2005100947744.5. Ramachandran, S.K., Ramakrishnan, V., Bang, S.S., 2001. Remediation of concrete using micro-organisms. ACI Mater. J. 98, 39. Ramakrishnan, S.K., Panchalan, R.K., Bang, S.S., 2001. Improvement of concrete durability by bacterial mineral precipitation. In: 11th International conference on Fracture, Turin, Italy. Ranalli, G., Chiavarini, M., Guidetti, V., Marsala, F., Matteini, M., Zanardini, E., Sorlini, C., 1997. The use of micro-organisms for the removal of sulphates on artistic stoneworks. Int. Biodeterior. Biodegrad. 40 (24), 255261. Ranalli, G., Matteini, M., Tosini, I., Zanardini, E., Sorlini, C., 1999. Bioremediation of cultural heritage: removal of sulphates, nitrates and organic substances. In: Ciferri, O., Tiano, P., Mastromei, G. (Eds.), Proc. of International Conference on Microbiology and Conservation Of Microbes and Art: The Role of Microbial Communities on the Degradation and Protection of Cultural Heritage. Florence, Italy, pp. 231245. Rivadeneyra, M.A., Delgado, R., del Moral, A., Ferrer, M.R., Ramos-Cormenzana, A., 1994. Precipitation of calcium carbonate by Vibrio spp. from an inland saltern. FEMS Microbiol. Ecol. 13 (3), 197204. Rivadeneyra, M.A., Parraga, J., Delgado, R., Ramos-Cormenzana, A., Delgado, G., 2004. Biomineralization of carbonates by Halobacillus trueperi in solid and liquid media with different salinities. FEMS Microbiol. Ecol. 48, 3946. Rodriguez-Navarro, C., Jimenez-Lopez, C., Rodriguez-Navarro, A., Gonzlez-Munoz, M.T., Rodriguez-Gallego, M., 2007. Bacterially mediated mineralization of vaterite. Geochim. Cosmochim. Acta 71 (5), 11971213. Rodriguez-Navarro, C., Rodriguez-Gallego, M., Ben Chekroun, K., Gonzlez-Munoz, M.T., 2003. Conservation of ornamental stone by Myxococcus xanthus-induced carbonate biomineralization. Appl. Environ. Microbiol. 69 (4), 21822193. Samonin, V.V., Elikova, E.E., 2004. A study on the absorption of bacterial cells on porous materials. Microbiology 73 (6), 696701. Saiz-Jimenez, C., 1997. Biodeterioration vs biodegradation: the role of microorganisms in the removal of pollutants deposited on historic buidlings. Int. Biodeter. Biodegr. 40 (24), 225232. Saroj, M., 2006. A process for preparing modied bioconcrete. C04B 28/02 (Indian patent). Shen, J., Cheng, X., 2008. Laboratory investigation on restoration of Chinese ancient masonry buildings using microbial carbonate precipitation. In: 1st BioGeoCivil Engineering Conference, Delft. Shinano, H., 1972. Studies of marine microorganisms taking part in the precipitation of calcium carbonate. Bull. Jpn. Soc. Sci. Fish. 38, 717.