Microbial Carbonate Precipitation Review

8/12/2019 Microbial Carbonate Precipitation Review

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Ecological Engineering 36 (2010) 118136

Contents lists available atScienceDirect

Ecological Engineering

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e c o l e n g

Review

Microbial carbonate precipitation in construction materials: A review

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

a Magnel Laboratory for Concrete Research, Dept. of Structural Engineering, Ghent University, Technologiepark Zwijnaarde 904, B-9052 Gent, Belgiumb 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

Article history:

Received 12 August 2008

Received in revised form 11 February 2009

Accepted 13 February 2009

Keywords:

Bacteria

Stone

Biomineralization

Biodeposition

Biomortar

BiocementBioconcrete

Calcite

Conservation

MICP

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 field of construction materials. One of the first patented applications concerned the protection ofornamental stone by means of a microbially deposited carbonate layer, i.e. biodeposition. The promisingresults of this technique encouraged different research groups to evaluate alternative approaches, eachgroup commenting on the original patent and promoting its bacterial strain or method as the best per-

forming. Thegoal of thisreviewis to providean in-depthcomparison of thesedifferent approaches. Specialattentionwas paid to theresearch background that could account forthe choiceof themicroorganismand

themetabolic pathway proposed. In addition, evaluation of thevarious methodologies allowed for a clearinterpretation of the differences observed in effectiveness. Furthermore, recommendations to improve

thein situfeasibility of the biodeposition method are postulated. In the second part of this paper, the useof 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. Cur-rent 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.

1. Introduction

Construction materials such as stone and concrete are sub-jected to the weathering action of several physical, chemical andbiological factors (Saiz-Jimenez, 1997; Le Metayer-Levrel et al.,1999; Warscheid and Braams, 2000).Because of their composition

and textural characteristics, carbonate stones (limestones, dolo-stones and marbles) are particularly susceptible to weathering.Progressive dissolution of the mineral matrix as a consequence ofweathering leads to an increase of the porosity, and as a result, a

decrease of the mechanical features (Tiano et al., 1999).In orderto decrease the susceptibility to decay, many conservation treat-ments have been applied with the aim of modifying some of thestone characteristics. Water repellents have been applied to pro-

tect stone from the ingress of water and other weathering agents.The use of stone consolidants aims at re-establishing the cohesionbetween grains of deteriorated stone. However, both conservation

treatments are subject to frequent controversy due to their non-reversibleaction and theirlimited long-term performance. Because

Corresponding author. Tel.: +32 092645522; fax: +32 092645845.

E-mail addresses: [email protected](W. De Muynck),

[email protected](N. De Belie), [email protected](W. Verstraete).1 Tel.: +32 092645976; fax: +32 092646248.

of problems related to incompatibility with the stone, both waterrepellents and consolidants have often been reported to acceler-

ate stone decay. (Clifton and Frohnsdorff, 1982; Delgado Rodrigues,2001; Moropoulou et al., 2003).

Organic treatmentscommonlyresult in the formationof incom-patible and often harmful surface films. Additionally, because large

quantitiesof organic solvents are used, they contribute to pollution(Camaiti etal.,1988; Rodriguez-Navarroet al.,2003). Inorganiccon-solidation may be preferable since stone materials and protectiveor consolidating materials share some physico-chemical affinity

(Rodriguez-Navarro et al., 2003).Some researchers have tried todevelop methods based on the reintroduction of calcite into thepores of limestone. The lime-water technique, i.e. application of asaturated solution of calcium hydroxide, has been proposed and

experimented both forwall painting mortars and forsome deterio-rated calcareous stones, in order to impart a slight water repellentand consolidating effect (Tiano et al., 1999).As of yet, little success

has been achieved in consolidating stone with inorganic materials.Some of the reasons for the poor performance of inorganic con-solidants are their tendencies to produce shallow and hard crustsbecause of their poor penetration abilities, the formation of soluble

salts as reaction by-products, growth of precipitated crystals andthe questionable ability of some of them to bind stone particlestogether (Clifton and Frohnsdorff, 1982).In the case of the calcitereintroduction methods, the latter is attributable to the production

0925-8574/$ see front matter 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.ecoleng.2009.02.006
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W. De Muynck et al. / Ecological Engineering 36 (2010) 118136 119

of many small crystallites, which are not chemically bound to theinternal surface of the pore and which are not able to bridge thepores (Tiano et al., 2006).

Recently, bacterially induced carbonate precipitation has beenproposed as an environmentally friendly method to protectdecayed ornamental stone. The method relies on the bacteriallyinduced formation of a compatible carbonate precipitate on lime-

stone, and unlike the lime-water treatment, the carbonate cementappears to be highly coherent (Le Metayer-Levrel et al., 1999).In addition, this technique has been explored for the improve-

ment of the durability of cementitious materials (Ramachandran et

al., 2001; Ramakrishnan et al., 2001; De Muynck et al., 2008a,b).

2. Microbially induced carbonate precipitation (MICP)

Like other biomineralization processes, calcium carbonate(CaCO3) precipitation can occur by two different mechanisms:biologically controlled or induced (Lowenstan and Weiner, 1988).

In biologically controlled mineralization, the organism controlsthe process, i.e. nucleation and growth of the mineral particles,to a high degree. The organism synthesizes minerals in a formthat is unique to that species, independently of environmental

conditions. Examples of controlled mineralization are magnetiteformation in magnetotactic bacteria (Bazylinski et al., 2007)and silica deposition in the unicellular algae coccolithophoresand diatoms, respectively (Barabesi et al., 2007). However, cal-

cium carbonate production by bacteria is generally regarded asinduced, as the type of mineral produced is largely dependenton the environmental conditions (Rivadeneyra et al., 1994)andno specialized structures or specific molecular mechanism are

thought to be involved (Barabesi et al., 2007). Different typesof bacteria, as well as abiotic factors (salinity and composi-tion of the medium) seem to contribute in a variety of waysto calcium carbonate precipitation in a wide range of different

environments (Knorre and Krumbein, 2000; Rivadeneyra et al.,2004).

Calcium carbonate precipitation is a rather straightforwardchemical process governed mainly by four key factors: (1) the cal-

cium concentration, (2) the concentration of dissolved inorganiccarbon (DIC), (3) the pH and (4) the availability of nucleation sites(Hammes and Verstraete, 2002).CaCO3precipitation requires suf-ficient calcium and carbonate ions so that the ion activity product

(IAP) exceeds the solubility constant (Kso) (Eqs.(1)and(2)).Fromthe comparison of the IAP with theKsothe saturation state () ofthe system can be defined; if > 1 thesystem is oversaturated andprecipitation is likely (Morse, 1983):

Ca2++CO32CaCO3 (1)

=a(Ca2+)a(CO32)/Kso with Ksocalcite,25 =4.810

9 (2)

The concentration of carbonate ions is related to the concentra-tion of DIC and the pH of a given aquatic system. In addition, theconcentration of DIC depends on several environmental parameters

such as temperature and the partial pressure of carbon dioxide (forsystems exposed to the atmosphere). The equilibrium reactionsandconstants governing the dissolutionof CO2in aqueous media (25

Cand 1 atm) are given in Eqs.(3)(6)(Stumm and Morgan, 1981):

CO2(g) CO2(aq.) (pKH= 1.468) (3)

CO2(aq.)+H2O H2CO3 (pK= 2.84) (4)

H2CO3H++HCO3

(pK1=6.352) (5)

HCO3 CO3

2+H+ (pK2=10.329) (6)

WithH2CO3= CO

2(aq.)+H2CO3.

Microorganisms can influence precipitation by altering almost

any of the precipitation parameters described above, either sepa-rately or in various combinations with one another (Hammes andVerstraete, 2002).However, the primary role has been ascribedto their ability to create an alkaline environment through various

physiological activities. Both autotrophic and heterotrophic path-ways are involved in the creation of such an alkaline environment

(for an extensive review, seeCastanier et al., 1999). While theenvironmental conditions of heterotrophic pathways are diverse

(aerobiosis, anaerobiosis and microaerophily), carbonateprecipita-tion always appears to be a response of the heterotrophic bacterialcommunities to an enrichment of the environment in organic mat-ter (Castanier et al., 1999).A first heterotrophic pathway involves

the sulphur cycle, in particular the dissimilatory sulphate reduc-tion, which is carried out by sulphate reducing bacteria underanoxic conditions. A second heterotrophic pathway involves thenitrogen cycle, and more specifically, (1) the oxidative deamina-

tion of amino acids in aerobiosis, (2) the dissimilatory reduction ofnitrate in anaerobiosis or microaerophily and (3) the degradationof urea or uric acid in aerobiosis. Another microbial process thatleads to an increase of both the pH and the concentration of dis-

solvedinorganiccarbonis the utilization of organic acids (Braissantet al., 2002), a process which has beencommonly used in microbialcarbonate precipitation experiments. The precipitation pathwaysdescribed above are general in nature, which accounts for the com-

mon occurrence of microbial carbonate precipitation (MCP) andvalidates the statement byBoquet et al. (1973)that under suitableconditions, most bacteria are capable of inducing carbonate pre-cipitation. In addition, carbonate particles can also be produced by

ion exchange through the cell membrane (Rivadeneyra et al., 1994;Castanier et al., 1999).

Besides changes induced in the macro-environment, bacteriahave also been reported to influence calcium carbonate precipita-

tion by acting as sites of nucleation or calcium enrichment(Morita,1980). Due to the presence of several negatively charged groups onthe cell wall, at a neutral pH, positively charged metal ions can bebound on bacterial surfaces (Douglas and Beveridge, 1998; Ehrlich,

1998). Suchboundmetal ions(e.g.calcium)maysubsequently reactwith anions (e.g. carbonate) to form an insoluble salt (e.g. calciumcarbonate). In the case of a sufficient excess of the required cationsand anions, the metal salt on the cell surface initiates mineral for-

mation by acting as a nucleation site. The anion (e.g. carbonate)in this reaction may be a product of the bacterial metabolism, orit may have an abiotic origin (Ehrlich, 1998).Furthermore, it hasbeen demonstrated that specific bacterial outer structures (glyco-

calyx and parietal polymers) consisting of exopolysaccharides andamino acids play an essential role in the morphology and mineral-ogy 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 bean unwanted and accidental by-product of the metabolism (Knorreand Krumbein, 2000) while others think that it is a specific process

with ecological benefits for the precipitating organisms (Ehrlich,1996; McConnaughey and Whelan, 1997).

The evidence of microbial involvement in carbonate precipi-tation has subsequently led to the exploration of this process in

a variety of fields. A first series of applications is situated in thefieldof bioremediation. In addition to conventional bioremediationstrategies 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 includethe 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).


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

Table 1

Characteristics of the various biodeposition processes.

Characteristic Process

Biodeposition

Definition Biologically induced deposition of a carbonate layer on the surface of building materials

Type Surface treatment

Goals Improvement of the durability, consolidation and decrease of water absorption

Where/when Applied on the surface of building materials such as stone, bricks and concrete

Mediator Microorganisms Organic matrix molecules (OMM) Activator mediumHow Spraying of bacteria and nutrients Spraying of OMM and carbonate rich solution Application of nutrient media

Application of poultice

Research group Calcite Bioconcept, Granada University, Ghent

University Biobrush consortium

Bioreinforce consortium Granada University

Added value Ecological, environmentally friendly, compatibility

Possibility of including pigments

(Current) Limitations Costs of bacteria and nutrients Costs of chemicals

Rather limited efficacy Long activation period required

Status of use The Calcite Bioconcept: laboratory andin situ

experiments

Laboratory andin situexperiments Laboratory andin situexperiments

Several research groups: laboratory experiments

Market Niche Restoration sector

Limestone, concrete Marble statues Limestone

Another series of applications aims at modifying the propertiesof soil, i.e. for the enhancement of oil recovery from oil reservoirs(Nemati andVoordouw, 2003;Nematiet al., 2005), plugging (Ferris

and Stehmeier, 1992)and strengthening of sand columns (DeJonget al., 2006; Whiffin et al., 2007).Moreover, microbially inducedprecipitation has been investigated for its potential to improve thedurability of construction materials such as limestone and cemen-

titious materials. The latter is dealt with in this review paper andcan be dividedinto processes for the deposition of a protective sur-face layer with consolidating and/or waterproofing 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 first to consider the useof microbially induced carbonate precipitation (MICP) for the pro-

tection of ornamental stone. They applied for a patent regardingthe use of calcinogenic bacteria on stone surfaces, as is discussed inSection 3.1.2. The promising results of this so-called Calcite Biocon-cept technique encouraged different research groups to evaluate

alternative approaches for the biomediated carbonate precipita-tion on limestone. These approaches can be mainly divided intothose falling within and those falling outside the specificationsof the patent byAdolphe et al. (1990),i.e. the application of cal-

cinogenic bacteria to a stone surface. The first series of approaches(Sections 3.1.33.1.6), those falling within the patent specifications,arecharacterized by the use of different microorganisms,metabolicpathways or delivery systems to overcome some of the poten-

tial limitations of the Calcite Bioconcept technique. The selectionof a microorganism by the different research groups was oftenbased on their experiences from previous studies on microbiallyinduced mineral precipitation. In the second series of approaches,

no microorganisms are applied to the surface. These approachescan be divided into studies where inducing macromolecules aresupplied to the stone together with a supersaturated solution ofcalcium carbonate (Section3.2)and studies which obtain carbon-

ate precipitation by the microbiota inhabiting the stone (Section3.3).In the latter, only nutrients are added to the stone.

3.1. Application of calcinogenic bacteria

Before going into detail on the different methodologies pro-

posed, a short chronological overview is given on the work

preceding the application of microbially induced carbonate pro-duction 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 limestonequickly acquires a protective skin (calcin) through dissolution ofcarbonates 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 comparedto the underlying layers. As a result of atmospheric pollutants,however, this layer slowly degrades, losing its protective role. The

discoverythat bacteria contributeto the formationof limestonehasled to the suggestion to use bacteria forthe re-establishment of this

calcin.Boquet et al. (1973)were among the first to demonstrate the

ability of soil bacteria to precipitate calcium carbonate under labo-ratory conditions. While previous research only concerned marinebacteria in liquid media (Drew, 1911; Shinano, 1972),the authorsinvestigated 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) andPseudomonas aeruginosawere observed to form crys-tals. The authors concluded that crystal formation is a function of

the medium, and that under suitable conditions most bacteria canform crystals.

In parallel with the work done by this Spanish research group,Adolphe andBilly (1974) succeeded in theformation ofcalcite in the

laboratory by bacteria isolated from tuff and travertine. Between1983 and 1987,Castanier et al. (1999)investigated the differentmechanisms responsible for the microbial formation of calciumcarbonate, evidencing the microbial origin of limestone.Adolphe

et al. (1989) further demonstrated the bacterial origin of the calcitecrustsin extremeclimates,suchas GreenlandandtheSahara desert.In addition, the team observed the great resistance of these lay-ers towards erosion. From the above findings,Adolphe et al. (1990)

applied fora patent for the treatment of artificial surfaces by virtueof a surface coating produced by microorganisms. In addition, acompany, 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


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Table 2

Overview of the different methodologies used for the deposition of a layer of calcium carbonate on stone and concrete (biodeposition).

Application Mediator Authors Organism/molecule Metabolisma Solutionb

Limestone Microorganisms Calcite Bioconcept (Le Metayer-Levrel et al., 1999) Bacillus cereus ODAA Growth mand Nutric

Tiano et al. (1999) Micrococcus sp. Bacillus subtilis ODAA OAU B4

Rodriguez-Navarro et al. (2003) Myxococcus xanthus ODAA OAU M-3, M-3P

Dick et al. (2006) Bacillus sphaericus HU SF

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

Organic matrixmolecules

Tiano (1995)andTiano et al. (2006) Mytilus californianusshell extracts n.a. Ammoniumethod o

supersatu

bicarbona

Aspartic acid

Bacillus cell fragments

Activator medium Jimenez-Lopez et al. (2007) Microbiota inhabiting the stone ODAA OAU M-3, M-3

Cementitious materials Microorganisms De Muynck et al. (2008a,b) Bacillus sphaericus HU SF

Authors Experimental methods

Inoculum Application procedure Septicity conditions

Bacteria Nutrients/chemicals Stone N/C

Calcite bioconcept Culture in exponential

phase: 107 to

109 cellsmL1

Spraying Spraying (5 times) NS NS

Tiano et al. Overnight culture:

106 cellscm2Brushing on water

saturated specimens

Wetting every day for

15 days

S S

Rodriguez-Navarro et al. 2% inoculum Immersion in growing bacterial culture (shaking or

stationary conditions) for 30 days

S S

Dick et al. 1% inoculum Immersion in growing bacterial culture (intermediate

wetting) for 28 days

S S

Biobrush 108 cellsmL1 Spraying In Carbogel NS NS

Tiano et al. n.a. n.a. Immersion in test

solution or spraying (in

situtests)

NS NS


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T

able2(Continued)

A

uthors

Experimentalmethods

Evalu

ationprocedures

Inoculum

Applicationprocedure

Septicityconditions

Bacteria

Nutrients/chemicals

Stone

N/C

Appl.

Jimenez-Lopezetal.

Microbiotainhabiting

thestone

Immersioninagrowingbacterialculturefor30days

SandNS

S

S

Stone

cohesion(sonicator

bath),weightincrease,X

RD

andS

EManalysis,porosimetry

analysis

D

eMuyncketal.

Overnightculture:107

to109cellsmL1

Immersionfor1day

Immersionfor4days

NS

NS

NS

Weightincrease,Water

absor

ptiongas,permeability,

chloridemigration,

Carbo

nation,Freezingand

thawing,Thinsections,SEM,

XRDanalysis

n

.a.:notapplicable,N

/C:nutrients/chemicals,App

l.:application,N

S:non-sterileandS:sterile

aODAA:oxidativedeaminationofaminoacids;

OAU:organicacidutilization,H

U:hydrolysisofu

rea.

bCompositionseeTable5.

to investigate the viability and performance of these bacteriainsitu. The technique was further optimized and industrialized as aresult of the collaboration between the University of Nantes, the

Laboratory for the research of historic monuments (LRMH) and thecompany Calcite Bioconcept (Le Metayer-Levrel et al., 1999).

The first step comprised the searchfor suitable microorganisms.Bacteria were isolated from natural carbonate producing environ-

ments and screened for their carbonatogenic yield, i.e. ratio of theweight of calcium carbonate produced to the weight of organicmatter input (OM). The highest performance was obtained withB.cereus, which showed a carbonatogenic yield of 0.6g CaCO3 g OM

1

(Castanier et al., 1999).Furthermore, sinceB. cereuscould be easilyproduced on an industrial scale, this organism was selected for insituapplications (Orial, 2000).

The second step comprised the optimization of a nutrient

medium and the frequency of feeding to meet industrial economi-cal constraints. The nutritional medium was designed to stimulatethe production of carbonate through the nitrogen cycle metabolicpathways, which are the only pathways to be activated in oper-

ational conditions, i.e. in aerobiosis and microaerophily. Morespecifically, the media contain a source of proteins for the oxida-tive deamination of amino acids in aerobiosis and a source ofnitrate for the dissimilatory reduction of nitrate in anaerobiosis or

microaerophily. In addition, a fungicide was added to prevent theunwanted growth of fungi present on the stone, or deposited fromthe air (Orial et al., 2002).

From preliminary experiments in the laboratory, a treatment

procedure for in situ applications was proposed. The treatmentconsists of first spraying the entire surface to be protected witha suitable bacterial suspension culture (Tables 1 and 2). Subse-quently, the deposited culture is fed daily or every 2 days with the

suitable medium in order to create a surficial calcareous coatingscale, the biocalcin. Usual industrial and economical constraintsrestrict the number of feeding applications to five, but treatment ofhistoric patrimony may be less restrictive. The frequency of feed-

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

frequency more suitable for fine-grained limestone and the 2-dayfrequence for coarse grained limestone (Le Metayer-Levrel et al.,1999).

The firstapplication in situ was carriedout in 1993 in Thouars onthe tower of the Saint Mdard Church. The treatment was appliedon an area of 50 m2 of Tuffeau limestone. The protective effect ofthe treatment was evaluated by means of macro- and microscopic

investigations, such as measurements of the permeability, evalua-tion of the roughness and colorimetry and SEM examination. SEMimages indicated the abundant development of calcinogenic bac-terial populations, illustrating the viability ofB. cereus on stone

surfaces.The presence of the biocalcin decreasedthe waterabsorp-tion rate to a significant extent (5 times lower) while retaining thepermeability for gas. Furthermore, no influence on the aesthetic

appearance could be observed (Le Metayer-Levrel et al., 1999).Long-term evaluation of the biocalcin layer has shown differ-

ences in the durability behaviour related to the orientation of thefacade and the micro-relief of the stone. The densest layers could

be observed inside the pores, while cracking of the biocalcin wasobserved at crystoballite protrusions. From these observations, itwas concluded that every 10 years a new treatment is needed torestore the protective effect of the biocalcin (Orial, 2000).

Similar experiments were applied on limestone statuarieswhich had been placed in different climatic environments. Exper-iments were performed on two types of limestone, Tuffeau andSaint-Maximim. The former is a fine-grained limestone character-

izedby a high porosity andsmall(10m) (Le Metayer-Levrel et al.,1999). The rural


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Table 3

Characteristics of the various biocementation processes.

Characteristic Process

Biocementation

Remediation of cracks Bacterial concrete Self-healing concrete Biomortar

Definition Generation of a biologically induced binder

Type Surface treatment Admixture Admixture Binder

Goals Improvement of the durabilityExternal crack repair Strength improvement Self-healing of cracks Strength development

formation of a

carbonate-binder-based

mortar

Where/when Applied in cracks on the

surface of concrete and mortar

Applied in the concrete

mixture

Application of mortar mixture to

repair broken limestone fragments

or to fill cavities in stone

How Application of bacteria with a

carrier/binder material

immersion in nutrient solution

Application of bacteria

in the mixture, lower

amount of bacteria

(1wt%admixture)

Application of bacteria and

nutrients in the mixture, lower

amount of bacteria

(1wt%admixture)

Application of bacteria and

nutrients in the mixture,

higher amount of bacteria

(25vol.%binder)

Added value Ecological, environmentally friendly

Changes in

microstructure

Self-healing capacity Compatibility

Status of use Laboratory experiments Laboratory

experiments

Laboratory experiments Small scale applications in

practice

(current) Limitations Costs of bacteria and nutrientsNone or low bacterial activity at high pH of cementitious materials immobilization is necessary -

Difficult to apply in practice Long-term viability of spores

Market niche Eco-friendly repair of cracks Repair of difficult to reach concrete Restoration sector

eco-friendly reuse of brick

and maritime environment appeared to be very aggressive, withalmost complete loss of the biocalcin after 4 years of exposure.However, in the urban environment, the biocalcin retained its pro-

tective effect, with treated statues showing littledamage comparedto untreated ones. Furthermore, after 4 years of exposure to urbanconditions, no undesired biological colonisation could be observed(Orial, 2000).

By adding natural pigments into the nutritional medium, it is

also possible to create a surficial patina with the biodepositiontreatment. The pigments are integrated into the biocalcin and thusgive a persistent light colouring to the stone. This technique has

been proposed to conceal some newly replaced stones on a monu-ment facade (Le Metayer-Levrel et al., 1999).

3.1.3. Procedure according to the University of Granada (Spain)

Rodriguez-Navarro et al. (2003) addressed two important

limitations of the calcite method. As the thickness of the biocon-solidating cement was limited to only a few microns, this methodseemed to be ineffective for in-depth consolidation. Moreover, theformation of a superficial film consisting of a mixture of biolog-

ical remains, plugged stone pores and provided no consolidation(Rodriguez-Navarro et al., 2003).Besides, the authors commented

on the potential drawback of the use ofBacillusin stone conser-

vation. According to these authors, the formation of endosporesmay lead to germination and uncontrolled biofilm growth underappropriate conditions (i.e. temperature, humidity and nutrient

availability).The authors, therefore proposed the use ofMyxococcus xanthus

for the creation of a consolidating carbonate matrix in the poroussystem of limestone.Their researchgroup previously demonstrated

the ability of this species to induce the precipitation of carbon-

ates, phosphates and sulfates in a wide range of solid and liquidmedia (Gonzlez-Munoz et al., 1993, 1996; Ben Omar et al., 1995,1998; Ben Chekroun et al., 2004; Rodriguez-Navarro et al., 2007),

being the first to describe struvite ((NH4)MgPO46H2O) formationby myxobacteria (Ben Omar et al., 1994).Furthermore, they wereable to obtainthe crystallizationof struvite andcalcite by dead cellsand cellular fractions ofM. xanthus(Gonzlez-Munoz et al., 1996).

The latter is an abundant Gram-negative, non-pathogenic aerobicsoil bacterium which belongs to a peculiar microbial group whosecomplex life cycle involves a remarkable process of morphogene-sis and differentiation. In the tested culture media, no formation

of a dormant stage was observed. Additionally, when applied onstone specimens, no fruiting bodies were observed upon drying.As a result of this cell death, no uncontrolled bacterial growth was

observed.

Table 4

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

Application Author Organism Metabolisma Solutionb

Biological mortar Calcite bioconcept Bacillus cereus ODAA Nutrical

Remediation of cracks inconcrete

Ramachandran et al. Bacillus pasteurii HU SF

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

Bacterial concrete Ramachandran et al. Bacillus pasteurii HU SF

Ghosh et al. Shewanella

Self-healing Jonkers et al. Bacillus pseudofirmus

Bacillus cohnii OAU Calcium lactate

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

b Composition seeTable 5;: not available.


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Table 5

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

Name Composition pHa References

Nutrients Conc.

Growth medium (CB) Peptone Orial (2000)Yeast extract

KNO3

NaClNutrical Growth medium + CaCl22H2O

Orial (2000)Actical

Natamycine

B4 Calcium acetate 0.25 wt% 8 Boquet et al.Yeast extract 0.4 wt%

Glucose 1 wt%

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

al. (2003)Ca(CH2COO)24H2O 1 wt%

K2CO3(1/2)H2O 0.2 wt%

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

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

(2008)Ca(CH2COO)24H2O 0.4 wt%

CaCl22H2O 0.1 wt%

NaHCO3 0.3wt%

Yeast extract 0.1 wt%

SFb ,c Nutrient broth 3 g L 1 6 Stocks-Fischer et al.

(1999)Urea 20 g L 1

CaCl22H2O 1.45.6 g L 1

NH4Cl 10 g L 1

NaHCO3 2.12gL1

Growth medium (DB) Yeast extract 20 g L 1 7 Whiffin (2004)Urea 20 g L 1

Biodeposition (DB) Urea 20 g L 1 7 De Belie and De

Muynck (2008)CaCl22H2O 50 g L 1

a pH was adjusted with HCl or NaOH.b Dick et al. used 7.5 g L1 CaCl22H2O.c De Muynck et al. used 25 g L1 CaCl22H2O or 26g L1 Ca(CH2COO)24H2O; : not available.

For the production of carbonate ions, the authors proposed a

medium containing a pancreatic digest of casein as the nitrogensource. Also, the effect of a phosphate buffer on the carbon-ate production was investigated. Biodeposition experiments wereperformed both under static and non-static conditions. Sterilized

calcarenite samples were submerged into a certain volume of M-3orM-3P(Table 5) whichwas subsequently inoculated(1%, v/v)with

M. xanthus. All experiments were performed at 28 C under sterileconditions.

The phosphate buffer had a profound effect on the bacterial cellyield and the carbonate productivity, as well as on the supersat-uration preceding the nucleation of carbonate crystals. A greaterbacterial production also led to a higher yield in calcite crystals.

Furthermore,the buffering effect of the phosphate prevented rapidlocal pH variations and, concomitantly the occurrence of a highsupersaturation. As a result, the deposited carbonate crystals wereshown to be strongly adhered to the surface of the pores, since

the newly formed carbonates were more resistant to mechani-cal stress in the form of sonication than the calcite crystals inthe stone. The authors attributed this to their epitaxial growthon pre-existing calcite crystals and to the incorporation of organic

molecules. Apparently, the presence of organic molecules causes amisalignment of different domains within a single crystal.

The authors observed carbonate cementation to a depth of sev-eral hundred micrometers (>500m) without the occurrence of

any plugging or blocking of the pores. Plugging is mainly a conse-quence of extracellular polymeric substance (EPS) film 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 findings are in sharp contrastwith the abundant production of EPS byM. xanthusdescribed bySutherland and Thomson (1975).The latter could be attributed todifferences in culture mediumcompositionand 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 Universityresearch team, proposed the microbial hydrolysis of urea as a strat-egy to obtain a restoring and protective calcite layer on degradedlimestone. 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 highamounts of carbonate within a short period of time.

The hydrolysis of urea is catalyzed bymeans of urease. As a con-

sequence, urea is degraded to carbonate and ammonium, resultingin an increase of the pH and carbonate concentration in the bacte-rial environment (Stocks-Fischer et al., 1999).One mole of urea ishydrolyzed intracellularly to one mole of ammonia andone mole of

carbamate(Eq.(7)), whichspontaneouslyhydrolyzesto onemoleofammonia and carbonic acid (Eq.(8)).These products subsequentlyequilibrate in water to form bicarbonate and two moles of ammo-nium and hydroxide ions (Eqs.(9)and(10)):

CO(NH2)2+H2O H2COOH +NH3 (7)

NH2COOH+

H2O

NH3+

H2CO3 (8)


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Fig. 1. Simplified representation of the events occurring during the ureolytic induced carbonate precipitation. Calcium ions in the solution are attracted to the bacterial

cell wall due to the negative charge of the latter. Upon addition of urea to the bacteria, dissolved inorganic carbon (DIC) and ammonium (AMM) are released in the micro-

environment of the bacteria (A). In the presence of calcium ions, this can result in a local supersaturation and hence heterogeneous precipitation of calcium carbonate on

the bacterial cell wall (B). After a while, the whole cell becomes encapsulated (C), limiting nutrient transfer, resulting in cell death. Image (D) shows the imprints of bacterial

cells involved in carbonate precipitation. A more in-depth representation can be found in Hammes and Verstraete (2002).

could be considered as a special kind of biodeposition treatment.Heselmeyer et al. (1991)obtained the complete removal of gyp-sum crusts from marble samples in laboratory conditions using

a strain ofDesulfovibrio vulgaris. The procedure was further opti-mized by Ranalli et al. (1997), who used sepiolite as a carrier

material forD. vulgarisandDesulfovibrio desulfuricans. The use ofsepiolite notonly provided anaerobic conditions and humidity, but

also enabled the authors to shorten the treatment time. Additionalimprovements were made byCappitelli et al. (2006)who reportedon the superiority of Carbogel as a delivery system for the bacte-ria. The use of Carbogel allowed for a higher retention of viable

bacteria and significantly decreased the time needed for entrap-ment of the microorganisms as compared to the use of sepiolite.In addition, methods were presented to avoid the precipitationof black iron sulfide. The optimized methodology appeared to be

superior to chemical treatments involving the use of ethylenedi-aminetetraaceticacid (EDTA),since no sodiumsulphatewas formed(Cappitelli et al., 2007).

AsCappitelli et al. (2006, 2007)were among the members of

the Biobrush consortium, the use of Carbogel was subsequentlyintroduced into the field of biodeposition. According to the consor-tium, these delivery systems could be used to control the possibleharmful side effects of bacteria to stone. In addition, it was noticed

that the application of calcinogenic bacteria by spraying alone onlyresulted in a limited change of the capillary water uptake of Port-land stone. According to the consortium, the latter is attributableto the limited colonization of the stone as a result of drying

out.Within the framework of the Biobrush project regarding biode-

position, bacteria isolated from a stream in Somerset (UK), andbacteria from culture collections that had been reported to have

calcifying activity, were screened for their ability to deposit calcitein solid and liquidmodified B4 media(Table 5). From the 10 isolates

that were retained and assessed for their ability to deposit calciteon stonesurfaces,Pseudomonasputida was chosen forfurther study

in field trials. The latter has a low risk to humans and is sensitiveto most of the tested antibiotics and precipitated calcite in a widetemperature range (May, 2005).

In these field trials, bacteria were applied to the stone by

brushing. Subsequently, the bacteria were covered with moistenedJapanese paper,above which a 11.5cm thick layer of Carbogel pre-pared with modified B4 was applied. TrisHCl buffer was added tothe Carbogel to adjust the low pH of this carrier. Finally, the gel

was covered with a polyethylene sheet. As a result of this treat-ment a decrease of the water absorption and open porosity by 1%and 5%, respectively, was obtained. In order for this treatment to beeffective 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 theformation of new minerals inside the stone. This was the result

of their experiments with Micrococcus spp. and Bacillus subtilisstrains on Pietra di Lecce bioclastic limestone. In these experi-

ments, bacteria were applied by brushing sterilizedspecimens thatwere soaked with distilled water, reaching a final concentration of106 cells cm2. Subsequently, the bacteria were feddailyby wettingwith a small amount ofB4 medium (Table 5) for a period of 15 days.

Experimentswere performedat 28 C undernon-sterile conditions.According to the authors, the decrease in water absorption after

a biodeposition treatment is for a large part attributable to thephysical obstruction of pores, rather than to the stable presence of

newly precipitated calcite. Furthermore, the authors commentedon some possible negative consequences, such as (1) the pres-ence of products of new formation, due to the chemical reactionsbetween the stonemineralsand someby-products originatingfrom

the metabolism of viable heterotrophic bacteria and (2) the forma-tion of stained patches, due to the growth of air-borne micro-fungirelated to the presence of organic nutrients necessary for bacterialdevelopment.

To avoid some of these problems, the authors proposed the useof natural and synthetic polypeptides to control the growth of cal-cite crystals in the pores. The first suggestions in this directionalready date from the time at which the Calcite Bioconcept treat-

mentwas developed.Tiano et al.(1992) and Tiano (1995) proposedthe use of organic matrix macromolecules (OMM) extracted from

Mytilus californianusshells to induce the precipitation of calciumcarbonate within the pores of the stone. The organic matrix was

shown to produce a more relevant and durable carbonate precipi-tation compared to the single use of calcium chloride or hydroxide.

This precipitation resulted in a slightdecreasein porosity andwaterabsorption by capillarity (Tiano, 1995).

However, the practical application was hindered by the com-plexityof the extraction procedureand the very lowyield of usableproduct (Tiano et al., 1999).Given this, the authors searched foralternative 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 suchas polyaspartic acid. Calcium and carbonate ions for crystal growth

were supplied by means of an ammonium carbonate and calciumchloride solution or a saturated solution of bicarbonate, and weresupplemented in some cases by calcite nanoparticles, in orderto maintain a saturated carbonate solution in the pore over a

prolonged period. Proteins, calcium ions and nanoparticles were


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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 theauthors, the method is most suitable for the use on marble statuesand objects of high aesthetic value where conservation is requiredwith the minimum change in thechemistryof the object. Field test

results, however,indicate that theeffects ofthe BIMtreatment wererather small. The consolidating effect and the decrease in wateruptake were very low compared to the use of ethylsilicates, i.e.15% over 12 mm depth compared to 30% up to 10mm 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 inbacteria has been proposed as an alternative way for the produc-

tion of inducing macromolecules. This was one of the objectivesof the European Bioreinforce (BIOmediated calcite precipitation

for monumental stones REINFORCEment) project. According to

the consortium, the genes responsible for crystal formation couldbe cloned and transferred to an appropriate expression vector,enabling the overproduction of the molecules inducing crystal for-

mation (http://www.ub.es/rpat/bioreinforce/bioreinforce.htm).Initially, the consortium searched for bacterial cell structures or

molecules able to induce and control the carbonate precipitationprocess. In this way, living cells would no longer be needed for the

biodeposition treatment. The authors demonstrated the ability ofautoclaved cells and cell fragments to induce calcite crystallizationin liquid media. Furthermore, they observed that dead cells fromactive calcinogenic strains (B. cereus and B. subtilis) showed a much

higher and/or faster production of CaCO3crystals than dead cellsfromless active strains (Escherichiacoli).Thisledthe authors tocon-clude that calcinogenic strains might have a subcellular structure,resistant to the methods used to kill cells (sonication, autoclav-

ing), able to promote CaCO3precipitation. The crystals induced bydead cells andBacilluscell fragments (BCF) had a more complexshape 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 pronouncedon high porosity stones such as Tuffeau. Again, this method onlyappeared to be useful for very delicate small calcareous stoneobjects, rather than for a monumental facade (Mastromei et al.,

2008).In addition,Barabesi et al. (2007)reported on a gene cluster

ofB. subtilisinvolved in calcium carbonate precipitation. From UVmutagenesis experiments, six mutants impaired in calcite crystal

formation were isolated. Sequence analysis of the mutated genesrevealed that in many cases their putative function was linked tothe fatty acid metabolism (Perito et al., 2000; Barabesi et al., 2007).Further experiments are ongoing to investigate the link between

this kind of metabolism and calcium precipitation.

3.3. Application of an activator medium (Spain)

Concerning possible changes of the activity and composition of

the autochthonous microbiota upon addition of an inoculated cul-ture media to ornamental stone,Rodriguez-Navarro et al. (2003)pointed out the possibility of a synergetic contribution of the for-

mer to the overall biodeposition process. In fact,Urzi et al. (1999)previously demonstratedthat the majorityof bacteria isolated frombuilding materialsare able to induce carbonateprecipitation underlaboratory conditions.

From the above, Jimenez-Lopez et al. (2007) proposed theapplication of a culture medium, able to activate the calcinogenicbacteria from the microbial community of the stone, as a moreuser friendly method for the in situconsolidation of ornamental

stone. In addition to their work on decayed limestone fragments(Jimenez-Lopez et al., 2007),this technique was recently proposedfor the treatment of new stones used for replacement purposes

(Jimenez-Lopez et al., 2008).Upon comparison ofthe microbialcommunity identifiedin non-

treated quarry stone andthat identified in the non-treated decayedstone, the authors observed for the latter the presence of microor-

ganisms related to the quarry from which the stone was extractedandmicroorganismsrelatedto the environmentand contaminationto which the stone was exposed.

Some of the identified bacteria,PseudomonasandBacillus, had

already been reported to produce calcium carbonate both inlaboratory conditions and in nature. These chemoorganotrophicorganismsare able to grow in culture media containing amino acidssuch as nutrient agar and tryptic soy agar (Jimenez-Lopez et al.,

2007).From these findings, the authors proposed the use of bacto-

casitone as a way to activate the calcinogenic bacteria from the
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stone microbial community. Bacto-casitone is a source of carbonand nitrogen, which favours alkalinisation due to the oxidativedeamination of amino acids. Furthermore, as no carbohydrates

were supplied, the probability of acid production, which is detri-mental to the stone, was believed to be minimal. According to theauthors, this procedure is much easier than the use of bacteriallyinoculated media, since difficulties linkedto theneed of specialized

persons and equipment to work with microorganisms or tech-nical requirements to ensure optimal growth conditions wouldbe avoided (Jimenez-Lopez et al., 2007). However, the fact thatmicroorganisms do not need to be introduced not only leads to a

decrease of the overall cost, butalso ensures that thismethod is notcovered by the claims of thepatent by Adolphe et al. (1990). Conse-quently, Gonzlez-Munoz et al. (2008) applied for a new patent forthe protection and reinforcement of construction and ornamental

materials by means of the application of an activator medium ableto induce the formation of calcium carbonate.

Sonication test results demonstrated that the new cement cre-ated by the microbial community and/or the combined action of

the microbial community andM. xanthuswas more resistant thanthat created by the sole action of eitherM. xanthusor the culturemedia. Furthermore,the authors didnotobserve any changes in theporosity of the stone. In addition, limited exopolysaccharide pro-

duction was observed (Jimenez-Lopez et al., 2007). The latter couldbe somewhat expected asRodriguez-Navarro et al. (2003)notedthat organic films are unable to attach to the stones under shak-ing conditions, as was the case in the work byJimenez-Lopez et al.

(2007, 2008).Dueto the timerequired forthe activation of the microbialcom-

munity,Jimenez-Lopez et al. (2007) proposed the additional use of

M. xanthusfor 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 SanJeronimo and Hospital Real, Granada). Preliminary results show the

effectiveness of the treatment in terms of colour changes (negligi-

ble) and surface resistance by means of a peeling test (Personalcommunication by Rodriguez-Navarro, 2008).

Although the formation of endospores was previously consid-

ered a potential drawback for the use ofBacillusin stone conserva-tion (Rodriguez-Navarro et al., 2003),spore forming bacteria, ableto germinate upon the application of the culture media, contributein large extent to the precipitation of carbonate by the method

described byJimenez-Lopez et al. (2007).Drawbacks to the use ofspore forming bacteria were related to the possible uncontrolledgrowth of bacteria upon germination. However,Le Metayer-Levrelet al. (1999)found that no increases in the microbial activity or

changes in the autochthonous microbiota were observed immedi-ately or 4 years after the application of calcinogenic bacteria.

The long activation times of this technique inspiredDe Muynck

et al. (2008c)to develop anin situenrichment of carbonate pro-ducing bacteria. For that purpose, different media were developedwhich allowed a rapid growth of carbonate producing strains uponexposure to the surrounding air. Among the different metabolicpathways under investigation, conditions optimal for carbonate

precipitation were most rapidly obtained upon the hydrolysis ofurea. Nevertheless, the rate of urea hydrolysis and biodepositionremained low compared to pure cultures ofB. sphaericus.

4. Biocementation

Besides the deposition of a layer of carbonate on the surface ofbuilding materials, MICP has also been used for the generation of

binder-based materials. Initial developments were mainly situated

in the field of geotechnical engineering, i.e. plugging, strengthen-ing and improvement of soils (Ferris and Stehmeier, 1992; ZhongandIslam,1995; Nematiand Voordouw, 2003;Whiffin et al., 2007).

Recent advances, however, indicate the potential use of this tech-nique for the remediation of cracks in building materials, strengthimprovement and self-healing of cementitious materials.

4.1. Biological mortar (France)

The knowledge and experiences obtained with the Calcite Bio-concept treatment for limestone, have resulted in the development

of a biological mortar for the remediation of small cavities onlimestone surfaces. The aim of the biological mortar was to avoidsome of the problems related to chemical and physical incom-patibilities of commonly used repair mortars with the underlying

material, especially in the case of brittle materials (Castanier,1995; Le Metayer-Levrel et al., 1999; Orial et al., 2002,Personalcommunication by Loubire (Chief of Calcite Bioconcept), 2008;http://www.calcitebioconcept.com/).

In general, a mortar refers to a workable paste consisting of abinder, aggregates and water to bind building materials togetherand to fill the gaps between them. In particular, a biological mortarrefers to a mixture of bacteria, finely ground limestone and a nutri-

tional medium containing a calcium salt. The term biological refersto the microbial origin of the binder, i.e.microbiologically producedcalcium carbonate. Similar to lime mortars, the produced calciumcarbonate cements the aggregates together. Cementation occurs as

a result of the nucleation and growth of carbonate crystals at thesurface of the aggregates, especially at the contact areas betweenthem.

The optimization of the mortar composition encompassed the

dosage and composition of the three main components, i.e. lime-stone powder, nutrients and bacterial paste. The mortars wereevaluated based upon their appearance (cohesion and colour),the presence of micro-cracks and the resistance towards fractur-

ing. Concerning the medium composition, some adjustments were

made to the initial method, as was used forbiodeposition purposes.The amount of nutrient solution introduced during the fabricationof the mortar was sufficient to support bacterial activity. Repeated

external applications of the nutrient solution were unable to com-pletelywetthe mortar. Furthermore, theyresultedin discolorationsat the surface andwere, therefore,rapidly omitted. Additionally, thebiological mortars necessitated the use of larger amounts of bacte-

ria and as a result the composition of the nutrient medium had tobe altered.

Based on the different evaluation parameters, best resultswere obtained with one part of bacterial paste (containing

109 cells mL1), one part of nutritional medium and two partsof limestone powder. Limestone powder with a granulometrybetween 40 and 160m was observed to be the most suited. The

technique has already been successfully tested on a small scale onsculptures of the Amiens Cathedral and on a portal of the churchof Argenton-Chteau (France). Visual observations 2 years afterthe 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 fields, where water is morereadily removed than the viscous oil, the ability to selectively plugporous rock to focus pumping energy in oil rich zones is highlydesirable (Hart et al., 1960; Lappin-Scott et al., 1988).Because of

the cost and unsatisfactory performance of some of the chemi-callycross-linkedpolymers, manyworkers suggestedthatinsoluble

biopolymers and biomass generated by injection of indigenous
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microorganisms can be used to selectively plug off zones of highwater permeability (Lappin-Scott et al., 1988; Jack et al., 1991;Gollapudi et al., 1995).In addition, the use of a microbial mineral

plugging system based on the precipitation of carbonates was sug-gested (Ferris and Stehmeier, 1992; Zhong and Islam, 1995).Whileinitial research on MICP in sand columns was mainly focused onthe decrease of porosity and permeability as a result of the physi-

calpresence of the newly formed carbonates (Ferris and Stehmeier,1992),recent investigations focus on the improvement of strengthas a result of the cementation of sand particles (Whiffin, 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 alkalinizethe environment. Furthermore, urea is an important organic nitro-

gen carrier in natural environments and is commonly used as anagricultural fertilizer (Nielsen et al., 1998).Moreover, the ability tohydrolyze urea is widely distributed among indigenous bacteria insoilsand groundwater systems (Mobley andHausinger, 1989; Fujita

et al., 2000).Urea-utilizing bacteria such asSporosarcina pasteuriiand Sporosarcina ureae are commonly isolated from soil, water,sewage and incrustations on urinals.

The participation ofS. pasteuriiin sand consolidation has been

demonstrated byKantzas et al. (1992).Gollapudi et al. (1995)fur-ther investigated the use ofS. pasteurii for the plugging of sandcolumns. 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 thecalcite precipitation by providing a nucleation site and by creatinganalkalineenvironmentwhich favouredthe precipitation of calcite.Zhong andIslam (1995)used theconsolidation of sand mixtures for

the remediation of cracks in granite. Cracks in granite were packedwith a mixture of bacteria, nutrients and a filler material. Amongthe different materials that were mixed with S. pasteurii, the silicafume (10%)andsand (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 inconcrete. The authors proposed MICP as an effective way to seal

cracks. The appearance of cracks and fissures is an inevitable phe-nomenon during the ageing process of concrete structures uponexposure to weather changes. If left untreated, cracks tend toexpand further and eventuallylead to costly repair. Specimens with

cracks filled with bacteria, nutrients and sand demonstrated a sig-nificant increase in compressive strength and stiffness values whencompared 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 thatS. pasteuriigrows more actively inthe presence of oxygen. Still, the highly alkaline pH (1213) of con-crete was a major hindering factor to the growth of the moderate

alkaliphileS. pasteurii, whose growth optimum is around a pH of9. In order to protect the cells from the high pH,Day et al. (2003)investigated the effect of different filler materials on the effective-ness of the crack remediation. Beams treated with bacteria andpolyurethane showed a higher improvement in stiffness compared

to filler materials such as lime, silica, fly ash and sand. Accordingto the authors, the porous nature of the polyurethane minimizestransfer limitations to substrates and supports the growth of bac-

teria more efficiently than other filling materials, enabling anaccumulation of calcite in deeper areas of the crack. No differ-ences could be observed between the overall performances of freeor polyurethane immobilized cells in the precipitation of carbon-

ate (Bang et al., 2001).In addition to this research,Bachmeier et al.(2002) investigated the precipitation of calcium carbonate with the

urease enzyme immobilized on polyurethane. The immobilization

was shown to protect the enzyme from environmental changes,as the immobilized urease retained higher enzymatic activities athigh temperatures and in the presence of high concentrations of

pronase. While the rate of calcite precipitation of the immobilizedenzyme was slower compared to that of the free enzyme, lowerconcentrations of the former where needed to obtain the theoreti-calmaximumprecipitation ina periodof 24h. Although theauthors

mentioned ongoing research on the use of immobilized urease inthe remediation of surface cracks in concrete, to our knowledge nopublished results are available at the moment.

As an extension to their research on biodeposition on cementi-

tiousmaterials, DeBelieandDeMuynck(2008) further investigatedtheuse ofmicrobially inducedcarbonate precipitationforthe repairof cracks in concrete. For the protection ofB. sphaericusfrom thealkaline pH conditions, bacteria were immobilized in a silica sol.

Upon the addition of a salt, a bioceramic material (biocer) wasformed, which was able to bridge the crack. Subsequent additionof a urea and calcium chloride solution resulted in the formation ofcarbonate crystals inside the pores of the biocer andconcomitantly

sealing of the crack. As a result, a decrease of the water permeabil-ity, similar to that obtained with traditional epoxy injections, wasobserved.

4.3. Bacterial concrete (USA, India)

Besides external application of bacteria in the case of reme-diation of cracks, microorganisms have also been applied in the

concrete mixture. Until now, research has mainly focused on theconsequences of this addition on the material properties of con-crete, i.e. strength and durability. Both properties depend on themicrostructureof the concrete.However, the effects of the presence

of the microorganisms and/or the microbially induced carbonateson the microstructure still need to be elucidated, especially theinteraction between the biomass and the cement matrix.

Ramachandran et al. (2001)investigated the use of microbio-

logically induced mineral precipitation for the improvement of the

compressive strength of Portland cement mortar cubes. This studyidentified the effect of the buffer solution and type and amount ofmicroorganisms, i.e.S. pasteuriiand P. aeruginosa, used. Further-

more, in order to study the effect of the biomass, the influenceof both living and dead cells was investigated. Before addition tothe mortar mixture, bacteria were centrifuged and washed twice.The final pellets were then suspended in either saline or phos-

phate buffer, which was subsequently added to the mixture. Afterdemolding, the mortar specimens were stored in a solution con-taining urea and calcium chloride for 7 days. Subsequently, thespecimens were cured in air until the measurement of the com-

pressive strength.At lower concentrations, the presence ofS. pasteuriiwas shown

toincreasethe compressivestrengthof mortarcubes.While the28-

day compressive strength of the control cubes amounted to about551 MPa, specimens treated with 103 cells cm3 had a compres-sive strength of about 651 MPa. The contribution ofP. aeruginosatothe strength was found tobe insignificant. FromtheX-raydiffrac-

tion (XRD) analysis, no significant increased amounts of calcitecould be found in mortar specimens treated with bacteria. Thiscould be attributed to the inhibition of the microorganisms by thehigh pH andthe lack of oxygen insidethe mortarmixture. The over-

all increase of strength, therefore, resulted from the presence of anadequate amount of organic substances in the matrix due to themicrobial biomass. However, an increase of the biomass, as deadcells in particular, resultedin a decreased strength.According to the

authors,this could be attributed to the disintegration of the organicmatter 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 PriceD /unit Dosage unit/m2 ProductD /m2 No. of applic. Prod.+applic.D /m2

Calcite bioconcept 2328a

3540

b

Growth medium 1Dg1 23 g 23 1

Nutrical 0.2Dg1 816 g(5) 24(5) 5Water repellents 2.54 DL1 0.51 L 1.254 1 1525

Consolidants 1015DL1 >1 L >10 1 >30

a Unaltered stone.b Sculptured and degraded stone.

Ramakrishnan et al. (2001)investigated the effect of this tech-nique on the durability of concrete. The presence of bacteria was

observed to increase the resistance of concrete towards alkali, sul-fate, freezethaw attack anddryingshrinkage; theeffectbeing morepronounced with increasing concentrations of bacterial cells. Theauthors attributed this to the presence of a calcite layer on the sur-

face, as confirmed by XRD analysis, lowering the permeability ofthe specimens. The best results were obtained with the phosphatebuffer.

Ghosh et al. (2005) demonstrated the positive effect of the

addition ofShewanella on the compressive strength of mortar spec-imens. Contrary to the aforementioned research, these authors didnot intend mineral precipitation, as these specimens were cured inair and not in a nutrient containing medium. An increase of 25%

of the 28 days compressive strength was obtained for a cell con-centration of about 105 cellsmL1 and a water to cement ratio of0.4. For these samples, the presence of a fibrous material inside thepores could be noticed. As a result, a modification of the pore size

distribution was observed.The positive effectof theaddition ofShe-

wanella improvedwith increasingcuring times.Fora concentrationof105 cells mL1, anincreaseof thecompressive strength of17%and

25% was observed after 7 and 28 days, respectively. However, noincrease of the compressive strength was observed with additionsofEscherichia coli to the mortarmixture. This ledtheauthors to sug-gest that the choice of the microorganism plays an important rolein the improvement of the compressive strength. More specifically,

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)andJonkers and Schlangen (2007) investigated the use of bac-

teria as self-healing agents for the autonomous remediation ofcracks 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 ensureminimal externally needed triggers. Therefore, the authors inves-

tigated the compatibility of different organic compounds with thecement matrix. Moreover, suitable bacteria should be able to sur-vive concrete incorporation for prolonged periods of time. For thatpurpose,alkali-resistantspore forming bacteriarelatedto thegenus

Bacillus,Bacillus pseudofirmusDSM 8715 andBacillus cohniiDSM6307, were selected. In addition, the bacteria were added as spores,as these are known for their ability to endure extreme mechanicaland chemical stress. On top of this, the authors decided to choose

a pathway different from the hydrolysis of urea for the productionof carbonate ions. In this way, possible negative effects of the pro-duced ammonia on the reinforcement corrosion and degradationof the concrete matrix (when further oxidized by bacteria to yield

nitric acid) could be avoided. Among the components selected, cal-cium lactate did not substantially affect the compressive strengthvalues. Furthermore, the addition of a high number of bacterialspores (108 cm3)resultedinadecreaseofstrengthoflessthan10%.

For the evaluation of the mineral producing capacity, healingagent-incorporated specimens and control specimens werebrokento pieces after 7 or 28 dayscuring, immersedin tap water for 8 days

and subsequently analyzedby ESEM. Whilea massive productionoflarger-sized CaCO3precipitates was observed for the 7 days curedspecimens, no differences could be observed between the healingagent incorporated specimensand control specimens after 28 days.The authors related this to a decrease of the viability of the spores

upon incorporation in the cement matrix. The decrease in viabil-ity appears to be linked with a decrease of the matrix pores sizediameter (Jonkers and Schlangen, 2007; Jonkers et al., 2008).

5. Cost evaluation

5.1. Biodeposition

Table 6gives an overview of the costs related to the application

of surface treatments to building materials (Personal communi-


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the time of wetting and thus the number of nutrient applicationscould be drastically decreased. Consequently, the outgrowth ofother microorganisms (e.g. fungi and heterotrophic bacteria) will

be highlyunlikely. This makes the hydrolysis of urea a very feasiblepathway for applicationsin situ. From the above, it is clear that thehydrolysis of urea could present some economical advantages overthe other pathways.

In addition to the feasibility, which is governed by the timerequired for production of carbonates to occur (see previous para-graphs), the efficiency of the biodeposition treatment has beenobserved to depend on the speed of precipitation.

Rodriguez-Navarro et al. (2003) reported on the importanceof the type and structure of the precipitated CaCO3 polymorphs(vateriteor calcite)on the efficiency of the biodeposition treatment.The presence of well developed rhombohedral calcite crystals

resulted in a more pronounced consolidating effect compared tothe presence of tiny acicular vaterite crystals.

Differences in size and morphology of the crystals can beattributed to differences in the saturation state of a system pre-

ceding nucleation, with large rhombohedral calcite crystals beingformedat relatively low supersaturationand vateritecrystals beingproduced under highly supersaturated conditions. From the above,the authors concluded that fast precipitation could resultin a lower

efficiency of the biodeposition treatment.According toRodriguez-Navarro et al. (2003),the presence of

a phosphate buffer could explain for the occurrence of rhombo-hedral crystals. They attributed this to the buffering effect of the

phosphate, preventing rapid local pH variations, and hence, rapidchanges in the saturation state of the system. However, from thepapers byJimenez-Lopez et al. (2007, 2008),it appears that thephosphate buffer is not the only compositional difference between

the M-3 and M-3P media (composition seeTable 5). From thegraphs indicating the removal of calcium ions from solution, it canbe clearly observed that the initial concentration of calcium ionswas much higher in the M-3 medium (50mM Ca2+) compared

to that of the M-3P medium (35mM Ca2+). The latter could be

attributed to the precipitation of calcium phosphate which wasremoved before the start of the experiment (Personal communi-cation byJimenez-Lopez et al., 2008).As a result, the M-3 medium

showed initially a higher saturation state compared to the M-3Pmedium.

In spite of the high speed of carbonate formation and calciumdosages used, De Muyncket al. (2009) obtainedan excellent water-

proofing and consolidating effect with an ureolytic biodepositiontreatment on Euville limestone. From SEM examinations, the pres-ence of rhombohedral crystals could be clearly observed.

Besides the hydrolysis of urea, most MICP treatments rely on

the production of ammonia for the alkalinization of the culturemedium. Because of the fact that atmospheric ammonia is beingrecognized as a pollutant, thein situuse of such treatments might

raise some issues of environmental concern. Atmospheric ammo-nia is known to contribute to several environmental problems,including direct toxic effects on vegetation, atmospheric nitro-gen deposition, leading to the eutrophication and acidification of

sensitive ecosystems, and to the formation of secondary partic-ulate matter in the atmosphere, with effects on human health,atmospheric visibility and global radiative balance (Sutton et al.,2008).

However, when the concentration of ammonia generating com-pounds does not exceed the concentration of the calcium salt, it ispossible to decrease the emission of ammonia to a great extent.DeMuynck 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 pHincrease as a result of the release of ammonia.

Nonetheless, even in the case of the ureolytic biodeposition

treatment the production of ammonium will be rather low com-pared to conventional sources of nitrogen pollution, i.e. agricultureand domestic waste water. The treatment of 1 m2 of building mate-rial with 1L of a biodeposition medium containing 10 g L1 urea,

results in the production of 4.7 g N. For comparison, from wastewater treatment plants it can be calculated that one person pro-duces between 6 and 16 g ofN per day (DeCuyper and Loutz, 1992).

The presence of ammonium might also present some risks to

the stone itself. First of all, the presenceof an ammonium salt mightpresent some risks related to salt damage. Depending on the typeof calcium salt used, ammonium acetate or ammonium chloridewill be present in the stone after treatment. To our knowledge, no

reports areavailable on the effect of these salts on stone. Therefore,future investigations should investigate the retention of these saltsin the stone.

Secondly, ammonium can be converted to nitric acid by the

activity of nitrifying bacteria, resulting in damage to the stone.However,Mansch and Bock (1998)observed that the initial col-onization of natural stone by nitrifying bacteria takes several years.In addition, the extent of colonization is mainlygoverned by the pH

of the pore solution, with a pH between 7 and 9 being optimal forgrowth. 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 carbonateproducing bacteria will

outcompete the nitrifying bacteria for oxygen during the precipita-tion process. As a result of the precipitation, however, the pH willdrop to a value of about seven. Therefore, in order to avoidnitrifica-tion in the long-term, the presence of large amounts of ammonium

salts should be avoided. From long-term observations on the effi-ciency of the Calcite Bioconcept treatment, however, no damagesto 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 mostcommonly applied methods for the removal of salts from build-ing materials (Woolfitt and Abrey, 2008; Carretero et al., 2006).

Upon wet application, the paste facilitates the dissolution of saltswithin stones and migration of ions to the outside, where theyrecrystallize and are retained. Once dry, the paste can be easilyremoved. Different types of pastes or combinations thereof have

been applied for such purposes: paper pulp, clay materials (sepio-lite, bentonite) and cellulose derivatives. As a result of their uniqueproperties, many of these materials have also been used for theimmobilization of microorganisms in a variety of fields. A com-

bination of these two applications has already been applied forthe removal of black crusts on stone artworks (Ranalli et al., 1997;Cappitelli et al., 2006).While Carbogel was observed to remove

about 42% of the calcium ions from a black crust, the combina-tion of the former with sulphate reducing bacteria led to a totalremoval efficiency of about 95%. Besides removing the producedammonium, the use of a paste will also protect the bacteria fromdrying out, enhancing the overall biodeposition treatment, as was

observed by the Biobrush consortium (May, 2005).As seen before,however, the use of a paste results in a higher cost for the treat-ment.

In their search for alternative approaches towards the CalciteBioconcept method, most researchers have focused on the use ofdifferent organisms or metabolic pathways. Little attention, how-ever, has been paid to the influence of the dosage (gm2) 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 illustratethe carbonate precipitation potential of the strain. An overview ofthe different concentrations of calcium salts used can be seen in

Table 5.The importance of the calcium dosage on the overall effective-

ness of the biodeposition treatment can be easily demonstratedby the following example. In the Calcite Bioconcept method, the

total calcium dosage amounts to about 5.5 g m

2

calcium chlo-ride (MW 147g mol1) or 1.5g m2 calcium. Theoretically, this willresult in an overall precipitation of about 3.74g calcium carbonate(MW 100gmol1) per square meter of stone surface. Assuming a

density of calcium carbonateof 2.71 g cm3 anda homogenous pre-cipitation over 1 m2 of a non-porous stone, this corresponds witha layer of calcium carbonate of about 1.38 m in thickness. In thecase of a porous stone, a smaller thickness is expected due to the

high surface area of the pores. In practice, however, bacteria willbe mainly retained in the pores of the stone, especially those poreswith a diameter larger than 1m. Consequently, precipitation willmainly occuraround large pores, and layer thicknessesgreater than

1m can be observed. This is in agreement with the findings ofLeMetayer-Levrel et al. (1999)andOrial (2000),who observed layerthicknesses of about 23m and 45m, respectively.

Letus nowconsider themethodologyas proposedby Rodriguez-

Navarroet al. (2003). In one of their experiments, the authorssubmerged limestone prisms of 2.5 by 4.5 by 0.5 cm in an Erlen-meyer flask containing 100mL of M-3 solution. As such, thetheoretical calcium dosage amounts to about 339 g m2 calcium

acetate (MW 230 g mol1)or59gm2 calcium, corresponding withan overall precipitation of about147g calcium carbonateper squaremeter. This will in theoryresult in a layer of calcium carbonatewitha thickness of about 54m on the surface of the limestone prisms.

Although the authors did not report on the thickness of the carbon-ate layer, cementation was found up to depths larger than 500m(Rodriguez-Navarro et al., 2003).

As a result of the more pronounced precipitation of calcium

carbonate in the case of the methodology proposed by Rodriguez-

Navarroet al. (2003),a larger consolidating effect can be observedcompared tothe Calcite Bioconcept treatment.In theirworkon con-solidation of sand columns by means of biocementation, Whiffin

et al. (2007) observed that a minimum amount of carbonateprecipitation per m3 of sand was required in order to obtain a sig-nificant consolidating effect. DeJong et al. (2006)observed thatthe cementing effect occurred as a result of the precipitated cal-

cite forming bonds at the particle-particle contacts of sand grains.With increasing concentrations of precipitated carbonate, increas-ing bond formation and hence consolidation can be obtained.

Therefore, increasing amounts of carbonate precipitates could

result in an increased protective effect of the biodeposition treat-ment. This was indeed observed byDe Muynck et al. (2009),whonoticed an increased waterproofing with increasing numbers of

treatments or increasing the concentration of the crystal precur-sors in one treatment. The latter is also known to play a role in thespeed, and hence, the type of crystals that are formed, affecting theglobal effectiveness of a treatment (Whiffin et al., 2007).

Anincreasein thedosage ofthe calcium salt could,however,leadtoan accumulationof salts inthe stone,whichcould depending onthe anion give rise to efflorescence or damage related to crystal-lization. As mentioned earlier, the use of a paste could prevent this

from happening Regarding the Calcite Bioconcept treatment, sev-eral tests did not reveal any problems related to salt damage. Thechlorides are rapidly washed away as a result of raining (Personalcommunication, Loubire, 2008).

Besides the dosage of the calcium salt used, the type of stonewill also have a major impact on the global performance of the

treatment. The porosity, and more specifically the pore size dis-

tribution could be considered as one of the most determiningfactors.Samonin and Elikova (2004) reported that for a maxi-mum adsorption of microbial cells, the adsorbent pores must be

25 times larger than the cells. Therefore, the amount of bacte-ria retained in high macroporosity stones will be higher than inhigh microporosity stones. As a consequence, carbonate precip-itation can occur at higher depths in macroporous stone. From

SEM analyses, precipitation has been observed at depths of about100m for the Calcite Bioconcept treatment (Personal communi-cation by Loubire,2008). As mentioned earlier, Rodriguez-Navarroet al. (2003)observed precipitation at depths greater than 500m

in a bioclastic calcarenite.De Muynck et al. (2008b)observed anincreased amount of biomass adsorption in mortar specimens withincreasing water to cement ratio (w/c). The authors attributed thisto the increasing amountof pores with a diameter largerthan 1m

in specimens with increasing w/c. Since the amount of capillarypores between 2 and 10m is ratherlimited in cementitious mate-rials, the authors concluded that for these types of materials, thebiodeposition treatment is mainlya surface phenomenon. This was

also observed from thin sections, where a layer of crystals withinthe range of 1040m on the surface was found, correspond-ing with the theoretical thickness calculated from the calciumdosage.

FromTable 1it is clear that the differences between the variousmethodologies are not limited to the mediator used for precipita-tion. In addition, different research groups used different dosagesof calcium salts and different application procedures on different

types of stone. Besides the differentmetabolicpathways andbacte-riaproposed,the difference in inoculum size could also account forthe differences in time required for precipitation to occur. Further-more, manyexperiments were performed under sterile conditions.

However, for applicationsin situgrowth and activity are requiredunder non-sterile conditions. This could potentially influence onthe microbial activity. From the above mentioned it shouldbe clearthat thiswill hamperany quantitative comparison between the dif-

ferent treatments. Additionally, such a comparison is even more

difficult due to the fact that different authors used different eval-uation parameters and procedures (Table 1).Some authors mainlyfocused on the waterproofing effect (Dicket al.,2006), while others

mainly investigated the strengthening effect (Rodriguez-Navarroet al., 2003; Jimenez-Lopez et al., 2007).In addition to these twoeffects,Tiano et al. (1999)further proposed the evaluation of thevisual aspect before and after treatment by means of colorimetric

analysis.Therefore, the next step in research regarding the application of

calcinogenic bacteria should be a qualitative and quantitative eval-uation of the different methodologies under identical conditions.

Besides the evaluation of the protective performance (strength-ening and waterproofing (incl. porosity)), the influence of thetreatment on the visual aspect should be investigated. From this,

theexactrole of the microorganism andthe metabolicpathway canbe distinguished among the other parameters contributing to theoverall effectiveness. An expanded knowledge on these factors willno doubt contribute to the added value of the biodeposition treat-

ment, which is an ecological, compatible surface treatment with ahigh protective effect.

7. Future perspectives

In 2010, the patent of Adolphe et al. (1990) will expire.This will certainly lead to further explorations of the biodepo-

sition technique by the different research groups. As a result,reports on experiences from life size experiments can be soon

expected.

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