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into fresh concrete, it is difficult to prove that they actively contribute to calcium carbonate precipitation and the effects on concrete strength are

1 Introduction beforemicrobialtechniqueswouldbeusedatalargerscale
Over the last decade, the application of bacteria for
construction purposes has become a topic of research 2 Microbially induced calcium carbonate
worldwide. Just like towards the human body micro precipitation
organisms can have pathogenic or probiotic effects, for
building materials their presence can be devastating or Thepositiveeffectthathasbeenstudiedbyfarthemost,is
useful [1]. Maybe nowadays, biodeterioration is still better theMicrobiallyInducedCaCO3precipitation(MICP).MICPin
recognized than possible positive effects microorganisms civil engineering has been studied mainly for application in
can have on construction materials [24]. Microorganisms the fields of surface protection of natural stone, crack
can cause mechanical erosion and internal pressures by remediationinconcreteandsoilimprovement.Alsostrength
growthofhyphae,aswellaschemicalsolution,precipitation enhancement by mixing bacteria into concrete has been
andalterationofmineralphases.Organicandinorganicacids investigated. For more information on carbonate precipi
producedbymicroorganismscaninduceextensiveleaching tation in construction materials the reviews ofCastanieret
effectsonconcrete.Certainacids,likesulfuricacidproduced al. [6] and De Muynck et al. [7] can be consulted. More
byThiobacillibacteriainsewersystems,haveanadditional recentinformationisincludedinanupdatedpaper[8].Many
aggressive effect. In this case the involved sulfates cause bacteria can mediate the formation of calcium carbonate
expansive ettringite and gypsum formation. Biofilms, according to various metabolic pathways if given suitable
extracellular polymeric substances excreted by micro conditions (Table 1). It seems that calcium carbonate,
organisms to protect themselves, can cause discoloration precipitated in heterotrophic processes, is more abundant
and scaling of surfaces by inducing changes in water than that from autotrophic processes. Autotrophs are
transport properties. According to a US estimation, the organisms that produce complex organic compounds, such
contribution of microorganisms to the deterioration of as carbohydrates, from simple substances, generally using
materialsasawholemaybeintherangeof30%[5]. energy from light (photosynthesis) or chemical reactions
It comes therefore as no surprise that engineers question (chemosynthesis);whereasheterotrophsareorganismsthat
thebeneficialeffectsmicroorganismsmayhaveonbuilding cannotfixcarbontoformtheirownorganiccompoundsand
materials.Nevertheless,manystudiesnowprovideprooffor need organic carbon sources for growth. The microbially
these positive effects, but some challenges still remain produced calcium carbonate forms a mineral layer that


N.DeBelie,RILEMTechnicalLetters(2016)1:5661 57

Thislayercangrowtosuchextentthatitcanfillporesand 3 Selectionofbacteria
The selection of the most suitable bacteria for MICP is
mostly based on their carbonate yield. Bacteria are mainly
isolated from natural carbonate producing environments,
such as calcareous stones [12], calcareous sludge [13],
of amino acids), which showed a carbonate yield of 0.6 g
With B. cohnii living cells (108 cells/mL) Wang [16] found
25 g/L CaCO3 production (1.7 g/(L.d)) with the highest
production in the first two days. In the same study [16], it
60 g/L CaCO3 within one day at optimal conditions,
illustrating the higher carbonate precipitation yield of the
ureolytic pathway. De Muynck et al. [17] have shown that
between various ureolytic strains even at lower

Figure 1. SEM micrograph of bacterial CaCO3 precipitation on temperatures of 10C the mesophilic Bacillus sphaericus
concretesamples(courtesyofLienStandaert). produced a higher amount of carbonate in the shortest
amount of time than other more cold resistant strains like
The pathway that has been studied most for engineering Sporosarcinapsychrophila.Ersanetal.[18]haveinvestigated
purposesisprobablythedecompositionofureabybacteria, MICP through denitrification, in a minimalnutrient
withtheaidofthebacterialureaseenzyme.Thisresultsin environment. In this study, CaCO3 precipitation
the production of ammonium (NH4+), dissolved inorganic performances of two newly isolated resilient strains
carbon (DIC), and an increase in pH favoring CaCO3 DiaphorobacternitroreducensandPseudomonasaeruginosa
precipitation.Furthermore, dueto the negativecharges on were optimized and the repetitive CaCO3 precipitation ofa
thebacterialcellwallandthehighsurfacetovolumeratioof singleinoculumwasinvestigated.CaCO3precipitationyields
bacteria,thebacterialsurfacehasahighcapabilityofbinding of14.1gto18.9gCaCO3/gNO3Nwereachievedin2d.
cationsandservesasanidealnucleationsite.Thebacterially Nevertheless,astraightcomparisonisdifficulttomake,since
catalyzedureahydrolysisisapproximately1014timesfaster there are many different factors governing the CaCO3
than the uncatalyzed rate [9]. Especially Bacillus pasteurii precipitation yield, that need to be optimized and that
andBacillussphaericushavebeenstudiedforthispurpose.A furthermore interact with each other [8], such as bacterial
downside of urea hydrolysis especially for the use in soil cell concentration, concentration of urea, organic acids or
improvement,isthebyproductammonia,thatneedstobe nitrate (for the above mentioned pathways), and calcium
removedbyflushing. concentration.
A pathway which does not produce this byproduct is the
oxidation of organic acids, where promising results have 4 MicrobialCaCO3forstrengthimprovement
been obtained with Bacillus cohnii [10, 11]. In alkaline
environments, organic compounds are degraded into CO2 Theliteratureonstrengthimprovementsobtainedbymixing
and H2O. In high pH environment, CO2 transforms to CO32 bacteria into concrete shows variable results. Several
andinthepresenceofCa2+,CaCO3canbeformed.Biological authors have mentioned increases of 28 d compressive
reduction of NO3 in the denitrification pathway also strength with 9 to 25% when mixing bacterial cells into
generates CO32 and HCO3 ions, which are necessary for mortar or concrete [1921]. Others mention both positive
CaCO3precipitation. andnegativeeffects,dependingonthebacterialstrain,cell

Autotrophicbacteria Heterotrophicbacteria
nonmethylotrophic Dissimilatorypathways
Aerobic Anaerobic
Process e acceptor Process e acceptor
Ammonification of amino
Respiration O2 NOxreduction NO3 /NO2
oxygenicphotosynthesis acids 2
Methane oxidation CH4/O2 Sulfatereduction SO4

N.DeBelie,RILEMTechnicalLetters(2016)1:5661 58

Hereby, it is very difficult to prove the actual influence of formate, ) as a calcium source for the bioprecipitation,
livingbacteria.Insidetheconcretematrix,duetothelackof whichmakesthemfearforlaterdamagetothestone.
oxygen, high pH and limited space availability, activity of Until now, the onsite bioconsolidation has been mainly
(aerobic)bacteriawouldbegreatlyinhibited. restricted to porous calcareous stones of cultural heritage
Achaletal.[24]noticedthattheaveragenumberofviable buildings. Nevertheless, several researchers have
bacteria decreased from 6106 to 4.5104 after 7 d, which successfully applied the MICP treatment on cementbased
meansmostbacteriadiedandbacterialgrowthwasgreatly materialsinthelaboratoryandinsmallonsitetests[20,30
inhibitedsincetherewasnodifferenceincellnumbersat7d 33]. Compared with porous limestone, concrete is denser,
and28d. which results in less retention of bacteria in the surface
Basaran [21] did find viable S. pasteurii cells in hardened layer. De Muynck et al. [31] have shown that the surface
cement paste samples that were as old as 330 d with deposition of calcium carbonate crystals on mortar
approximately2%viabilityretention,and50%oftheviable decreasedthewaterabsorptionwith65to90%depending
cellsdetectedweredefinedasvegetativecells,whichcould on the porosity of the specimens. As a consequence, the
bemetabolicallyactive.Nevertheless,itisdifficulttoprove carbonationrateandchloridemigrationdecreasedbyabout
that the bacteria would actively contribute to calcium 2530% and 1040% respectively. An increased resistance
carbonate precipitation at the high pH of 13 inside the towardsfreezingandthawingwasalsonoticed.Theresults
concretematrix. obtained with the biodeposition treatment were similar as
The measured increase in calcite content, occurring within
cement paste when the bacterial medium was used [21],
wallsortotheureaseenzymeremainingactiveeveninthe 6 Microbial CaCO3 for selfhealing of cracks in
deadcells. concrete
active. These nutrients are added to the mortar mix or Theprincipleofbacteriabasedselfhealingconcreteisthat
providedbyimmersionofthemortarsamplesintonutrient carbonate precipitating bacteria are added into concrete
solutions. The nutrients could also affect the mortar during the mixing process. When cracking occurs, the
strength. Wang et al. [25] investigated the influence of bacteriawillbeactivatedtoprecipitateCaCO3toinsituheal
calcium nitrate (as calcium source) and yeast extract (as concrete cracks. This selfhealing property results in a
nutrient) on the cement hydration with isothermal recovery of watertightness, and hence limits the
calorimetry. It was found that Canitrate can accelerate penetrationofcorrosivesubstancesintoconcretestructures
cement hydration, whereas yeast extract (0.85% versus andimprovesconcretedurability.Toapplybacteriaforself
cement weight) significantly delayed the hydration and healingofconcretecracks,theyshouldsustaintheirviability
resulted in a lower hydration degree at 7 d of age and a until crack formation. Therefore, researchers are generally
reducedcompressivestrengthat3months. proposing spores instead of vegetative cells for this
5 MicrobialCaCO3forsurfaceconsolidation source, calcium chloride should be avoided due to the
corrosive effect on the reinforcement and calcium nitrate,
calcium acetate, calcium formate orcalcium lactate can be
The first onsite application of the bioconsolidation
and bioprecipitation, especially in an unfavorable
environment, such as at low temperatures (10C) and in
presence of high concentrations of Ca2+. This may be very
Maastricht limestone by means of the drilling resistance
important for practical application since the optimal
measurement system (DRMS Cordless SINT Technology,
Italy). They were able to homogeneously strengthen
to 30C, whereas lower temperatures that are relevant in
limestone up to depths of 30 mm with a similar or better
many reallife situations retard the germination of spores
performance than that of traditional surface treatments
free and entailed both a significant protective and To ensure bacterial survival during concrete mixing and
consolidating effect after two spray applications within the hardening, encapsulation of bacteria in suitable carriers is
sameday:thedrillingresistancewasmorethandoubled.By preferable.Toreducethenegativeeffectofthenutrientson
optimizing the concentrations of the urease and carbonate the mechanical concrete properties, also encapsulation of
precursor solutions, the reseachers were able to lower the thenutrientshasbeenproposed.
costofthetreatmentwithintherangeofthatoftraditional Several encapsulation strategies have been tried for the
consolidants.However,onepointofconcernbypractitioners bacteria (or nutrients): silica gel or polyurethane in glass
is the use of hygroscopic salts (calcium chloride, acetate, capsules [35], expanded clay [36, 37], diatomaceous earth
N.DeBelie,RILEMTechnicalLetters(2016)1:5661 59

[38],melamineformaldehydebasedmicrocapsules[25,39], thattheexpectedservicelife(basedoncorrosioninitiation)
syntheticorbiobasedsuperabsorbentpolymers(hydrogels) of concrete structures containing cracks of 100300 m
[4042].Thehydrogelshavethedoublebenefitofprotecting decreases to one fifth of the value expected for a sound
the bacterial spores and of acting as water reservoir for structure. Taking into account the current selfhealing
spores germination and bacterial activity when cracking efficiency of systems based on encapsulated polyurethane
occurs. Recently, selfprotected nonaxenic mixed cultures (so no bacterial selfhealing), they estimated that the
have been tested [43]: Cyclic EnRiched Ureolytic Powder averageservicelifecouldbedoubledbyintroducingthisself
(CERUP), and Activated Compact Denitrifying Core (ACDC). healing technology. In case of perfect selfhealing, the
CERUP is a ureolytic community protected by its high salt servicelifeofanoncrackedstructurecouldbeapproached.
content and obtained from the further processing of side Similar durability tests and service life calculations for
streams from vegetable industry. ACDC is a denitrifying microbialselfhealingconcreteshouldbeperformed.
microbialcommunityprotectedbyvariousbacterialpartners Regarding rebar corrosion, it should be considered that
and obtained in a sequential batch reactor by applying microbial urea hydrolysis and aerobic oxidation of organic
selectivestressconditions.Silva[44]statedthatnonaxenic carbon require O2 as final electron acceptor to initiate
cultureshave,duetotheeliminationoftheneedforsterile and/or to keep the microbial activity. This may result in
production conditions, much lower production costs when oxygen consumption in the concrete, which may pose a
comparedwithaxeniccultures(atleastafactor10cheaper limitation for rebar corrosion. Yet, for crack healing in
forCERUPversusproductioncostsofBacillussphaericusor concrete, when O2 availability in the deeper parts of the
Bacillus cohnii spores). In theabove mentioned references, cracks is limited, this will in turn inhibit the bacterial
generally cracks of upto 500 m in width could be healed precipitationprocesses[47].Moreover,CaCO3precipitation
within3to14weeks(exampleinFigure2). itselfentrapsthebacterialcellsandmayinhibitO2diffusion
Mosttypesofencapsulationmaterialswillnegativelyaffect into cells [48]. These considerations have led to the
thecompressivestrengthofconcrete,especiallyiftheyare investigation of nitrate reducing bacteria for selfhealing.
added in dosages exceeding 1% versus cement weight. Biological NO3 reduction takes place during the microbial
Diatomaceous earth (DE) is an exception, since this has a oxidation of organic matter by use of NO3 as an electron
positive effect on strength due to its pozzolanicity; but it acceptorinsteadofO2.SolubilityofO2inwater(9.1mg/Lat
cannot be considered as a real encapsulation technique 20C) is 102 to 105 times lower than for NO3 as electron
since the bacteria are adsorbed on the DE instead of acceptor. In the presence of calcium ions, NO3 reduction
penetrating into its pores (which are too small for this inducesCaCO3precipitation.Ourresearchhasrevealedthat
purpose). throughNO3reduction,evenenhancedCaCO3precipitation
With regards to bacterial selfhealing, several questions performances could be achieved in nutrientpoor
remain open for further study. The viability of the self environmentswhichmakesthemechanismfeasibleforself
healingadditiveintimeisyetunknownandwillbedifferent healingconcrete[18,49].Nitratereductioncanalsoleadto
foreachspecificbacterialstrainandprotectionmethod.Also the intermediate production of NO2 which is known as
ithasnotbeenprovenwhethersporesthathavegerminated corrosioninhibitor.Itwillthereforeprotectthesteelsurface
andactivelycontributedtocrackhealing,willsporulateagain fromcorrosivesubstancesduringthehealingperiod.Itwas
andremainindormantstateuntiltheiractivityisoncemore shown that the nonaxenic culture named activated
needed. In addition, the selfhealing efficiency needs to be compact denitrifying core (ACDC) induced passivation of
proven for concrete elements, since until now most proof steel in corrosive electrolyte solution (0.05 M NaCl) by
ofconcept tests were carried out in mortar specimens. producing57mMNO2in1week[50].Recently,accelerated
Whenkeepingthedosageofthebacterialadditiveconstant corrosion experiments have been carried out on cracked
relativetothecementweightwhenupscalingfrommortar (300mcrackwidth)mortarspecimenswithanembedded
to concrete, this results in a significant dilution of the steel rod subjected to 0.4 M chloride solution to mimic
additive. However, when maintaining the same dosage in chlorideconcentrationsinseawater(Fig.2).
proportion to the total volume, an unacceptable strength

7 Bacterial selfhealing to increase concrete


Cracks in cementitious materials have a negative influence

chloride penetration, Maes [45] states that 10 m is the
critical crack width, above which cracks provide a

preferential and fast way for chloride ingress. Based on Figure 2. Micrographs of a selfhealing (containing bacterial ACDC
chloride penetration tests in submersed conditions and granules)mortarspecimenwith300mwidecrackbefore(left)and
service life modelling, Maes and De Belie [46] have found after (right) 28 d submersion in a 0.4 M chloride solution, showing
N.DeBelie,RILEMTechnicalLetters(2016)1:5661 60

These preliminary tests (data unpublished) revealed that References

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Application of hydrogel encapsulated carbonate precipitating