Title no.
98-M1
Remedlatlon of Concrete Using Mlcro ..Organisms
by Santhosh K. Ramachandran, V. Ramakrlshnan, and Sookle S. Bang
Thls paper describes an lnnovative blotechnology uttttzing micro-
btologlcalty-lnduoed mineral prectpitation lar concrete remedia-
(/011. Ca/cite precipita/ion lnduced by Baoillus pasteurii was
studled In two types 01portland cement mortar speolmens: olle
prepared from mlxlng wlth mlcro-organtems, and the other wlth
simulated cracksfilled wlth mtorobtal mixtures. The study showed
that there was a significant lncrease tn compressive strength of the
portland cement menar cubes contalnlng lower concenirations of
ttve cells. Compressive strengths of the el/bes containlng llve 01'
dead cell mass, however, decreased as cell conoentrattonsand cur-
illg lime lncreased, suggestlng the tnterference of'mortar inlegl'ly
by biomass. Cracks fllled wlth bacteria and sand demonstrated a
stgnifcant increase /11 compressive strength and stljJnes$ values
when compared IVI/h those wuhout cells. Soanntng electron micro-
graphs tdentlfted that mtcrohtotogtcat cale/le prectpitatlon
occurred matnly clase 10 the ~lIrface reasuf the crack, where a
dense growth of ca/cite crystals embedded with cells was observed.
Keywords: oalcte: compresslve 8lrenglh; cracking: s(iffnl)!)s.
INTRODUCTION
In natural environments, chemical CnCOJ preciptaton
(Ca2+ + coi- ~ CaC03.}) is accornpanied by biological
~TocesseB.both ofwhich ofien OCCUl' simultaneously or sequen-
tullIy, An endospore-forming son micro-organism, Bacillus
pastCl/rii, participares in enlcite precipitatiol1 in the cllviroll
ment by producing the urease cnzyme. Ureas e (urea ami-
dohydrolase, E.C. 3.5.1.5) catalyzos urea to produce CO2
and ammonia, I'esulting in an increa:;t: ofpH in the surround-
ings where mineral ions (Ca2+ and COl~) precipitate as
CaC03
Microbial CaC03 precipitation is a compLex mechanism.
?u~ stu.dy O? kin:etic details has indicatod that calcite precip-
ItatlOn I~ a tunctlo!, ofthe cell ~?ncent.ration. ionie strongth,
and pH In the medlum,l In addlllon, rnlcro-organisms whose
Ilet cell surface charge i5 neJl.ative also draw cations from the
envronment, including Ca ,to deposit 011 the cell surface.
The following equations surnmarize possible biochemical
l'eactions in a medium to preciptate CaCOJ al the cell sur-
face that serves as a nucleation slte
Ca2+ + Cell ~ Cell-C(+
Cell-Ca2+ + COl 2- -t Cell-CaC03.}
A llovel tcchnique for the remediation of damaged struc-
~ral fo~mat?ns has been developed by employing a selec-
tlvo IDlcroblal plugging process in which microbial
metabolic activities promote precipitation of calcium car-
bonate in the fonn of calcite?,3 As a microbial sealant
CaC~3 exhibited its positive potential to selectively consoli~
d~te slmulaled fractures and surface fissures in granites,2 Sig-
ntfican~e of l~icrobial calcite precipitation was quantified by
x-ray dlffractton (XRD) analysis and visualized by sconning
ACI Materlals Journal/January-February 2001
electrn mioroscopy (SEM),1 In the authors' studes on sand
consolldetion, morobiologically-lnduced calcite constituted
more than 30% of oonsolidated materials in which microbial
calcite pluggng was highly selectivo. The efficiency of mi-
crobial plugging was affected by (he porosity of'the mdium,
the number o cells present, and the total volume of'nutrient
added, In crack rernedlation of granite, un average crack
width of 2.7 mm (0.106 in.) and El mixture of slica fume
(10%) and sand (90%) showed the highest compressve
strength in microbial remediation of granite.4 Barler, Kant-
zas el 81.3 reponed that sand oonsolidation by B. pasteurli re-
duced porosity by up to 50% and permeabilty by up to 90%
in thc arcas whcre the cementaton took place.
The concept of using mcrobial mineral precpitatfon in
theremediaton of cracks has been applied in mcroblal en-
hanced o! recovery (MEOR), which has beneflted the oil in-
dustry by effectlvely utlllzing all fue mineral resources from
a particular digging site, It Is important to realize that micro-
organisms plug the pores of rock not only by aoting as small
partcles but also by adhering to the avalable surfaces
through extracellular organlo compounds, Biotechnology of
microbial crack remediation is therefore equalLy applicable
to those ofman-made structures, including concrete.
N
Cracking of concrete is a common phenomenon. Wthout
immediate and proper treatmel1ts, cracks in concrete struc-
tures tend to expand further and eventually require costly re-
pairo Even though It is possihle to reduce the oxtent of
cracking by available modern technology, remediatioll of
crncks in concrete has been the subject of research for many
years, Tllere are a large number of products availnble com-
mercially foc repairing crack s in concrete: structural epoxy,
resins, epoxy mortar, and otller syl1thelio mixtures,s Current-
Iy, these type.'l ofsynthetic filler agents are extcnsively used
in concrete crack ropa ir. Because cracking in concrete stl'UC-
tures continuos over a long pedod of time, this type of one-
time, quick remedy ShOllld also be applied repeatedly as
needed.
Use of bacteria in concrete remediation ls an unorthodox
coneep! in current concrete ..esearch. lt s, however, a new
approach to all old idea Ihat a microbial mineral deposit con-
(1) stantLy oocurs in natural environments, Specifically, micro-
bologically-nduced caleite is envirol1mentally innocuous,
(2) compared to synthetic polymccs currently used for concrete
~epairs. The highly alkaline pH of concrete is a major hindcr-
mg factor lo the growth of a mooerate alkaliphile, B. pasteuri/,
whosc optimum pH for growth is around nine. B. pasteurlt,
however, has an ability to produce the endospore, a dormant
form ofthe cell, to endure extreme environment. Our prelim-
.fel I,{,,(erlal. JOllntal, V. 98. No. 1, anlUlry-Fcbrutry 2001.
MS No. 99-234 rwelved DIlcombcr 13, 1999, .nd rQvlowcd undcr In6litllt~ publica-
tlon palldas. CoPyright 1:1 2001. Alllorcan Concrete InJlilute. AH rihts ,eoorved
ncl~ding Iho mnkinof oopios IInl""9ponnls41on1801>1.100<1 from Iha copyright pto:
prlolou.l'cninchl d,scussloll wlll ha J!u~l~h....t in Ihu Nowmbur DCCI>mbcrZ~OI tiC!
Material. JOllnral if received by August 1,2001.
3
 .--- -.
SAII!hsh1(. HlIlII.thIlIlOl'Ah I.f (1 graduot research assstant ln te Deparlllwnt of
Cvtl ond NlJVllwll/ldll/nl ttlJghlMl'lllg 111 '/w SOIl(It Dao/a Scttoo! of MIIIM and Toch-
III)/ogy (Sl)SML~n.Raptd Clly. S, Dule.
V. H"ltlllkrl!hn"", FACI, ts Dlsttngnlsed Proff'.1sOl'a! Sf.lSM&1: lle recolved !tls
PI,D 111 cMI ellgl"e.,,'llIgjro/llI,OlldOl' Ullly.nIIY. UK, 11M re.,,"rc' tuterests ncludc
stecl and 1J'/II/lct/c flber COIU'IWOC,)/IIpo,llIc.<, and md,ulldwl'cd .w~Jfibr.r 1/1 bOII,
COI1CNrO and shotcrote.
Soo!dc S, llolIllls a professor ofblo/lIgy al SDSM&T, S/~ recctved Iwl' PhI) IIJmicro-
bI"I"8}' from 1/101 Onlv~rsil)' tif Ca/I/om/a at Davls. ller rosearch tntorast lile/mM
,'h.lrollltlcll(<1/m/croblollJgy, btotecnology, alld b1oL'heml.wy,
inary study on using micro-organsms in 11 concrete mixture
was somewhat inconclusive in demonstrating the increase of
compressive strength of'mortar, duo to a complex internoton
between the bacteria and the cement mntrix.6,7 The long-
term goal of the authcrs' rcscarch in microbial concrete re-
modiatlon is o undersnnd the slgniflcancc of micro-organ-
isma in the concrete structurc, It is of great importance to
identify thc microbial behavior in an extremo envronrnent of
concrete. The objcctive of this study 18 to investigate ihe ef-
fects of experimental parameters of mlcroblologlcsl mineral
precpi tation on the compressve strength and stiffuess of ce-
ment mortar.
RESEARCH SIGNIFICANCe
The signi ficol1cCof Ihis research is the use o tl1icro~ol'gan-
3m3 in l'emediatioll o concrete. T11estudy has idenlificd the
cffect oflhe type aud 81110\lnlofbiomass (micl'o-organisms)
on the comprcssive strcnglh of porlland eomont mOl'tl1l'
cubes, Furthennore, comparisoll of Ihe valnes of sliffness
and compressivc strength of mortal' beams and cubes with
variatiol1s In crack deplhs and microbial eell cOllcenlrations
has been made to evaluale the pel'tonnance ofmicrobiologi-
cal remcdiatioll of concrete cracks. The development of Ihe
micl'Obiologically-indueed cnlcite as a tool oC concrete fepair
will provide the basis for un ultel'1lativc and hgh quality COIl-
crete sealant that i6 cost effective and enviromnentaJly safe,
MATERIAlS
Mlcroorganisms and chernicals
Bacillus pasteurli ATCC 11859 and Pseudomonas aerug-
nosa ATCC 27853 werc used fol' this study.
Cement, fIne aggregate, and water
Type I/II normal portland cement was used fol' all mix-
tures, The cement mccls ASTM e 150 requiremcnts. Natural
sand satisfying lhe gradatiol1 requirements of ASTM C 33
was used as tne aggl'egate. Glass-distilled water was used
fol' the pl'eparatioll ofmedia and buffersolutions, AII mortal'
mixing was done with lap water.
EXPERIMENTAL PROCEDURES
Micro-organisms and growth conditlons
A stock culture of B, pas(eul'/i was maintaned in a solid
medium containing: 10 g trypensc; 5 g yeas\ extraet; 4.5 g tri-
cinc; 5 g (NH4hS04; 2 g ghltamic acid; 10 g urca per L, pH
8.6, which was filter-stcrilized; and El final conccntration of
1.6% agar, whicll wns nUloclaved scpal'ately IInd added aflor-
wards. For qllsntily culture, B. pasteuri was grown in yeast
extraet (YE) broth medium. Dclals of YE mediulll contcllt
nd preparation were published previously. I P. aeruglnosa
wel'e maintained in llutrient agar for stock cultures and
gl'own in nutrient brolh for mass cultures. Routinely,both
cultures of B. pasteurii and P. ae/'uglnosa were grown at 30
4 ACI Matarais JournallJanuary-February
C. Cells wcre grown in the water-bath shuker for 20 lo 24 h
to reach a fale exponential stago. The growth cultures were
centrifuged to harvest cells, Cell pellets were washed twice
with satine containing 0.85% NaCI 01' with phosphate buffer
containing 50 mm sodlum phosphate, pH 7.5, The final cell
pelleta were suspended in either salino or phosphate bu fer lo
make il total volume of 100 ml.,
Absorban ce of the cell suspension was read at 600 nm
(Spec 20). By comparing optical density readings and plate
cell-counting at the corresponding ntcrvals during the cell
growth, tite cell concentration was determined accordug to
the equatlon: Y = 8.59 x 101 XJ.3627, where X = readlng al
OD 600' and y = concentration of cells per mL. Thls expres-
sien was used o determine the concentraton of cells sus-
pended in a buffer solution.
PreparaUon of portland cement mortar cubas In
salina and phosphate buffer
A mixture of240 g cement, 660 g sand, and 116.4 mL of
salino 01' phosphete buffer was used to cast 12 portland ce-
ment mortal' cubos of dlmenslons of 50.8 x 50.8 x 50,8 mm
(2 x 2 x 2 in.). The mortal' was mixed and compllcted aceol'd-
ng to ASTM C 305-94. B. pasteu/'i was suspended in two
solutions (saline ll11dphosphate buffer), separateLy, to final
concentrationsofO, 3.0 x 107,6,0 x l07, and l.2 x 108 cells
per em3 The mortar cubes prepared with each solution COI1-
talning no celIs were used as control, AH samples were pre-
pared in sels of rour. Cement mortal' cubos with or without
cells were dcmolded afier 24 h and plnced in 3 L of Urea-
CaCI2 medium swirled with 2 g of lime. The speclmens I'e-
mained in Urca-CaC12 medillm fol' 7 days. Afier l'Cmoval
from 1he medium, the surfaces oi' the cubos were completely
dried prior lo compressive s11'engthtests,
Preparation of portland cement mortar cubes
contalnlng biomass in different concentratlons
POl'tland eement mortal' cubes were prepare<! according to
ASTM e 305-94. The contents of the cement mixture were
the same as descrlbed previollsly, except 11sing phosphate
buffer fol' mortal' mixing. To study the eftect of biolllass on
portland coment mortal" micro-ol'ganisms were prepared as
forms of living 01' k!lled by autoclave. Two sets of micro-or-
ganisms were used: one sel with a pure culture of B, pasteurU
and tlle other with a mixture ofequal concenl;mtions of B. pas-
leuri and p, aemginosa. Live or dead biomass ofthesc micro-
ol'ganlsms werc suspended in El phosphutc buffer to obtnin Ci-
nal concen1rntions of7.6 x 103,7.6 x 105, and 7.6 x 107 cells
per em3, Samples in each concentratioll were prepared in
triplica te. Cell-Iaden mortal' cubes and control cubes without
cells were submerged in Ul'ea-CaCl2 medium and curcd at
room temperature. The compressive strengths of sample
cubes were determillcd afier 7 and 28 dnys.
Stiffness testlng: simulated cracks with variable
depths in portland cement mortar beams
Portland cement mortar bcams of dimensions 25.4 x 25.4
x 152 mm (1 x I x 6 in,) containing no cells were cost as per
the ASTM C 305-95 procedufe, in which the same amount
of water was lIsed insteod of phosphate buffer, The speci-
mens were cUl'ed in water for 28 days and t110n10ft exposed
to air, The width of tlle cut was malntained constant at
3.175 mm (0.125 in.), while the depths ofthe cutwere 3.175
nd 9,$25 mm (0.125 and 0.375 in.). For each crack dcptb, a
total of 10 spccmens were prepared, The first five speci-
2001
mens were used 1Is control wthout filling in the 01'(\Ok8 and
left exposed t ar. rhe cracks in the remuining five speci-
mens were filled with II mixture of sand and !J. pasteurll, The
sand used passcd through the U .S. standard seve No. 16, but
was retained 011 the V,S. standard sieve No. 40, The sand
mlxed wlth a bacteria SlISpCl1S1oll to a final concentratlon of
3.8 x 109 cells per cm3 was forced lnto the crack wth a thin
knife-edge. Then tho specmen bcams with bacteria were
placed in a tray contalning the Uren-CaC12 medum and
cured for 28 days, separately. The mdium was replaced af-
ter 14 days, Extreme oare was takcn not to disturb the precip-
ita tlon of the oalcium carbonate on thc specimens durlng the
renewal ofthe medium. The beams with and wlthout bacteria
were tested for thelr stiffne;s aer 28 days ustng a three-
polnt loadlng method wHh a span of 127 mm (5 in.). Load-
deflecton relatlonshps were recorded using a data acquisi-
tion system, . Fig. J--Effect$ 01saline and phosphate buffer 0/1 compres-
Ocmpreaslve strength testlng: slmulatsd cracks
wlth varlabte depths In portland coment mortar
cubas
Portland cement mortal' cubes of a dimcnsion of 50,8 x
50.8 x 50.8 111m(2 x 2 x 2 in.) were cast according to the
ASTM C 305-<)4 procedure. The specimens were cured in
water for 28 days and then 10ft exposed to ni!'. TIJe cubes
were provded with a cut to slmulate a crack. The width of
the cut was kept at nn average of3.175 mm (0.125 n.) and the
depths were 12.7, 19,05, and 25.4 mm (0,5, 0.75, and 1.0 in.).
For each crack depth, 10 specimens were prepared: the flrst
five were used as control and the remaning vc for test spec-
mens with bacteriu, l'espectively, The cracks in the control
speeimells were flled with natural sand )!lssing through the
U.S. standard sieve No. 16 and l'olalned 011 the U.S. standard
sieve No. 40) and water. The cracks in the other Ove speci-
mens were filled wlth sand and bacteria, to n final cOl\centra~
tion of3.8 x 109 cells per cm3 Both sets ofcubes were cUl'ed
in Urea-CaC12 broth fOi' 28 days, The mediutll was replnced
after 14 days, The specitnens \Vere tested fOl' their compres-
sive strengths using a compresslol1-lcsting machi ne.
Compresslve strength testlng: slmulated craoks In
portland mortar cubos wlth variable cell
concentrattons
Porttand cement mMar cubes of a dimcnsion of 50.8 x 50,8
x 50.8 mm (2 x 2 x 2 in.) were cast as per the procedure previ-
ously described, The cubos were provided with a cut to SilllU"
late a crack. The width and depth ofthe cut wece maintained
conslllnt as 3.175 aud 25.4 mm (0..125 amI 1.0 in.), l'espectivc-
Iy. A total of 1811lortar cube specimells, each wth a cut of the
game size, were prepared for tlle test. AH specimens, including
three scts of controls, were prcpared in triplicate. Three speci-
mens in tho firat set of controls contnined 110 I1l1ingand were
keptex.posed to airo Cracks in three specmens ofilie second set
wcre filled with Il mixture of sand nnd water and cured in watel.
Cracks in three specimens of the third set wel'e t1lled with a
mixture of salld and water and cured in Urea-CaClz mediuOl.
Microbial plugging ofthe concrete cracks was eXllmlned with
three different concen!ratiOllsof B, pC1SJUl'ii mixed with salld,
Final concentratiotls ofcells \Vere 2.6 x (O7, 5,2 x 107, and 2,6
x 108 celia per cm3 AH seIs of specimens conlaining cells were
cured separately in the medium. Tlle medium was changed af-
ter 14 days, The cubes were tested fol' theit compression
strengths after 28 days.
ACl Materlals Journal/January-February 2001
c:::: SoU",
ll'WA:I pholvl,>l' h.lfu
10
o 3x 107
Cell Concenlraton (cc1ls/om)
slve strength 01 portland cement mortar cubes contatnlng
vario us concentrations o/B, pasteurii,
SEM examlnatlon
Fractlons of remediated crack s were prepared for SEM
analyss as publlshed prevously.' Samples were coated
with carbon prior to cxamination by SEM. which was
equipped with an energy-dlspersive x-ray analyzer for ele-
mental analysls.
XRD analysis
Samples subjected to XRD analyss lncluded Ihree pOl't-
land cemenl mortal' cubes: 1) mixed snd cured in water; 2)
mixed in phosphate buffer and cured in Urea-CaCl2 medum;
and 3) mixed wilh B.pasteurli suspended in phosphate buff.
er and ClIred in Uren-CaCl2 medium. Eacll sample was
cl'ushed and pulverizad to an average partiole size of less
than 10 microns, nud thCll mounted onto n gloss fiber filter
using a tubular aerosol suspension chamber (TASC). The
mass absorption coefficient of the mler was determined pl'ior
to uso. Then the moss absol'ption coefficient of tite sample
was detel'lnined by x-tay transmission. Tite XRD pnttern was
obtained by scannng from 5 to 60 degrees, 2 theta using a
vel'tical x-rny di ffraclometer. The components of the sllInple
wel'O identified by comparing them wilh standards estab-
Iished by the Internotional Ccnter fol' Diffractioll Data. The
quunHtative analysis was perfofmed USillg quantitative XRD
analysls.8
RESUL TS ANO DISCUSSION
Effects of type of buffer solutlon on compresslve
strength of portJand cement mortar cubes wlth
various concentratlons of bacteria
Compressive strength vaIues of cubes prepal'ed in phos"
phate buffer and salino solutOll are compal'ed in Fg. 1, Port-
Isud coment mortal' cubes prepared in saline have ShOWll an
ovel'all deorease in compressive strength in the presenco of
cells. rhe decrease in compressi ve strength of he cubes con-
tailling satine may be due to the presence of cllloride lons in
the solution, which is kl10wn lo weaken lhe integrity ofthe
cernen! matrix,9 The compressive strength of mortar cubes
prepared in the phosphate buffer was consistently highel'
than the stl'engUl of saline-prepared cubes at various concen-
trations of cells laden in the cube. Thus, phosphate buffer so-
lution was uscd in al1 the other tests. The cubes prepated with
5
 ., Tabla 1-Comprasslve strC:trlgthvalues of 7 and 28-day tests with portland
cement mortar cubas mlxod wlth dlfferant types of blomass
7.oy oomprcsslvo 28-doy compreesivc
Micro-orgnnlsms, cellslcm3 strcngth, MPo strengrh, M I'u

B.pnrlel/I'/I P. aeniginosa Average

. Standard
deviatlon
. Avcrngo'
Standard
devlation
.
- COI\('o! O
3.8 x 103
O
3.8 x lO)
47
55
1.38
2.39
55
62
1.27
3.23
Uva 3.8 x lOS 3,8>: lOS 54 1.55 63 1.97
3.8 x 107 3.8 x J01 57 1.27 65 0.87
l8)( 10l 3.8 x 103 53 3.73 56 2,36
Killod 3,8 x lOS ),8 x JOS 59 3.46 55 2.42
s.a K 107 3.8x lO? 60 1.81 61 0.92

7,6 x 101 O 46 2,08 65 0.81

Uvo 7.6 x I~ O 50 2.73 55 2.33
7.6 x 107 O 5S 1.28 55 0.44
1.6 x l()l O 57 1,47 59 3.19
Kllcd 'J.6 x lOS O 61 2.88 56 2.57

7.6x 107 O 66 1.41 58 0.79

Avcrugo "lid stnndard dcvlmlon VAlvas obteined from triplicafo eoml'k a.

prcssve strength wth the increase of cell concentrations in

lIII1Il lve Oa,UII",ndPevt1._,,,,
both forms 'of the Bacillus and Pseudomonas mixtures,
20
.... lv./Jacm,u whereas an opposte effect was shown in both fOl111S of the
lIII1Il kllled l1a<//I",
and I'_d."", ...
lj lE!ll9 kllle<lDa<flf" Bacillus-only group.
Changos of the comprcssive strength vales between 7-
~ 10 and 28-day tests of Baclllus cells ami mixtures of equal con-
1 centratlons of Bacillus and Pseudomonas are presented in

1.. .s
1------
10
Fig. 2~Changs 111compressive strength values from 7- to
2B-day tests of cement mortar cubes oontainlng different
concentrations 01microbial biomass,
phosphate buffer, however, showed a tendenoy 10 decrease
slightly in strength at higher concentrations of cells,
Effects of blomass on compresslve strength of
portland cement mortar cubas
The main objective of lhilj test wns to study the effeet of
the bacterial biomnss, Iive or dead, on the compressive
strength of coment morlar cubes. Table 1 summarizes the 7-
and 28-day compl'essive strenglh values of cubes containing
different concentrations of live and dead forms of Baci/lus
celIs and Uve and dead forms of Bacillus and Pseudomonas
mixtures of equal concentrations. P. aeruginosa has becn
chosen for the study mainly becouBe the organism is ubiqui-
tous and ofteu accumulatcs cxtracellular polysacchadde.
Ovcrall. the cQmpressive strength incrcased in the mortar
cubes Ihot containcd uHforms ofbiomass, The 7-day compres-
sive strength increased as the cell cOllcentratkms incl'eased in
cement cubes regardless of thelr forms. The 28-day strength
test results of cubes with Iive cells showed an increase in COnl-
Fig. 2. It s olear that live Bacillus cells, at lower concentra-
tons, increased the eompressive strength of cement rnortar with
a longer incubatlon periodo Initially, extracellular material pro-
duced by Pseudomonas was expected lo conrlbute to the
strength of cement mortar, The resulta indioate, however,
that hc role of Pseudomonas in strengthening the cement
matrlx is not significant. As shown in Pg. 2 and Table 1, tite
lncrease of'the strength in cubes with the mixture of Baclllus
and Pseudomonas appcars to depend on the ooncentratlon of
Bacillus, not that of Pseudo monas. At lower concentrations,
Bactllus continuos to metabol Ize to induce cal cite precipita-
tion during the lncubation perlod,
The oyeran rend of a decrease in compress ve strength at
28 days might be attributed 10 the behavior ofbiomass wthin
the mortar matrlx, During the initial curing period, the live
cells obtained good nourishment, because the portland ce-
ment mortal' was still porous. Upon cell growth, calcite
would precipitnte 011 the cell surface as well as within the
portland cement mortar matrix. Tllus, the portland cement
mortar became less pomus and permeable. Once mUlly ofthe
pores in the l11atl'ix were pluggcd, the flow of nutricnts atld
oxygell to the baclerial cells stopped and evelltunlIy the cel1s
eithcl' died or turned nto endospores. It i8 also possible tha
as the pH of the cemen! remailled high, cells prematurely
died. As a l'esult, the dead cells simply remained in the mortar
matrix "ud acted as an organic fiber, increasing the compres-
sive slrengLh of the cubes. This explains the behavior of the
compressve strength to rcmain Almos!constant al the age of
28 days in cement mortar cubes prepared with live forms of
both o the mixture of Bacillus and Pseudomonas alld Bacil-
lus alone. The organie mattel' ofthe dead celia would eventu-
ally disintegrute wth age. In a similar fashion, biomass that

6 ACI Materlals JournalfJanuary-February 2001

 11UU ...-""-.-----~~-~_._-------- ----.~-,

1150 1::::1 wltho~I~II>1

W.aW[thC6I1s
S!mplo l 1100
1000 .1. lOSO

! ,~ A " .k.

SalU{lle 2
-----
850

J t.....~\
o
1lQ
OIQOOIMo
100 .L.. -l.~ .._.

I
QUnlll
SI\1lWlc 1 <)'lOlI 3.m 952S
1000 (.',.ck ".I"1t (mm)
Ii.{ {' Porllandll
o A 1 lt ..~~ Fig. 4--Comparson of stiffttess vales of portland c~mellt
\:;;1 !b !b al ab oh ~ 40" 4~ ~ :/$ t:;b mortar beams with cracks 01 dtfferent depths filled with B.
pnsteurll,
Fig. 3-X-ray dtffracon patterns for cement mortal' sam-
pies: Sample I=cast and cured in water; Sample 2-cast ln
phosphate btiffer and cured In Urea-Cacl- medium: (111.1 Table 2-XRD quantltatlve analysls of portland
Sample 3-cast in phosphate buffer containlng D, pasteurn cement mortar samples
and el/red in Urea-Cae! 2 medium. Oligoclese
(Na. Ca) CaJaitcl
Portlanditc Qunrlz AI(AI, Calclle Quartz +
was killed prior to mixing would Herveas an organic fiber in Ca(OH)z 8i02 Si)SI206 CaCO) Oligocluse
the initlal days but dlsintegrnte toward the later curng perl- Sample l' 0,22 0.62 0,03 0,13 0,20
od, making the matrix porous and rcsulting in the reduction
Snmple 2t 0.19 0,61 0,02 0,18 0.29
of compressive strength, Figure 2 supports the notlon that
compressivc strength of portland ,cem~llt mortar contalnng Snmple 3t 0.26 0.54 0,03 0,17 0.30
dead biomass decreases as the cunng tune and concentraton 'SnmplB J: porlland ccmcnt morar mtxcd and curcd In water, ,
of biornass iucrease. ISnmplo 2: portlnnd ccmcnt menar rnlxcd in phosphnte buffer nnd cured tn Urca-
CaCI~ mcctum,
Figure 3 depicts the profiles of XRD patterns of three port- ISamplo 3: portland cemcnt morter mlxed wllh 8, posteurlt suspended in phosphatc
land cement mortar samples. AH cement mortar cubes tested buffer nnd cured in Ur~n.CnCI2 rncdlum.
contained similar proportons of the major cement material,
portlandite, quartz, olgoclase, and calcite, Crystal fractions in
cement mortar samplcs and the ratio of calcite to a total of 811d a bcst-fit line was drawn, (he slope of'which was taken
quartz and oligoclase are included in 'l'able 2. Because varia- as the stiffness valu. Figure 4 provdes a comparson of
tlons of fractions of cement components are inevitable. t s stlffness values for the two depths of cut for both control and
meaningful lo compare the ratio of calcite to quartz and olgo- speolmens wilh bacteria ami sand in their cracks, T~e stiff-
clase, both ofwhich orginate from sand, not from portland ce- ness values of beams filled with sand and bacteria were
mento In the absence of bacteria, the average ralio valne of found to be hgher than the values of the contl"Olspecimens
calcile lo these sand components was 0,20 and 0.29 for the in both cases. As expected, beams with a deeper cut showed
sample cured in water and in Urea-CaCl2 medum, respective" a (ower sfifflless value when compared to thoso with 8 shal-
Iy, In the presence ofbacteria, the rotio from the samplc ~on. lowel' cut. The results from this test showed a significant ,in-
(Bining B, pastel/rU, which WIlS cured in Urea.CaC~ medmlll, crease in the stiffhess ofthe benms with 3.175 mm (0.125 111,)
was 0.30. The resulls indicate that XRD aUl\lysis detected 8n depth of cut. T11epresence of 8, pasfeurU showed more efft:c-
jnsignificant inecease of catcite precipitarian in the cell~laden live remediation in sballowcr cracks Ihan deeper ones, 10-
creasing the stlffness ofbeams by 9.4% with a 3,175 mm cut
portland cement mortar. The compressive strength, however,
and by 4,8% in 9.525 mm cut beams compared to the con-
increased considerably, Although the biomass of cells has
trols. UpOtl physical examination of the specimens. it was
contrbuted to the incl'ease in compressive strenglh of portland lloticed Ihat tlle sand partic10s were held togethel' by the pre-
cement mortal' to somo oxtont, hgh pH and lack of oxygen are
cipitation of calcium carbonate, Even aner the beam was
suspected to have inhibited the growth of micro-organisms,
fractured, the sand particles l'emained toget?er, This ,shows
resulting in an insigniflllllt amount of calcite preclpitadon, that the preeipitation of calcium carbonate tn the vOlds be-
Furthcr investigatiol1 is necessary to understand the precise tween Ihe sand particles had stuck the sand particles togeth-
role ofmicro,ol'ganisms in retation to the ealejte precipitation el' which could in turn mt the crack, It was not possible to
in portland cement matrixes and the compressive strength of adcmately determine the quality of bond between the sand
portland coment mortar.
pal'tic1cs and the surfllce of the cut.
Effects Of micrObiological calcita preclpltatlon In Effects of microbiological cal cite precipitation of
cracks wlth various depths on stlffness values In crack!!; of various depths on compressive strength
port~ndcementmortarbeams values In portland cement mortar cubes
The stiffness values (N/mm) wel'e obtained fOl'all beams In this study, experiments wcre artied out to ,identi~ t~le
cured fol' 28 days. A load"deflection relatiol1ship was plotted effect of the precpitated calcillll1 carbonate Jl1 artifiCial

ACI Materials Journal/January-February 2001 7

I .

12.70 IM$ 23.40

CrackDo~th(mm) Cell Conccntratlon (C<lJl/clIl')

Fig. 5--Increase ofcompressivestrengthof portland cement Fig. 6-Cowpl'essive strengths oj portland cement mortal'
mortar el/hes wtth cracks 01 different depths in presence 01 cubes wfth cracks filled wtth various concentrattons olB.
B. pasteuri. pasteurii,

Tabla 3~Compressiv strength values of portland Tabla 4-Compresslve strength values of portland
cernent mortar cubas wlth cracks of dlfferent cement mortar cubes with cracks containlng
depths remedlated by B. pastur/i various concentratlons of B, pasteurii
Comprcsslvc slren_~lh,MPn Conceutraton Compreuaive streugth, MPa
Sand + water Sand+ cells of oclls, Standard
Standnrd Standard oells/om3 Crack lltng Curad in Average" devlatlou"
Depth, mm Average' devtatlon" Average' devlauon" O None Air 36 0.56
12.70 49 2.27 49 1.84 O Sand +wnter Water 34 1.98
19.05 42 2.99 45 ~.49 Urea-CnClz
0.74
O Sand+wnlcr mdium 42
23.40 21 6.08 34 2.64
'VnlllQS of'uverngc und standard dcvlntlon obtnincd frorn four snmple a. Sand + phos- Uron-C"C~ 2.30
2.6 x 107 40
phate buffer mdium
Sand +phos- Urea-CoCJ:
cracks on the compressve strength in cement mortal' cubes. 5.2 x 107 46 1.53
pbate buffer mdium
Compressive strengths of cement mortar cubes with cracks Sand+ phos- Uroa-CaC~
of different depths are compared in Table 3, in which cracks 2.6 x 108 phute buffer 44 1.37
medlum
filled with bacteria and sand show higher strength values 'Vnlues oft>vom&o nnd standard devlntion obraincd from trlpltcate samplcs,
than those wth sand only. As expected, the overall compres-
sive strength of portland cement mortal' cubes decreased
cracks fllled with different concentrations of cclls. From thc
with an increase in the crack depth, whether micro-organ-
tested microbial concentratious, a concentration of 5.2 x 107
isms were present or not. Differences of the test values be- c0l18por cm3 has been found to induce the mximum com-
tween controls containlng no oells and cell-laden crack pressve strength,
specimens are graphed in Fg, 5. An increase of the strength
in the prcsence of B. pasteurii was 1110stsignificant in the
SEM analysls
cubes prepared with the deepest crack (25.4 mm), whose mi-
To determine whether the increase in compressive
orobial remediation Incrcased the compressive strength by strength of the specimens with bacteria and sand in thelr
approximately 61 % of that of the control. craoks could be auributcd to the mcrobial calcte precipita-
tion, the crack samples with the highest strength vales were
Effects of diffcmmt ccncentratlons of bacteria examlncd undor SEM. Figure 7(1l)is a sCllnning electron mi-
usad for crack remedlatlon on compresslve crograph of the matrix of bacteria-free portland cemen!
strength of portland cement mortar cubas morar. Figure 7(b) through (d) include selected micro-
This stlldy aimed nt investigating the effeet o the vadation graphs of the specimells taken froln the area clase to Ihe
in the bacterial cOl1centration on the compressive strength surface and in the interior of the crack. rhe sample taken
nnd also obtaining an opllnum cOllcentra!ion oflhe bacterial from the arca close to the surface showed calcite cl'ystals
cclls for futme studies. Compl'essive strengths affected by grown all over the sand particles and precpitated between
the presence of B. pasteurli are compal'ed in Table 4. Two of ).
tllem (Fig. 7(b On closer observatioll (lf the arca contain-
lhe thl'ee sets with bacteria showed higher compl'cssive ing dense calcite pl'CClpttion,it wos fOtlnd that the calcium
stl'ength than tlle control specimells. It ia noteworlhy Ihal, carbonatc crystals were well developednear the surface of
among the control groups without cells, the cubcs 0\11'00 in he cmck (Fig. 7(c)). They had distinct and sharp cdges, in-
microbial growlh medium were strongcr than those cured In dicuting a full growth uf lhe ;rystals. As noticed in the figure,
water or left in airo The ionic strength ofUrea-CaCl2 medium calcte cl'ystals are seen with rod-shaped holes, which are
nppeul'ed to enhance the strength of the morlar cubes. Figure 6 presumably the spacc occupied by lhe bacteria. Much less
depicts tlle profiles ofthe compressive strength of cubes with consolidation, however, WIlS observedIn the sample pre-

8 ACI Materials Journal/January-February 2001

 I
Flg, 7-8cannlng electron mlcrographs showing microbto-
lag/cal ca/cite precipita/ion in concrete cracks: (u) matrix 01
portland cement mortar prepared wtthout bacteria, Bar, 100
pm; (b) sample taken near surface area of remediated
crack, showlng dense calcite precipttatton between and 011
surface 01sand particles, Bar, 100 ..tn: (e) enlarged portio n
of ca/cite crystals found in b, showlng calotte crystals with
empty holes once housed by S, pasteuril Bar 10 .m: and
(d) sample token 11'0m tnterior 01 concrete crack showlng lit-
tle calcite precipita/ion, Bar, 100 .$Ir.
pared from an interior area of the crack (Fig. 7(d). The
sparsc growth of calcite crystals in the deepcr arca of the
cracks also supports the results obtained by XRD analysis
that detected lttle increase o calcite precpitation in the
presence of B. pasteurti in the portland cement matrlx,
R. pasteurti is facultatively anaerobic, They are abte to
grow elther aerobically or anaeroblcally. They prefer the
presence of oxygen, Itowevel'. snd are metabolically active
via aerobic rcspil'ation. ft is therefore expected to have more
calcite precipitation in areas (lIoso to the surfllce in crack ro-
mediation. This behavior, evidenced by SEM exuminution,
suggests that microbial remediation is in general more etrec-
tive in shalIow cracks.
CONClUSIONS
The following conclusions are dmwn from the obselVa-
tion5 macle throughout tbe investigation.
1. The presence of chloride ons (en in portland cement
produces a negativo mpaot on the compressive strength on
portland cement mortar. Chloride ions present in microbial
medium should be washed out complotely before beng add-
ed in cement mixture. Sodillm phosphate solution performed
better thsn waler in enhancing tbe strength of portland ce-
ment mortar;
2. At tower conccntrutioDs. he presence of B. pasteul'/I jn~
crellses Ihe oompressive strength of' porttalld cement m011ar
cubes. An ncrease ofmicrobal biomass, as dead forms in par-
ticular, bowcver, reduces the strength of cement mortar cubcs;
3. In au extremely alkaline environment of cement matrix
(greater than pH 12), neither B. pasfellrli Ilor P. ael'uginosa
grow aclively. Therefore, the increase of overalt strength o
mortal' when Ilxed witlt micro-organisms resulled from the
presenee of en adequate amount of organic substance in the
ACI Materials Journal/January-February 2001
matrix duo lo the microblal biomass, but 1101from the prcsence
of calcite induced by morobial growth;
4. Mcrobologicnlly-induoed caleite ls more effective in the
remedlaton of cracks. Resulta obtained from SEM examina-
tion conrm that calcite precipitatcd during microbial growth
111the main substance that ncreases the compressive strength
of cracked mortar cubos; and
5. Microhiologcal remediation 18 more efflclent In shallow
cracks than in deeper cracks primarily because the mcro-or-
ganisms grow more actlvely in the presence of oxygen,
In summary, calcite precipitated by B. pasteurll 15effec-
tlve in crack remedlation, but not in strength enhancernent of
the cement mortar mixture. The importance of this study is
[O use micro-organisms, which are found eommon in soil, in
crack remediation of concrete. In addtlon, this new microb-
al sealant containing calcte s not onLyenvironmcntally safc
but also persists in the natural cnvironment for an extended
period of time. Purther detailed lnvestigaton, however, ls
necessary for ldenti fication of the specifc basis for tite qual-
ity performance of mlcroblal sealant,
ACKNOWLEDGMENTS
The sfUdy wss supported by the National Scleneo Foundation (CMS-
9802121).Tite uurhorswould likc lo thank the I:ltlllineering and Mltllnll Exper-
ment SlatiQn lit SD$M&;T for providing technlcal asslstancc in XRD and SBM.
CONVERSION FACTORS
I in. - 2.54 cm - 25.4 mm
1 lb '" 0.45 leg "" 4.45 N
1 MPa '" 145 psi
I mL = 0.061 it\?
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9