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98-M1
Thls paper describes an lnnovative blotechnology uttttzing micro- electrn mioroscopy (SEM),1 In the authors' studes on sand
btologlcalty-lnduoed mineral prectpitation lar concrete remedia- consolldetion, morobiologically-lnduced calcite constituted
(/011. Ca/cite precipita/ion lnduced by Baoillus pasteurii was more than 30% of oonsolidated materials in which microbial
studled In two types 01portland cement mortar speolmens: olle calcite pluggng was highly selectivo. The efficiency of mi-
prepared from mlxlng wlth mlcro-organtems, and the other wlth
crobial plugging was affected by (he porosity of'the mdium,
simulated cracksfilled wlth mtorobtal mixtures. The study showed
that there was a significant lncrease tn compressive strength of the the number o cells present, and the total volume of'nutrient
portland cement menar cubes contalnlng lower concenirations of added, In crack rernedlation of granite, un average crack
ttve cells. Compressive strengths of the el/bes containlng llve 01' width of 2.7 mm (0.106 in.) and El mixture of slica fume
dead cell mass, however, decreased as cell conoentrattonsand cur- (10%) and sand (90%) showed the highest compressve
illg lime lncreased, suggestlng the tnterference of'mortar inlegl'ly strength in microbial remediation of granite.4 Barler, Kant-
by biomass. Cracks fllled wlth bacteria and sand demonstrated a zas el 81.3 reponed that sand oonsolidation by B. pasteurli re-
stgnifcant increase /11 compressive strength and stljJnes$ values duced porosity by up to 50% and permeabilty by up to 90%
when compared IVI/h those wuhout cells. Soanntng electron micro- in thc arcas whcre the cementaton took place.
graphs tdentlfted that mtcrohtotogtcat cale/le prectpitatlon The concept of using mcrobial mineral precpitatfon in
occurred matnly clase 10 the ~lIrface reasuf the crack, where a
dense growth of ca/cite crystals embedded with cells was observed. theremediaton of cracks has been applied in mcroblal en-
hanced o! recovery (MEOR), which has beneflted the oil in-
Keywords: oalcte: compresslve 8lrenglh; cracking: s(iffnl)!)s.
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
INTRODUCTION
partcles but also by adhering to the avalable surfaces
In natural environments, chemical CnCOJ preciptaton
(Ca2+ + coi- ~ CaC03.}) is accornpanied by biological through extracellular organlo compounds, Biotechnology of
~TocesseB.both ofwhich ofien OCCUl' simultaneously or sequen- microbial crack remediation is therefore equalLy applicable
tullIy, An endospore-forming son micro-organism, Bacillus to those ofman-made structures, including concrete.
pastCl/rii, participares in enlcite precipitatiol1 in the cllviroll N
Cracking of concrete is a common phenomenon. Wthout
ment by producing the urease cnzyme. Ureas e (urea ami- immediate and proper treatmel1ts, cracks in concrete struc-
dohydrolase, E.C. 3.5.1.5) catalyzos urea to produce CO2 tures tend to expand further and eventually require costly re-
and ammonia, I'esulting in an increa:;t: ofpH in the surround- pairo Even though It is possihle to reduce the oxtent of
ings where mineral ions (Ca2+ and COl~) precipitate as cracking by available modern technology, remediatioll of
CaC03 crncks in concrete has been the subject of research for many
Microbial CaC03 precipitation is a compLex mechanism. years, Tllere are a large number of products availnble com-
?u~ stu.dy O? kin:etic details has indicatod that calcite precip- mercially foc repairing crack s in concrete: structural epoxy,
resins, epoxy mortar, and otller syl1thelio mixtures,s Current-
ItatlOn I~ a tunctlo!, ofthe cell ~?ncent.ration. ionie strongth,
and pH In the medlum,l In addlllon, rnlcro-organisms whose Iy, these type.'l ofsynthetic filler agents are extcnsively used
Ilet cell surface charge i5 neJl.ative also draw cations from the in concrete crack ropa ir. Because cracking in concrete stl'UC-
tures continuos over a long pedod of time, this type of one-
envronment, including Ca ,to deposit 011 the cell surface.
The following equations surnmarize possible biochemical time, quick remedy ShOllld also be applied repeatedly as
l'eactions in a medium to preciptate CaCOJ al the cell sur- needed.
face that serves as a nucleation slte 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-
Ca2+ + Cell ~ Cell-C(+ (1) stantLy oocurs in natural environments, Specifically, micro-
bologically-nduced caleite is envirol1mentally innocuous,
Cell-Ca2+ + COl 2- -t Cell-CaC03.} (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/,
A llovel tcchnique for the remediation of damaged struc-
whosc optimum pH for growth is around nine. B. pasteurlt,
~ral fo~mat?ns has been developed by employing a selec-
tlvo IDlcroblal plugging process in which microbial however, has an ability to produce the endospore, a dormant
metabolic activities promote precipitation of calcium car- form ofthe cell, to endure extreme environment. Our prelim-
bonate in the fonn of calcite?,3 As a microbial sealant
CaC~3 exhibited its positive potential to selectively consoli~ .fel I,{,,(erlal. JOllntal, V. 98. No. 1, anlUlry-Fcbrutry 2001.
d~te slmulaled fractures and surface fissures in granites,2 Sig- 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
ntfican~e of l~icrobial calcite precipitation was quantified by 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!
x-ray dlffractton (XRD) analysis and visualized by sconning Material. JOllnral if received by August 1,2001.
deflecton relatlonshps were recorded using a data acquisi- Cell Concenlraton (cc1ls/om)
tion system, . Fig. J--Effect$ 01saline and phosphate buffer 0/1 compres-
slve strength 01 portland cement mortar cubes contatnlng
Ocmpreaslve strength testlng: slmulatsd cracks vario us concentrations o/B, pasteurii,
wlth varlabte depths In portland coment mortar
cubas
Portland cement mortal' cubes of a dimcnsion of 50,8 x SEM examlnatlon
50.8 x 50.8 111m(2 x 2 x 2 in.) were cast according to the Fractlons of remediated crack s were prepared for SEM
ASTM C 305-<)4 procedure. The specimens were cured in analyss as publlshed prevously.' Samples were coated
water for 28 days and then 10ft exposed to ni!'. TIJe cubes with carbon prior to cxamination by SEM. which was
were provded with a cut to slmulate a crack. The width of equipped with an energy-dlspersive x-ray analyzer for ele-
the cut was kept at nn average of3.175 mm (0.125 n.) and the mental analysls.
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 XRD analysis
five were used as control and the remaning vc for test spec- Samples subjected to XRD analyss lncluded Ihree pOl't-
mens with bacteriu, l'espectively, The cracks in the control land cemenl mortal' cubes: 1) mixed snd cured in water; 2)
speeimells were flled with natural sand )!lssing through the mixed in phosphate buffer and cured in Urea-CaCl2 medum;
U.S. standard sieve No. 16 and l'olalned 011 the U.S. standard and 3) mixed wilh B.pasteurli suspended in phosphate buff.
sieve No. 40) and water. The cracks in the other Ove speci- er and ClIred in Uren-CaCl2 medium. Eacll sample was
mens were filled wlth sand and bacteria, to n final cOl\centra~ cl'ushed and pulverizad to an average partiole size of less
tion of3.8 x 109 cells per cm3 Both sets ofcubes were cUl'ed than 10 microns, nud thCll mounted onto n gloss fiber filter
in Urea-CaC12 broth fOi' 28 days, The mediutll was replnced using a tubular aerosol suspension chamber (TASC). The
after 14 days, The specitnens \Vere tested fOl' their compres- mass absorption coefficient of the mler was determined pl'ior
sive strengths using a compresslol1-lcsting machi ne. to uso. Then the moss absol'ption coefficient of tite sample
was detel'lnined by x-tay transmission. Tite XRD pnttern was
Compresslve strength testlng: slmulated craoks In obtained by scannng from 5 to 60 degrees, 2 theta using a
portland mortar cubos wlth variable cell vel'tical x-rny di ffraclometer. The components of the sllInple
concentrattons wel'O identified by comparing them wilh standards estab-
Porttand cement mMar cubes of a dimcnsion of 50.8 x 50,8 Iished by the Internotional Ccnter fol' Diffractioll Data. The
x 50.8 mm (2 x 2 x 2 in.) were cast as per the procedure previ- quunHtative analysis was perfofmed USillg quantitative XRD
ously described, The cubos were provided with a cut to SilllU" analysls.8
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- RESUL TS ANO DISCUSSION
Iy. A total of 1811lortar cube specimells, each wth a cut of the Effects of type of buffer solutlon on compresslve
game size, were prepared for tlle test. AH specimens, including strength of portJand cement mortar cubes wlth
three scts of controls, were prcpared in triplicate. Three speci- various concentratlons of bacteria
mens in tho firat set of controls contnined 110 I1l1ingand were Compressive strength vaIues of cubes prepal'ed in phos"
keptex.posed to airo Cracks in three specmens ofilie second set phate buffer and salino solutOll are compal'ed in Fg. 1, Port-
wcre filled with Il mixture of sand nnd water and cured in watel. Isud coment mortal' cubes prepared in saline have ShOWll an
Cracks in three specimens of the third set wel'e t1lled with a ovel'all deorease in compressive strength in the presenco of
mixture of salld and water and cured in Urea-CaClz mediuOl. cells. rhe decrease in compressi ve strength of he cubes con-
Microbial plugging ofthe concrete cracks was eXllmlned with tailling satine may be due to the presence of cllloride lons in
three different concen!ratiOllsof B, pC1SJUl'ii mixed with salld, the solution, which is kl10wn lo weaken lhe integrity ofthe
Final concentratiotls ofcells \Vere 2.6 x (O7, 5,2 x 107, and 2,6 cernen! matrix,9 The compressive strength of mortar cubes
x 108 celia per cm3 AH seIs of specimens conlaining cells were prepared in the phosphate buffer was consistently highel'
cured separately in the medium. Tlle medium was changed af- than the stl'engUl of saline-prepared cubes at various concen-
ter 14 days, The cubes were tested fol' theit compression trations of cells laden in the cube. Thus, phosphate buffer so-
strengths after 28 days. lution was uscd in al1 the other tests. The cubes prepated with
1.. .s
1------
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,
10 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
Fig. 2~Changs 111compressive strength values from 7- to Bacillus, not that of Pseudo monas. At lower concentrations,
2B-day tests of cement mortar cubes oontainlng different Bactllus continuos to metabol Ize to induce cal cite precipita-
concentrations 01microbial biomass, tion during the lncubation perlod,
The oyeran rend of a decrease in compress ve strength at
phosphate buffer, however, showed a tendenoy 10 decrease 28 days might be attributed 10 the behavior ofbiomass wthin
slightly in strength at higher concentrations of cells, the mortar matrlx, During the initial curing period, the live
cells obtained good nourishment, because the portland ce-
Effects of blomass on compresslve strength of ment mortal' was still porous. Upon cell growth, calcite
portland cement mortar cubas would precipitnte 011 the cell surface as well as within the
The main objective of lhilj test wns to study the effeet of portland cement mortar matrix. Tllus, the portland cement
the bacterial biomnss, Iive or dead, on the compressive mortar became less pomus and permeable. Once mUlly ofthe
strength of coment morlar cubes. Table 1 summarizes the 7- pores in the l11atl'ix were pluggcd, the flow of nutricnts atld
and 28-day compl'essive strenglh values of cubes containing oxygell to the baclerial cells stopped and evelltunlIy the cel1s
different concentrations of live and dead forms of Baci/lus eithcl' died or turned nto endospores. It i8 also possible tha
celIs and Uve and dead forms of Bacillus and Pseudomonas as the pH of the cemen! remailled high, cells prematurely
mixtures of equal concentrations. P. aeruginosa has becn died. As a l'esult, the dead cells simply remained in the mortar
chosen for the study mainly becouBe the organism is ubiqui- matrix "ud acted as an organic fiber, increasing the compres-
tous and ofteu accumulatcs cxtracellular polysacchadde. sive slrengLh of the cubes. This explains the behavior of the
Ovcrall. the cQmpressive strength incrcased in the mortar compressve strength to rcmain Almos!constant al the age of
cubes Ihot containcd uHforms ofbiomass, The 7-day compres- 28 days in cement mortar cubes prepared with live forms of
sive strength increased as the cell cOllcentratkms incl'eased in both o the mixture of Bacillus and Pseudomonas alld Bacil-
cement cubes regardless of thelr forms. The 28-day strength lus alone. The organie mattel' ofthe dead celia would eventu-
test results of cubes with Iive cells showed an increase in COnl- ally disintegrute wth age. In a similar fashion, biomass that
! ,~ 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
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-
3. In au extremely alkaline environment of cement matrix 8. DIIvla. 13.L., ''Q1I6n!ttvc Phnae Al1lIlysiswi!h Rcfe~ Inlertslly RaIjos,"
N(1//M(t/ [mllMe / Slmldards alkl TecJmolagy SjJ<JC/a/Pllb/lcalioll 846,P~d
(greater than pH 12), neither B. pasfellrli Ilor P. ael'uginosa . inga oftho Intcmational Conference on Accumcy In Powdet' Dltremctlot\ 11,Galt1\
grow aclively. Therefore, the increase of overalt strength o ernburg.Md., May 1992,pp. 1-16.
mortal' when Ilxed witlt micro-organisms resulled from the 9. llerke, N. S.: l'feifer, 1). W.: amI Weil, T. O.. "fuMlioll agalnsl Chloride
presenee of en adequate amount of organic substance in the Induced Gorrosioo," COllcmleTlllemallollaI.V.10.No. 12,Dcc. 1988,pp. 45-55.