83

i) Professor, Tokai University, Shizuoka, Japan (fukue＠scc.u-tokai.ac.jp).ii) ditto.iii) ditto.

The manuscript for this paper was received for review on September 28, 2009; approved on October 1, 2010.Written discussions on this paper should be submitted before September 1, 2011 to the Japanese Geotechnical Society, 4-38-2, Sengoku,Bunkyo-ku, Tokyo 112-0011, Japan. Upon request the closing date may be extended one month.

Fig. 1. Carbonate precipitations by the catalysis due to the enzymesproduced by ureolytic bacteria

83

SOILS AND FOUNDATIONS Vol. 51, No. 1, 83–93, Feb. 2011Japanese Geotechnical Society

CEMENTATION OF SANDS DUE TO MICROBIOLOGICALLY-INDUCEDCARBONATE PRECIPITATION

MASAHARU FUKUEi), SHIN-ICHI ONOii) and YOSHIO SATOiii)

ABSTRACT

In this study, microbial precipitation of carbonate was observed using high microbial urease activity, and it wasfound that the ratio of Mg/Ca aŠected the types of crystals produced. Without Mg2＋, calcite was produced using onlyCaCl2, while the presence of Mg produced Mg-calcite, magnesite and/or possibly dolomite of round, spherical or ˆ-brous shapes, depending on reaction time, pH and Mg/Ca ratio. The carbonate produced contributed to the develop-ment of cementation for sands. The presence of Mg showed a relatively strong cementation of the carbonate.

Key words: carbonate, cement, microbes (IGC: K2/K6/K14)

INTRODUCTION

It was shown that marine deposited soils have cementa-tion due to carbonate (Fukue et al., 1999). The studyshowed that the intensity of the cementation due to car-bonate content was very strong. The analysis showed thatfor silty clays, one percent carbonate will increase the un-conˆned compressive strength by about 60 kPa. About a1 m thick sand rock layer was found in the Narita sandformation which was formed by the precipitation of car-bonate. The sand rock layer had an unconˆned compres-sive strength of about 19 MPa and contained 24 percentcarbonate by mass.

Carbonate is a salt or ester of carbonic acid, containingthe chemical group CO3. Typical carbonates are calciumcarbonate (calcite, aragonite and vaterite as minerals),CaCO3, magnesium carbonate (magnesite as a mineral),MgCO3, iron carbonate (siderite as a mineral), FeCO3,copper carbonate, CuCO3, etc. Thus, calcite, aragoniteand vaterite are calcium carbonates but they have diŠer-ent crystalline forms. In nature, there are many aspectsconcerning carbonates. For example, dolomite mineral(CaMg(CO3)2) can form Ca and Mg carbonate. The for-mation of dolomite is geologically complicated. In thisstudy, carbonates containing Ca and/or Mg are mainlydiscussed.

The artiˆcial precipitation of calcite in soils has beenstudied to examine the clogging of sand (Hart et al., 1960;UpdegraŠ, 1982; Macleod et al., 1988; Stocks-Fischer etal., 1999; Bang et al., 2001, Bachmeier et al., 2002; Ferriset al., 2003; Fujita et al., 2008). Stocks-Fischer et al.(1999) have investigated the microbiological remediationof concrete cracks and demonstrated the improvement of

compressive strengths of cement mortar cubes in thepresence of microorganisms.

Most of the studies on microbial cementation werebased on urease activity. Urease activity is the hydrolysisof urea due to an enzyme produced by microorganisms orplants such as jack beans. It is also noted that the enzymeinduces the hydrolysis by catalysis. The hydrolysis ofurea produces CO3

2－ which can react with Ca2＋. As aresult, calcite is produced. The process is illustrated inFig. 1. Therefore, up to now, many studies have been fo-cused on microbes which produce urease enzyme. The mi-crobes are called ureolytic bacteria. The importance ofthe hydrolysis of urea is that the ammonium produced bythe process of the hydrolysis increases the pH. It is knownthat calcite precipitation occurs under a pH value higherthan 7.

84

Photo 1. Calcite crystals produced by microbial urease

Fig. 2. The nucleotide sequence of 16S ribosomal DNA of NO-A10 strain used in this study

84 FUKUE ET AL.

Calcite precipitation induced by ureolytic bacteria wasstudied by Ciurli et al. (1996), Le M áetayer-Levrel et al.(1999), Tianoa et al. (1999), Ferris et al. (2003), Whi‹n etal. (2007), De Muynck et al. (2008), Lian et al. (2006) andJimenez-Lopez et al. (2008). These studies can be catego-rized into urease activity, calcite precipitation and theirapplication to engineering purposes. Note that calcite is atypical calcium carbonate and is more stable than othercalcium carbonates, such as aragonite.

Many studies concerning calcite precipitation havebeen performed in the laboratories. The diŠerences be-tween the conditions in the laboratory and under naturalconditions are as follows;a) duration of the chemical reactions,b) presence of impurities, andc) di‹culty in controlling pH.

In the laboratory, pure calcite can be created by inject-ing ureolytic bacteria into a CaCl2 solution, as shown inPhoto 1. The details are described in terms of urease ac-tivity in Section Experimentation. However, it is doubt-ful that this type of crystal can make a strong cementa-tion between soil particles. From the authors' ex-periences, fast reaction is not important for the cementa-tion between particles concerned. This property of calcitecan be improved by adding impurities which can changecrystal forms as found in nature (Moore, 2001). For ex-ample, the presence of Mg can create Mg-calcite whosecrystals are diŠerent from those of calcite. Folk (1974)showed that a small amount of Mg can interfere with thegrowth of calcite crystals and can help crystal growth into

the diŠerent direction. The amount of Mg used for thecrystals is less than 4z. Larger amounts of Mg possiblycontribute to the formation of dolomite or magnesite, be-cause of the high Mg/Ca ratio. Dolomite (CaMg(CO3)2)and magnesite (MgCO3) can resist against acid more thancalcite (Fukue et al., 2010).

In this study, microbiologically-induced calcite, Mg-calcite, dolomite and magnesite were observed using highactivity ureolytic bacteria. Furthermore, the cementationeŠects due to calcite and Mg-calcite were also examinedusing the ureolytic bacteria. The main purposes of thisstudy are to examine the capacity of the ureolytic bacteriaand the eŠect of impurities on carbonate precipitation forengineering applications.

MATERIALS AND EXPERIMENTALPROCEDURES

MicroorganismsTo isolate bacteria which have strong urease activity,

boring core samples were collected from eleven sites atdiŠerent locations throughout Japan. First, diŠerent con-centrations of CaCl2 or Ca(OH)2 were used to screen thebacteria in the soil samples. This approach was used toisolate the bacteria which are tolerant to Ca ions. Forthis, approximately 10 g of collected soil samples werekept for a few days in CaCl2 or Ca(OH)2 solution. Afterscreening, living strains were cultivated on an agar.Throughout these procedures, about 150 strains were iso-lated and their capacity for urease activity was examined.Finally, one of the most active strains, Sprosarcina sp.which is a new species and an alkalophilic strain waschosen. This strain was named as NO-A10, where ``NO''means the name of location for the sampling site,Niwase, Okayama City, and ``A10'' shows alkalophilic10th strain isolated.

Figure 2 shows the nucleotide sequence of 16Sribosomal DNA of NO-A10 strain. DNA consists of twolong chains of nucleotides twisted into a double helix andjoined by hydrogen bonds between the complementarybases adenine (A) and thymine (T) or cytosine (C) andguanine (G). The sequence of nucleotides determines in-dividual hereditary characteristics, which can be used toidentify and specify the species of the strain. A compari-son between NO-A10 and Bacillus pasteurii which arerecognized as ureolytic bacteria showed an accordance of

85

Fig. 3. Growth curves of NO-A10 strain and NO-N10

Fig. 4. A calcite-acid reactor to determine calcite content in soils

Fig. 5. Relationship between CO2 gaseous pressure and calcium car-bonate used as a calibration

85MICROBIAL CEMENTATION BY CARBONATES

93z (632/673).In this study, NO-N10 which is a parent strain of NO-

A10 was also examined. The two strains used in this studywere cultivated with EDC (electron donor compound) atpH 8.6. The EDC cannot be used as a usual culture medi-um but can be used for an application, because the EDCis more economical than the usual culture medium suchas nutrient medium. It is noted that the alkalophilic NO-A10 strain should be cultivated at pH higher than 7. Forcomparison, NO-N10 which is ureolytic was also used inthis experiment. NO-N10 is the same species but a diŠer-ent strain from NO-A10. NO-N10 strain can be cultivatedunder a pH range of 6.5 to 8.0.

The growth curves of both strains using proper culturemediums are shown in Fig. 3, where the vertical axis indi-cates the concentration of bacteria in terms of the absor-bance of light, i.e., optical density at a wave length of 600nm. Note that 1O.D.600 empirically corresponds to 8×108 cells/mL. Figure 3 shows that NO-A10 strain shows arelatively low growth rate, in comparison to NO-N10.However, this does not necessarily mean that the ureaseactivity of NO-A10 strain is weaker. The urease activityof NO-A10 is much higher than NO-N10, as describedlater.

ExperimentationMeasurement of carbonate content

The carbonate content can be measured using the dis-solution reactions of carbonates with acid. The carbonatecontent was determined by measuring the CO2 gaseouspressure produced from the following reaction;

CaCO3(s)＋2H＋ªCa2＋＋H2O＋CO2(aq) (1)

Subsequently, according to Henry's law, the dissolvedcarbon dioxide will be in equilibrium with the gaseousCO2.

CO2(aq)ªCO2(g) (2)

To investigate the reaction, a calcite-acid reactor wasused (Fukue et al., 1999). The device is schematically

shown in Fig. 4. The device consists of a reactor cham-ber, pressure meter and valve for the exhaust gas.

The calibration curve obtained using calcium car-bonate agent is shown in Fig. 5. The carbonate content Cis then deˆned as:

C＝carbonate

dry mass of soil×100(z) (3)

The hydrochloric acid concentrations used were 0.05,0.1, 0.5, 1.0, and 3.0 M. The experiment was carried outat 259C using an incubator. Carbonate samples of 0.1 gwere used and the CO2 gaseous pressure produced fromthe reaction was measured over time.

Urease activity in the presence of calcium ionsBoth strains, A10 and N10, were cultivated with a cul-

ture medium under the same conditions, except for pH.The cultivated bacteria were centrifuged to separate themfrom the medium. After the supernatant was removed, acertain volume of 5z NaCl solution was added to thebacteria. This is called the bacterial solution (B solution)in this study. Urea and calcium chloride solution with asimilar molar concentration was prepared using thebuŠer solution (10 mM NH4OH＋NH4Cl). This is calledthe reaction solution (R solution). It is noted that for theR solution, Mg ions can also be added. The concentra-tions of urea and CaCl2 ranged from 0.8 to 3.0 M. At theˆnal stage, the same volumes of the B and R solutions

86

Photo 2. Experimental setup for the microbial cementation of sands

Fig. 6. Drawing of the experimental setup

86 FUKUE ET AL.

were mixed. Therefore, the concentrations of urea andCaCl2 ranged from 0.4 to 1.5 M. A 5 mL aliquot of B andR solution was put into 5 mL glass test tubes and theamount of carbonate (calcite) precipitated with time wasmeasured by the following procedures.

After a certain time, the liquid in the test tubes wasˆltered with a ˆlter (1 mm). The ˆlter with solid residueswas dried in an oven (at 1109C). The carbonate (calcite inthis case) amount in the liquid was determined by sub-tracting the mass of the ˆlter from the total weight of theˆlter with carbonate. Most of the precipitated calcite ad-hered to the wall of the tube. After the liquid was re-moved, the tube was dried in an oven (at 1109C) until themass became constant. The precipitated calcite on thewall was determined by subtracting the mass of the tubefrom the total dry mass of the tube with calcite.

In this case, the precipitation of carbonate occurs asfollows:

CO(NH2)2＋2H2Oª2NH4＋＋CO3

2－ (4)Ca2＋＋CO3

2－ªCaCO3æ (5)

The reaction is promoted by catalysis due to the ureaseenzyme produced by the bacteria. To examine magnesiteprecipitation, the preparation and procedures used aresimilar to the case of calcite precipitation. In this case, thefollowing reaction occurs.

CO(NH2)2＋2H2Oª2NH4＋＋CO3

2－ (6)Mg2＋＋CO3

2－ªMgCO3æ (7)

Furthermore, under the presence of Mg and Ca ions, car-bonate precipitation may occur in a more complex man-ner. However, the reaction is ideally as follows:

CO(NH2)2＋2H2Oª2NH4＋＋CO3

2－ (8)1/2Mg2＋＋1/2Ca2＋＋CO3

2－ª1/2MgCa(CO3)2æ (9)

The product is called ``dolomite'' which is also a car-bonate. Reactions (6) to (9) were examined using bothMgCl2 and CaCl2.

Examination of microbial carbonate precipitation underdiŠerent Mg/Ca ratio

As a variation of reactions (8) and (9), experimentswere performed using diŠerent mixing ratios of Mg/Ca(0:10, 1:9, 2:8, 3:7, 5:5, 6:4, 7:3, 8:2, 9:1 and 10:0). If it isnot speciˆed, Mg/Ca means the molar ratio of concentra-tions. Two sets were prepared for diŠerent time dura-tions. In this test series, the ˆnal solution with a volumeof 5 mL was used for the precipitation experiment. Basi-cally, the same volumes of B and R solutions were pre-pared individually, and the two types of solutions weremixed just before the experiment starts. At 72 hours andone week after the experiment started, the concentrationsof precipitated carbonate were determined in the mannermentioned earlier.

Examination of cementation strength of sandThe biologically-produced cementation due to car-

bonate was examined using the B and R solutions. Themixed solution was applied to the sand specimens consist-

ing of sand layers in acrylic pipes. The strain, NO-A10used was cultivated in the EDC solution (10 g/L). Forthis experiment, the mixture of 0.5 M MgCl2 and 0.5 MCaCl2 and 1 M CaCl2 were used to produce carbonates inthe sand layers. To maintain a high pH, the buŠer solu-tion (10 mM NH4OH＋NH4Cl) was used. The concentra-tion of other additives such as urea was also 1 M.

The experimental setup for biological cementation ispresented in Photo 2. Sand specimens were prepared inacrylic pipes with an inner diameter of 43 mm and aheight of 180 mm, as shown in Fig. 6. Coarse sand(0.5–2.0 mm) layers with a thickness of about 20 mmwere set at both the top and bottom of the column, andˆne sand (0.3–0.5 mm) layer with a thickness of 90 mmwas set between the two coarse sand layers, as shown inFig. 6. Both the sands were settled under water and werein a loose state. The void ratio of the coarse sand was0.81, while it was 0.93 for the ˆne sand. Paper ˆlters werelaid between the coarse and ˆne layers.

A 150 mL solution involving 1 M-urea, Ca2＋ and/orMg2＋, the buŠer solution (10 mM) and the strain (NO-A10) were inˆltrated from the top of coarse sand in theacrylic pipes. The features and test conditions of speci-mens from Nos. 1 to 6 are presented in Table 1. In Table1, ``applied reagents'' means applied metal ions, Mg

87

Table 1. Curing conditions during precipitation of carbonates

No. Appliedreagents

Totalconc.(M)

Ca/(Ca＋Mg) Mg/CaSpecimenvolume(cm3)

1 porevolume(cm3)

Flowvolume/onetime (mL)

Timesof ‰ow

1 Mg＋Ca 1 0.5 1 130.6 60.4 150. 1

2 Mg＋Ca 1 0.5 1 130.6 60.4 150 2

3 Mg＋Ca 1 0.5 1 130.6 60.4 150 3

4 Mg＋Ca 1 0.5 1 130.6 60.4 150 4

5 Ca 1 1 0 130.6 60.4 150 2

6 Ca 1 1 0 130.6 60.4 150 3

Fig. 7. Microbial precipitation rates of calcite

87MICROBIAL CEMENTATION BY CARBONATES

and/or Ca. ``Mg＋Ca'' indicates that both Mg and Cawere applied, while ``Ca'' indicates calcium only. Theconcentration of the metal solutions used is 1 M (mol/L).Ca/(Ca＋Mg) and Mg/Ca show the respective ratio.Each solution was allowed to be drained initially from thebottom. The drain faucet was turned oŠ when the solu-tion level reached the surface of the top sand layer. Thespecimens were left for 24 hours. Except for specimenNo. 1, the pore liquids were replaced with new solutionsin a similar manner. The solution was poured from thetop of the sand specimen while the drain faucet wasturned on. The faucet was turned oŠ again when thepoured volume reached 150 mL, where the pore volumeof the sand specimen was approximately 60 mL, as indi-cated in Table 1. The volume equal to the pore volume iscalled one pore volume (1 PV). Two pore volumes istherefore a double portion (2 PV). Since 1 PV-solutioncan remain in the sand specimen, the carbonate precipita-tion can be 1 M times 1 PV. It increases proportionallywith the time of ‰ow.

The experimental conditions shown in Table 1 were setto examine the following items.a) EŠects of Mg for cementation development,b) EŠects of the time of ‰ow on the strength,c) Strength characteristics of calcite and Mg–Ca car-

bonate, andd) Dissolution characteristics of calcite and Mg–Ca car-

bonate.The pH value of the drained solution was 6.7 after 24

hours, though the initial pH value was 9. This may be dueto the buŠer capacity of sands (Yong et al., 2002) and theeŠect of the products of the chemical reactions. After 48hours from the beginning, the pore liquids were replacedagain with new solutions for specimens Nos. 3, 4 and 6.The pore liquid of No. 4 was replaced again after 72hours. The specimens were kept for about one month.The strength and carbonate content on the sand speci-mens were measured, as described later. Note that theoriginal carbonate content of the sands used is negligible.

RESULTS AND DISCUSSION

Precipitation Rate of CalciteCalcite precipitation was examined using diŠerent con-

centrations of urea and CaCl2 in the test tubes. The initial

concentrations of urea and CaCl2 were kept the same, be-cause 1 M-urea produces 1 M calcite, as indicated byreactions (4) and (5). The temperature was maintained at209C. The microbial precipitation of carbonate initiatedby NO-A10 was plotted against the curing time, as shownin Fig. 7. The legend shows the type of strain and the con-centration of CaCl2 solution used. For example,``A10–0.4 M'' means that the type of strain is NO-A10and the CaCl2 concentration is 0.4 M. For a comparison,the cases of NO-N10 are also shown. Although some bac-teria lose urease activity under the presence of Ca ions(Wi‹n, 2004), NO-A10 showed an ideal reaction, i.e.,100z production of calcite. Therefore, the precipitationrate of calcite by NO-A10 in Fig. 7 demonstrates directlythe urease activity, i.e., the hydrolysis of urea under thepresence of Ca ions. Figure 7 shows that NO-A10 showeda higher (faster) urease activity even for 1.5 M CaCl2. Onthe other hand, the carbonate precipitation by NO-N10 isrelatively slow. This means that the urease activity byNO-N10 is relatively low. Figure 7 also shows that thereaction rate by NO-A10 was almost independent of theconcentrations of CaCl2 and the reaction was completedwithin 25 hours for any concentration of CaCl2 up to 1.5M, while the reaction by NO-N10 is not completed after100 hours. Thus, NO-A10 demonstrated a very highurease activity as well as precipitation rate of calcite.

Precipitation Rates of Carbonate with MgThe precipitation rate of carbonate with Mg was exam-

ined using the mixture of 0.5 M MgCl2 and 0.5 M CaCl2.

88

Fig. 8. Microbial precipitation rates of Mg–Ca carbonate and magne-site

Photo 3. SEM photographs of Mg–Ca carbonate at diŠerent reaction time

88 FUKUE ET AL.

The concentrations of urea and buŠer reagents are 1 Mand 10 mM, respectively. The precipitation rate of mag-nesite was also examined using 0.5 M-MgCl2 and 0.5 M-urea. The precipitation of carbonates was observed using5 mL solution in the test tubes. The amount of theprecipitated carbonates was determined by the methodused for the calcite precipitation.

The precipitation rates of carbonates are shown in Fig.8. The concentrations of MgCa(CO3)2 and MgCO3 weremeasured by the method used for calcite shown in Fig. 7.The ultimate amount of Mg–Ca carbonate producedfrom the mixture of 0.5 M MgCl2 and 0.5 M CaCl2 does

not reach 1 M, but was approximately 0.75 M. It seemsthat the presence of Mg inhibits slightly the carbonationreaction. However, the precipitation rate is similar to cal-cite precipitation.

The products using the mixture of 0.5 M-MgCl2 and0.5 M-CaCl2 were observed with time. The SEM photo-graphs of the products are shown in Photo 3. The shapeof the products is quite diŠerent from the calcite shown inPhoto 1 and varies with time, as shown in Photo 3. Theprecipitation occurred within 24 hours, and the shape wasˆrst round (Photo 3, D-1). In 9 hours after the experi-ment started, the products grew slightly, and the shapechanged a little (Photo 3, D-3). However, at 33 hours,the rounded products started bursting (Photo 3, D-5).After bursting, the ˆbrous products covered the original-ly rounded products (Photo 3, D-7). The shape maychange until the most stable crystal is obtained, as a resultof crystallization or re-crystallization under a pH change.The carbonates produced in this experiment contained0.25 M Mg and 0.5 M Ca. The Mg/Ca ratio is 0.5. Dolo-mite formed in nature is the most stable when the Mg/Caratio is around 0.5, which is known in the skeletal struc-ture of organisms, such as the calcareous algae Gonioli-thon (Chilingar, 1957). The reaction although may nothave completed, continues to be more stable. The authorsconsider the ultimate products to be dolomite, howeverthis may take extended periods of time.

It was conˆrmed that magnesite crystals are producedusing 0.5 M-MgCl2. The concentration was up to 75z of

89

Fig. 9. EŠects of Mg/Ca ratio for precipitation of carbonates

89MICROBIAL CEMENTATION BY CARBONATES

0.5 M at 68.5 hours, but after that, hydration occurs (Fig.8).

EŠects of Mg/Ca Ratio on Precipitation of CarbonateEŠects of Mg/Ca ratio on precipitation of carbonate

were examined to determine the morphological aspects ofcarbonate, i.e., transform from calcite to magnesitethrough dolomite. The experimental procedures were al-ready described. The amount of precipitates was meas-ured in 72 hours and one week after the experiment beganin a manner similar to that previously described.

In Fig. 9, it was assumed that all calcium ions appliedwere used to produce carbonates, i.e., calcite, Mg-calcite,dolomite and/or Ca-magnesite. In a geological study,Faust (1949) concluded that the combination of calciteplus magnesite does not occur in nature, and the productsare calcite plus dolomite or magnesite plus dolomite.Other researchers reported that MgCO3 content in calciteis less than 4z (Goldsmith et al., 1955). Chave (1952)recognized that the magnesium content falls to 1 or 2zwithin a few tens of millions of years, because of the lackof stability.

In Fig. 9, the total amount of precipitation decreaseswith increasing Mg content until the applied Ca/(Ca＋Mg) ratio becomes 0.3, where the Mg/Ca ratio ofprecipitates is unity. In Fig. 9, the horizontal axis ofCa/(Ca＋Mg) was conveniently used to deˆne the Ca–Mgratio when Ca＝0 and Mg＝0. Below an applied Ca/(Ca＋Mg) ratio of 0.3, magnesite precipitates dominantly. Ifcalcite exists, magnesite precipitation does not occur(Faust, 1949). Mg ions must be used for other types ofminerals, such as dolomite, at a high Mg/Ca ratio.

In the absence of Ca ions, the precipitated concentra-tion of magnesite was 0.95 M one week later, as shown inFig. 9. It was only 0.7 M at 72 hours. Therefore, the mag-nesite might be produced until the concentration became1 M.

On the basis of Ca/Mg weight ratios, Chilingar (1957)recognized the following groups of dolomites: (1) mag-nesium dolomite (Ca/Mg＝1.0–1.5), (2) dolomites(Ca/Mg＝1.5–1.7), (slightly calcitic dolomites (Ca/Mg＝1.7–2.0), (4) calcitic dolomites (Ca/Mg)＝2.0–3.5). The

weight ratio can be converted to mole ratio, as

ØMgCa »

W＝

atomic weight of magnesiumatomic weight of calcium

×ØMgCa »

M

＝0.606ØMgCa »

M(10)

ØMgCa »

M＝

ØMgCa »

W

0.606＝

1

0.606×Ø CaMg»W

(11)

Where (Mg/Ca)M is the mole ratio of Mg/Ca, which isused in this study, and (Ca/Mg)W is the weight ratio ofCa/Mg used by Chilingar. It is noted that a pure dolomitedeˆned by Chilingar has a (Mg/Ca)M of 1.0. The dolo-mite is expressed by Ca0.5Mg0.5(CO3). In Fig. 9, this con-dition was obtained at a Ca/(Ca＋Mg) ratio of 0.3, i.e.,(Mg/Ca)M of 2.33.

The carbonates produced were removed from the testtubes and observed with an electron microscope. Themicrophotographs are shown in Photo 4. The indicationby A, B and C are points where the electron beams wereapplied for the electron probe micro analysis (EPMA).The penetration depth of the beams was approximately 5mm. The results of EPMA are indicated under the photos,respectively. It is noted that the measurement error by theEPMA is approximately one percent.

Photograph 4(a) shows a calcite crystal precipitated us-ing 1 M CaCl2. The EPMA shows that the Ca content is95z. The crystals are typical of those of calcite.

An addition of Mg2＋ changes the shape of the crystalof calcite (Folk, 1974). The calcite with a small amount ofMg can be called Mg-calcite. Photograph 4(b) is a goodexample of the transformation of the crystal in thepresence of a small amount of Mg ions. The EPMAshowed a relatively low content of Mg to Ca, though theapplied Mg/Ca ratio was 0.25. This can be interpreted bya lack of the calcite stability under a relatively high Mgcontent (Chave, 1952). The minerals in Photo 4(b) can becharacterized by Mg-calcite, basically calcite with Mg.Magnesium inhibits the crystal growth along the a and bcrystal axes but allows growth along the c axis (Folk,1974), as shown in Fig. 10. Under this situation, an ex-treme condition, this indicates that the crystal can growin one direction and will produce needle/thorn type ofcrystals (Fig. 10(b)). In Photo 4(b), thorn-shapedprecipitations are seen on the surfaces of calcites. In thephotograph, it is seen that calcites stick together. Thismay be because the crystal growth of calcite was inhibitedby Mg.

Photographs 4(c) and (d) show that dolomite-like par-ticles were produced for applied Mg/Ca molar ratios of0.67 and 1.0. The EPMA showed a measured Mg/Caweight ratio of approximately 0.14–0.33 (0.23–0.54 inmolar ratio), while Fig. 9 indicated a measured Mg/Caweight ratio of 0.3–0.5 (0.5–0.825 in molar ratio) for anapplied Mg/Ca molar ratio between 0.67 and 1.0.

The X-ray diŠraction pattern was observed on the

90

Photo 4. SEM photographs of the carbonate productions using diŠerent Mg/Ca ratio. (a)–(f): applied Mg/Ca ratio, (A), (B) and (C): measuredCa and Mg concentrations

Fig. 10. Crystalline axes for orthorhombic crystal and illustration ofthe eŠect of Mg ion

Photo 5. Fibrous crystal growth of MgCO3 without Ca

90 FUKUE ET AL.

precipitated carbonates within one week. The result ob-tained using a Mg/Ca ratio of 1 showed the presence ofcalcite, aragonite and possibly chlorartinite [Mg2(CO3)Cl(OH)･3(H2O)], but not dolomite. The ˆrst three miner-als are likely to be produced. It is also considered that cal-cite and aragonite are primary products of dolomite inthe presence of magnesium. Therefore, a long term ex-periment is required to conˆrm the ˆnal or ultimateproducts for engineering applications.

When the Mg/Ca ratio is high, the products are spheri-cal, as shown in Photos 4(e) and (f). In fact, the surfaces

of the spherical magnesite are covered with ˆbrous crys-tals, as shown in Photo 5. As was mentioned, the crystalis possibly growing. It seems that from Fig. 9, theproduction of magnesite (MgCO3) is unstable with timeand this is because of the hydration.

91

Photo 6. Needle penetration device (Soft rock penetrometer)

Table 2. Unconˆned compressive strength and conditions for experiments

No. Applied ions Timesof ‰ow Conc.

Calculatedcarbonatecontent

Measuredcarbonatecontent

Penetrationresistance

Penetrationdepth

Gradient ofpenetration

Uniaxialcompressive

strength

(M) (z) (z) (N) (mm) (N/mm) (MPa)

1 0.5M-Mg＋0.5M-Ca 1 0.75 1.9 1.4 0 10 0 0

2 0.5M-Mg＋0.6M-Ca 2 1.5 3.8 3.4 20 10 2 0.8

3 0.5M-Mg＋0.7M-Ca 3 2.25 5.6 5.6 55 10 5.5 2.05

4 0.5M-Mg＋0.8M-Ca 4 3 7.4 8.9 90 10 9 3.4

5 1M-Ca 2 2 6.2 8.5 25 10 2.5 1

6 1M-Ca 3 3 9.4 12.7 62 10 6.2 2.4

sand: initial density 1.42 g/cm3, void ratio＝0.86

91MICROBIAL CEMENTATION BY CARBONATES

Cementation of Sands due to CarbonatesThe sand layers in the acrylic pipes were cemented us-

ing the microbial process. The preparation of the speci-mens was described earlier. Specimen Nos. 1 to 4 werecemented with Mg and Ca (applied Mg/Ca＝1, but themeasured Mg/Ca ratio＝0.5) and specimen Nos. 5 and 6were cemented with 1 M calcite (without Mg).

The cementation strength of the specimens was exam-ined with the soft rock penetrometer (needle penetrationshown in Photo 6). The needle was penetrated into thesand specimens after microbiologically cemented, and thepenetration resistance (kPa) and penetration distance(mm) were measured. From the results, the unconˆnedcompressive strength of the sands was estimated using thecorrelation between unconˆned compressive strength andso called ``penetration gradient (N/mm)'' which can bedetermined penetration and penetration resistance of theneedle, by

log (qu)＝0.978x＋2.599 (12)

where x is the logarithm of ``the penetration gradient'',when the logarithm of qu is the unconˆned compressivestrength. The relationship is presented as a calibration ofthe penetrometer. In the instrument manual, it is de-scribed that the relationship was conˆrmed by a correla-tion coe‹cient more than 0.9, for a su‹cient number of

natural soft rock samples and improved soils with ce-ment.

The results obtained in this study are a slightly complexbecause of the diŠerent speciˆc gravities for calcite(CaCO3) and Mg0.5Ca0.5(CO3) carbonate. The correctionsand values presented in Table 2 are explained as follows:a) When Mg/Ca＝0.5, the calculated concentration is

0.75 times the applied salt concentration (M). Themolecular mass for carbonate (Mg/Ca＝0.5) is 77.

b) For calcite, the molecular mass is assumed to be 100.It was assumed that for calcite (CaCO3), 1 M calciumions in the pore space will produce 1 M CaCO3. Themass of carbonate can be obtained from the concen-tration, the molecular mass of CaCO3, the density ofsand particles and pore volume. In this case, withsand and carbonate initially separated, it is con-venient to use the following deˆnition of carbonatecontent modiˆed from (3) and evaluate the C value.

C＝carbonate

carbonate＋dry mass of soil×100(z) (13)

c) Another way to obtain the carbonate content is tomeasure using the device shown in Fig. 4. For Mg–Cacarbonate, the carbonate content was ˆrst determinedas calcite and it was multiplied by 0.77, i.e.,dolomite/calcite mass ratio (77/100), where Mg/Caratio and carbonate ions were used for the calcula-tion.

The carbonate contents of the specimens are presentedin Table 2. The measured carbonate content was ob-tained using the device shown in Fig. 4. The calculatedcarbonate content was determined using the concentra-tion of the solution used. The measured one is a little low-er than the calculated value for the ˆrst ‰ow (No. 1). Theamount of precipitation was less at a lower pH than ex-pected, because of the buŠer capacity of the sand.Though the initial pH of the solution was adjusted to 9.0with the buŠer solution, it decreased to 6.7 the followingday. The other data in Table 2 show that the measuredMg–Ca carbonate content is almost equal to the calculat-ed value. For calcite the measured carbonate content wasconsiderably higher than the calculated value.

92

Fig. 11. Comparison of unconˆned compressive strength for calciteand dolomite

Table 3. Reduction of unconˆned compressive strength by acid wash-ing

SpecimenMg-Ca carbonate

(No.3)Calcite (No.6)

Unconˆned compressivestrength before washing(MPa)

2.05 2.4

Unconˆned compressivestrength after washing(MPa)

1.35 1.42 0.97 0.40 0.53 0.90 1.45

Average strength (MPa) 1.25 0.819

Reduction due todissolution (z) 39.0 65.9

92 FUKUE ET AL.

In Fig. 9, it is likely that 0.5 M Mg and 0.5 M Ca rea-gents produce Mg–Ca carbonates (calcite, aragonite andpossibly chlorartinite) with a Mg/Ca ratio of 0.5. Be-cause of the crystal type, the calcite precipitated usingCaCl2 solution has a lower cementation eŠect on sandparticles. Therefore, the unconˆned compressive strengthis higher for Mg–Ca carbonate than calcite for a similarcarbonate content. At a carbonate content of about 8z,the strength of Mg–Ca carbonate (possibly dolomite) isapproximately three times higher than calcite, as shownin Fig. 11.

Dissolution of Carbonates with AcidAcid rain or ground water with a low pH may result in

the dissolution of carbonates in soils. Therefore, theeŠect of washing on the cemented sands was investigatedusing acetic acid. Specimens Nos. 3 and 6 were used forthe experiment. The acrylic pipes of the specimens Nos. 3and 6 were turned upside down and 70 mL of 0.5 M aceticacid solution was poured from the top and allowed to‰ow into the sand. The solution was drained out from thebottom freely. When the acid solution was poured, gasbubbles were emitted from the sand. It took about 5minutes to wash the sand with the acid. After washingwith the acid, the specimens were washed with 70 mL oftap water and kept for one hour.

The coarse sand layer shown in Fig. 6 was removed outfrom the pipes until the ˆne sand layer was exposed. Theneedle penetrometer shown in Photo 6 was used to evalu-ate the decrease in unconˆned compressive strength of theˆne sand layers washed with acid. The needle waspenetrated in the ˆne sand layer. The penetrationresistance and penetration depth were measured. Thegradient of penetration, deˆned as the penetrationresistance divided by the penetration depth, was deter-mined. The unconˆned compressive strength of the ˆnesand layers was estimated from the gradient of penetra-tion using the relationship in Eq. (12).

The results were presented in Table 3. The unconˆnedcompressive strengths of the specimens, Nos. 3 and 6, be-fore and after washing with acid were evaluated from the

needle penetration tests. The average strengths afterwashing are also presented. The comparison between theunconˆned compressive strengths before and after wash-ing was made to see the dissolution characteristics forMg–Ca carbonates (No. 3) and calcite (No. 6). SpecimenNo. 3 shows about 40z reduction in unconˆned com-pressive strength, while No. 6 shows about 66z reduc-tion. This indicates that the presence of Mg in carbonatedecreased the dissolution of carbonate. Thus, it is consis-tent that from the observation of crystals produced,strength characteristics, and washing eŠect with acid, theaddition of Mg can promote the cementation and inhibi-tion of the dissolution of crystals by acid. It is possiblethat other metals also may have potential as an inhibitoror a promoter in addition to Mg.

CONCLUDING REMARKS

Ureolytic strains were isolated from the natural soils.The study examined the urease activity of the strains interms of microbial carbonate precipitation (MCP). Thestrain showed a very high urease activity at high concen-trations of Ca and/or Mg ions. Their tolerance to Ca andMg was su‹cient.

To develop a feasible MCP for cementation of soils,various microbial minerals were produced using diŠerentMg/Ca ratios. The results showed that the concentrationof Mg in‰uenced the amount of carbonate precipitationand produced the diŠerent microbial minerals, whichwere possibly calcite, Mg-calcite, Ca-dolomite, dolomiteMg-dolomite and magnesite.

The cementation of sand due to MCP was examined interms of carbonate content and the types of minerals.The cementation due to the Mg/Ca ratio of 0.5 wasstronger than calcite without magnesium. The resistanceagainst acetic acid is stronger for the cementation with aMg/Ca ratio of 0.5 than calcite without magnesium. Theunconˆned compressive strength of the cemented sandwith carbonate content of 8 percent was estimated to be3.2 MPa at Mg/Ca of 0.5 and 1 MPa for calcite withoutMg.

9393MICROBIAL CEMENTATION BY CARBONATES

ACKNOWLEDGEMENTS

The X-ray diŠraction analysis was performed by Dr. I.Sakamoto (Associate Professor of Tokai University).The experimental study was also conducted with some as-sistance from N. Uehara and Y. Takahashi. The authorswould also like to thank and acknowledge Prof. C. N.Mulligan (Concordia University, Canada) for her usefulcomments.

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