Cementation of Sands Due to Microbiologically-Induced Carbonate Precipitation

SOILS AND FOUNDATIONS Japanese Geotechnical Society

Vol. 51, No. 1, 83–93, Feb. 2011

CEMENTATION OF SANDS DUE TO MICROBIOLOGICALLY-INDUCED CARBONATE 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 was found that the ratio of Mg/Ca aŠected the types of crystals produced. Without Mg2＋, calcite was produced using only CaCl2, while the presence of Mg produced Mg-calcite, magnesite and/or possibly dolomite of round, spherical or ˆbrous shapes, depending on reaction time, pH and Mg/Ca ratio. The carbonate produced contributed to the development 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) compressive strengths of cement mortar cubes in the presence of microorganisms. Most of the studies on microbial cementation were based on urease activity. Urease activity is the hydrolysis of urea due to an enzyme produced by microorganisms or plants such as jack beans. It is also noted that the enzyme induces the hydrolysis by catalysis. The hydrolysis of urea produces CO23－ which can react with Ca2＋. As a result, calcite is produced. The process is illustrated in Fig. 1. Therefore, up to now, many studies have been focused on microbes which produce urease enzyme. The microbes are called ureolytic bacteria. The importance of the hydrolysis of urea is that the ammonium produced by the process of the hydrolysis increases the pH. It is known that calcite precipitation occurs under a pH value higher than 7.

INTRODUCTION It was shown that marine deposited soils have cementation due to carbonate (Fukue et al., 1999). The study showed that the intensity of the cementation due to carbonate content was very strong. The analysis showed that for silty clays, one percent carbonate will increase the unconˆned compressive strength by about 60 kPa. About a 1 m thick sand rock layer was found in the Narita sand formation which was formed by the precipitation of carbonate. The sand rock layer had an unconˆned compressive strength of about 19 MPa and contained 24 percent carbonate by mass. Carbonate is a salt or ester of carbonic acid, containing the chemical group CO3. Typical carbonates are calcium carbonate (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, aragonite and vaterite are calcium carbonates but they have diŠerent crystalline forms. In nature, there are many aspects concerning carbonates. For example, dolomite mineral (CaMg(CO3)2) can form Ca and Mg carbonate. The formation of dolomite is geologically complicated. In this study, carbonates containing Ca and/or Mg are mainly discussed. The artiˆcial precipitation of calcite in soils has been studied to examine the clogging of sand (Hart et al., 1960; UpdegraŠ, 1982; Macleod et al., 1988; Stocks-Fischer et al., 1999; Bang et al., 2001, Bachmeier et al., 2002; Ferris et al., 2003; Fujita et al., 2008). Stocks-Fischer et al. (1999) have investigated the microbiological remediation of concrete cracks and demonstrated the improvement of i) ii) iii)

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

Professor, Tokai University, Shizuoka, Japan (fukue＠scc.u-tokai.ac.jp). ditto. 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. 83

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Calcite precipitation induced by ureolytic bacteria was studied by Ciurli et al. (1996), Le M áetayer-Levrel et al. (1999), Tianoa et al. (1999), Ferris et al. (2003), Whi‹n et al. (2007), De Muynck et al. (2008), Lian et al. (2006) and Jimenez-Lopez et al. (2008). These studies can be categorized into urease activity, calcite precipitation and their application to engineering purposes. Note that calcite is a typical calcium carbonate and is more stable than other calcium carbonates, such as aragonite. Many studies concerning calcite precipitation have been performed in the laboratories. The diŠerences between the conditions in the laboratory and under natural conditions are as follows; a) duration of the chemical reactions, b) presence of impurities, and c) di‹culty in controlling pH. In the laboratory, pure calcite can be created by injecting ureolytic bacteria into a CaCl2 solution, as shown in Photo 1. The details are described in terms of urease activity in Section Experimentation. However, it is doubtful that this type of crystal can make a strong cementation between soil particles. From the authors' experiences, fast reaction is not important for the cementation between particles concerned. This property of calcite can be improved by adding impurities which can change crystal forms as found in nature (Moore, 2001). For example, the presence of Mg can create Mg-calcite whose crystals are diŠerent from those of calcite. Folk (1974) showed that a small amount of Mg can interfere with the growth of calcite crystals and can help crystal growth into

Photo 1.

Calcite crystals produced by microbial urease

Fig. 2.

the diŠerent direction. The amount of Mg used for the crystals is less than 4z. Larger amounts of Mg possibly contribute to the formation of dolomite or magnesite, because of the high Mg/Ca ratio. Dolomite (CaMg(CO3)2) and magnesite (MgCO3) can resist against acid more than calcite (Fukue et al., 2010). In this study, microbiologically-induced calcite, Mgcalcite, dolomite and magnesite were observed using high activity ureolytic bacteria. Furthermore, the cementation eŠects due to calcite and Mg-calcite were also examined using the ureolytic bacteria. The main purposes of this study are to examine the capacity of the ureolytic bacteria and the eŠect of impurities on carbonate precipitation for engineering applications. MATERIALS AND EXPERIMENTAL PROCEDURES

Microorganisms To isolate bacteria which have strong urease activity, boring core samples were collected from eleven sites at diŠerent locations throughout Japan. First, diŠerent concentrations of CaCl2 or Ca(OH)2 were used to screen the bacteria in the soil samples. This approach was used to isolate the bacteria which are tolerant to Ca ions. For this, approximately 10 g of collected soil samples were kept for a few days in CaCl2 or Ca(OH)2 solution. After screening, living strains were cultivated on an agar. Throughout these procedures, about 150 strains were isolated 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 was chosen. This strain was named as NO-A10, where ``NO'' means the name of location for the sampling site, Niwase, Okayama City, and ``A10'' shows alkalophilic 10th strain isolated. Figure 2 shows the nucleotide sequence of 16S ribosomal DNA of NO-A10 strain. DNA consists of two long chains of nucleotides twisted into a double helix and joined by hydrogen bonds between the complementary bases adenine (A) and thymine (T) or cytosine (C) and guanine (G). The sequence of nucleotides determines individual hereditary characteristics, which can be used to identify and specify the species of the strain. A comparison between NO-A10 and Bacillus pasteurii which are recognized as ureolytic bacteria showed an accordance of

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

MICROBIAL CEMENTATION BY CARBONATES

Fig. 4.

Fig. 3.

85

A calcite-acid reactor to determine calcite content in soils

Growth curves of NO-A10 strain and NO-N10

93z (632/673). In this study, NO-N10 which is a parent strain of NOA10 was also examined. The two strains used in this study were cultivated with EDC (electron donor compound) at pH 8.6. The EDC cannot be used as a usual culture medium but can be used for an application, because the EDC is more economical than the usual culture medium such as nutrient medium. It is noted that the alkalophilic NOA10 strain should be cultivated at pH higher than 7. For comparison, NO-N10 which is ureolytic was also used in this experiment. NO-N10 is the same species but a diŠerent strain from NO-A10. NO-N10 strain can be cultivated under a pH range of 6.5 to 8.0. The growth curves of both strains using proper culture mediums are shown in Fig. 3, where the vertical axis indicates the concentration of bacteria in terms of the absorbance of light, i.e., optical density at a wave length of 600 nm. Note that 1O.D.600 empirically corresponds to 8× 108 cells/mL. Figure 3 shows that NO-A10 strain shows a relatively low growth rate, in comparison to NO-N10. However, this does not necessarily mean that the urease activity of NO-A10 strain is weaker. The urease activity of NO-A10 is much higher than NO-N10, as described later.

Experimentation Measurement of carbonate content The carbonate content can be measured using the dissolution reactions of carbonates with acid. The carbonate content was determined by measuring the CO2 gaseous pressure produced from the following reaction; CaCO3(s)＋2H＋ª Ca2＋＋H2O＋CO2(aq)

(1)

Subsequently, according to Henry's law, the dissolved carbon dioxide will be in equilibrium with the gaseous CO2. CO2(aq)ª CO2(g)

(2)

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

Fig. 5. Relationship between CO2 gaseous pressure and calcium carbonate used as a calibration

shown in Fig. 4. The device consists of a reactor chamber, pressure meter and valve for the exhaust gas. The calibration curve obtained using calcium carbonate agent is shown in Fig. 5. The carbonate content C is then deˆned as:

C＝

carbonate ×100(z) dry mass of soil

(3)

The hydrochloric acid concentrations used were 0.05, 0.1, 0.5, 1.0, and 3.0 M. The experiment was carried out at 259C using an incubator. Carbonate samples of 0.1 g were used and the CO2 gaseous pressure produced from the reaction was measured over time. Urease activity in the presence of calcium ions Both strains, A10 and N10, were cultivated with a culture medium under the same conditions, except for pH. The cultivated bacteria were centrifuged to separate them from the medium. After the supernatant was removed, a certain volume of 5z NaCl solution was added to the bacteria. This is called the bacterial solution (B solution) in this study. Urea and calcium chloride solution with a similar molar concentration was prepared using the buŠer solution (10 mM NH4OH＋NH4Cl). This is called the reaction solution (R solution). It is noted that for the R solution, Mg ions can also be added. The concentrations of urea and CaCl2 ranged from 0.8 to 3.0 M. At the ˆnal stage, the same volumes of the B and R solutions

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86

were mixed. Therefore, the concentrations of urea and CaCl2 ranged from 0.4 to 1.5 M. A 5 mL aliquot of B and R solution was put into 5 mL glass test tubes and the amount of carbonate (calcite) precipitated with time was measured by the following procedures. After a certain time, the liquid in the test tubes was ˆltered with a ˆlter (1 mm). The ˆlter with solid residues C). The carbonate (calcite in was dried in an oven (at 1109 this case) amount in the liquid was determined by subtracting the mass of the ˆlter from the total weight of the ˆlter with carbonate. Most of the precipitated calcite adhered to the wall of the tube. After the liquid was removed, the tube was dried in an oven (at 1109C) until the mass became constant. The precipitated calcite on the wall was determined by subtracting the mass of the tube from the total dry mass of the tube with calcite. In this case, the precipitation of carbonate occurs as follows: 2－ CO(NH2)2＋2H2Oª 2NH＋ 4 ＋CO3 2＋ 2－ Ca ＋CO3 ª CaCO3æ

Photo 2.

Experimental setup for the microbial cementation of sands

(4) (5)

The reaction is promoted by catalysis due to the urease enzyme produced by the bacteria. To examine magnesite precipitation, the preparation and procedures used are similar to the case of calcite precipitation. In this case, the following reaction occurs. 2－ CO(NH2)2＋2H2Oª 2NH＋ 4 ＋CO3 2＋ 2－ Mg ＋CO3 ª MgCO3æ

(6) (7)

Furthermore, under the presence of Mg and Ca ions, carbonate precipitation may occur in a more complex manner. However, the reaction is ideally as follows: 2－ CO(NH2)2＋2H2Oª 2NH＋ 4 ＋CO3 2＋ 2＋ 2－ 1/2Mg ＋1/2Ca ＋CO3 ª 1/2MgCa(CO3)2æ

(8) (9)

The product is called ``dolomite'' which is also a carbonate. Reactions (6) to (9) were examined using both MgCl2 and CaCl2. Examination of microbial carbonate precipitation under diŠerent Mg/Ca ratio As a variation of reactions (8) and (9), experiments were 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 is not speciˆed, Mg/Ca means the molar ratio of concentrations. Two sets were prepared for diŠerent time durations. In this test series, the ˆnal solution with a volume of 5 mL was used for the precipitation experiment. Basically, the same volumes of B and R solutions were prepared individually, and the two types of solutions were mixed just before the experiment starts. At 72 hours and one week after the experiment started, the concentrations of precipitated carbonate were determined in the manner mentioned earlier. Examination of cementation strength of sand The biologically-produced cementation due to carbonate was examined using the B and R solutions. The mixed solution was applied to the sand specimens consist-

Fig. 6.

Drawing of the experimental setup

ing of sand layers in acrylic pipes. The strain, NO-A10 used was cultivated in the EDC solution (10 g/L). For this experiment, the mixture of 0.5 M MgCl2 and 0.5 M CaCl2 and 1 M CaCl2 were used to produce carbonates in the sand layers. To maintain a high pH, the buŠer solution (10 mM NH4OH＋NH4Cl) was used. The concentration of other additives such as urea was also 1 M. The experimental setup for biological cementation is presented in Photo 2. Sand specimens were prepared in acrylic pipes with an inner diameter of 43 mm and a height of 180 mm, as shown in Fig. 6. Coarse sand (0.5–2.0 mm) layers with a thickness of about 20 mm were set at both the top and bottom of the column, and ˆne sand (0.3–0.5 mm) layer with a thickness of 90 mm was set between the two coarse sand layers, as shown in Fig. 6. Both the sands were settled under water and were in a loose state. The void ratio of the coarse sand was 0.81, while it was 0.93 for the ˆne sand. Paper ˆlters were laid between the coarse and ˆne layers. A 150 mL solution involving 1 M-urea, Ca2＋ and/or Mg2＋, the buŠer solution (10 mM) and the strain (NOA10) were inˆltrated from the top of coarse sand in the acrylic pipes. The features and test conditions of specimens from Nos. 1 to 6 are presented in Table 1. In Table 1, ``applied reagents'' means applied metal ions, Mg

MICROBIAL CEMENTATION BY CARBONATES Table 1.

87

Curing conditions during precipitation of carbonates Mg/Ca

Specimen volume (cm 3)

0.5

1

0.5

1

0.5

1

Ca Ca

1 pore volume (cm3)

Flow volume/one time (mL)

Times of ‰ow

130.6

60.4

150.

1

130.6

60.4

150

2

1

130.6

60.4

150

3

0.5

1

130.6

60.4

150

4

1

1

0

130.6

60.4

150

2

1

1

0

130.6

60.4

150

3

Total conc. (M)

No.

Applied reagents

1

Mg＋Ca

1

2

Mg＋Ca

1

3

Mg＋Ca

1

4

Mg＋Ca

5 6

Ca/(Ca＋Mg)

and/or Ca. ``Mg＋Ca'' indicates that both Mg and Ca were applied, while ``Ca'' indicates calcium only. The concentration 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 the bottom. The drain faucet was turned oŠ when the solution level reached the surface of the top sand layer. The specimens were left for 24 hours. Except for specimen No. 1, the pore liquids were replaced with new solutions in a similar manner. The solution was poured from the top of the sand specimen while the drain faucet was turned on. The faucet was turned oŠ again when the poured volume reached 150 mL, where the pore volume of the sand specimen was approximately 60 mL, as indicated in Table 1. The volume equal to the pore volume is called one pore volume (1 PV). Two pore volumes is therefore a double portion (2 PV). Since 1 PV-solution can remain in the sand specimen, the carbonate precipitation can be 1 M times 1 PV. It increases proportionally with the time of ‰ow. The experimental conditions shown in Table 1 were set to 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 carbonate, and d) Dissolution characteristics of calcite and Mg–Ca carbonate. The pH value of the drained solution was 6.7 after 24 hours, though the initial pH value was 9. This may be due to the buŠer capacity of sands (Yong et al., 2002) and the eŠect of the products of the chemical reactions. After 48 hours from the beginning, the pore liquids were replaced again with new solutions for specimens Nos. 3, 4 and 6. The pore liquid of No. 4 was replaced again after 72 hours. The specimens were kept for about one month. The strength and carbonate content on the sand specimens were measured, as described later. Note that the original carbonate content of the sands used is negligible. RESULTS AND DISCUSSION

Precipitation Rate of Calcite Calcite precipitation was examined using diŠerent concentrations of urea and CaCl2 in the test tubes. The initial

Fig. 7.

Microbial precipitation rates of calcite

concentrations of urea and CaCl2 were kept the same, because 1 M-urea produces 1 M calcite, as indicated by reactions (4) and (5). The temperature was maintained at C. The microbial precipitation of carbonate initiated 209 by NO-A10 was plotted against the curing time, as shown in Fig. 7. The legend shows the type of strain and the concentration of CaCl2 solution used. For example, ``A10–0.4 M'' means that the type of strain is NO-A10 and the CaCl2 concentration is 0.4 M. For a comparison, the cases of NO-N10 are also shown. Although some bacteria 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 precipitation rate of calcite by NO-A10 in Fig. 7 demonstrates directly the urease activity, i.e., the hydrolysis of urea under the presence of Ca ions. Figure 7 shows that NO-A10 showed a higher (faster) urease activity even for 1.5 M CaCl2. On the other hand, the carbonate precipitation by NO-N10 is relatively slow. This means that the urease activity by NO-N10 is relatively low. Figure 7 also shows that the reaction rate by NO-A10 was almost independent of the concentrations of CaCl2 and the reaction was completed within 25 hours for any concentration of CaCl2 up to 1.5 M, while the reaction by NO-N10 is not completed after 100 hours. Thus, NO-A10 demonstrated a very high urease activity as well as precipitation rate of calcite.

Precipitation Rates of Carbonate with Mg The precipitation rate of carbonate with Mg was examined using the mixture of 0.5 M MgCl2 and 0.5 M CaCl2.

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The concentrations of urea and buŠer reagents are 1 M and 10 mM, respectively. The precipitation rate of magnesite was also examined using 0.5 M-MgCl2 and 0.5 Murea. The precipitation of carbonates was observed using 5 mL solution in the test tubes. The amount of the precipitated carbonates was determined by the method used for the calcite precipitation. The precipitation rates of carbonates are shown in Fig. 8. The concentrations of MgCa(CO3)2 and MgCO3 were measured by the method used for calcite shown in Fig. 7. The ultimate amount of Mg–Ca carbonate produced from the mixture of 0.5 M MgCl2 and 0.5 M CaCl2 does

Fig. 8. Microbial precipitation rates of Mg–Ca carbonate and magnesite

Photo 3.

not reach 1 M, but was approximately 0.75 M. It seems that the presence of Mg inhibits slightly the carbonation reaction. However, the precipitation rate is similar to calcite precipitation. The products using the mixture of 0.5 M-MgCl2 and 0.5 M-CaCl2 were observed with time. The SEM photographs of the products are shown in Photo 3. The shape of the products is quite diŠerent from the calcite shown in Photo 1 and varies with time, as shown in Photo 3. The precipitation occurred within 24 hours, and the shape was ˆrst round (Photo 3, D-1). In 9 hours after the experiment started, the products grew slightly, and the shape changed 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 originally rounded products (Photo 3, D-7). The shape may change until the most stable crystal is obtained, as a result of crystallization or re-crystallization under a pH change. The carbonates produced in this experiment contained 0.25 M Mg and 0.5 M Ca. The Mg/Ca ratio is 0.5. Dolomite formed in nature is the most stable when the Mg/Ca ratio is around 0.5, which is known in the skeletal structure of organisms, such as the calcareous algae Goniolithon (Chilingar, 1957). The reaction although may not have completed, continues to be more stable. The authors consider the ultimate products to be dolomite, however this may take extended periods of time. It was conˆrmed that magnesite crystals are produced using 0.5 M-MgCl2. The concentration was up to 75z of

SEM photographs of Mg–Ca carbonate at diŠerent reaction time

MICROBIAL CEMENTATION BY CARBONATES

89

weight ratio can be converted to mole ratio, as

Ø »＝ Mg Ca

W

Ø »

atomic weight of magnesium Mg × atomic weight of calcium Ca

Ø » Ø »＝ ＝ Mg Ca

＝0.606

Ø » Mg Ca

Fig. 9.

EŠects of Mg/Ca ratio for precipitation of carbonates

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

EŠects of Mg/Ca Ratio on Precipitation of Carbonate EŠects of Mg/Ca ratio on precipitation of carbonate were examined to determine the morphological aspects of carbonate, i.e., transform from calcite to magnesite through dolomite. The experimental procedures were already described. The amount of precipitates was measured in 72 hours and one week after the experiment began in a manner similar to that previously described. In Fig. 9, it was assumed that all calcium ions applied were 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 calcite plus magnesite does not occur in nature, and the products are calcite plus dolomite or magnesite plus dolomite. Other researchers reported that MgCO3 content in calcite is less than 4z (Goldsmith et al., 1955). Chave (1952) recognized that the magnesium content falls to 1 or 2z within a few tens of millions of years, because of the lack of stability. In Fig. 9, the total amount of precipitation decreases with increasing Mg content until the applied Ca/(Ca＋ Mg) ratio becomes 0.3, where the Mg/Ca ratio of precipitates is unity. In Fig. 9, the horizontal axis of Ca/(Ca＋Mg) was conveniently used to deˆne the Ca–Mg ratio when Ca＝0 and Mg＝0. Below an applied Ca/(Ca ＋Mg) ratio of 0.3, magnesite precipitates dominantly. If calcite exists, magnesite precipitation does not occur (Faust, 1949). Mg ions must be used for other types of minerals, such as dolomite, at a high Mg/Ca ratio. In the absence of Ca ions, the precipitated concentration of magnesite was 0.95 M one week later, as shown in Fig. 9. It was only 0.7 M at 72 hours. Therefore, the magnesite might be produced until the concentration became 1 M. On the basis of Ca/Mg weight ratios, Chilingar (1957) recognized the following groups of dolomites: (1) magnesium 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

Mg Ca

M

W

0.606

M

(10) M

1

Ø »

Ca 0.606× Mg

(11) W

Where (Mg/Ca)M is the mole ratio of Mg/Ca, which is used in this study, and (Ca/Mg)W is the weight ratio of Ca/Mg used by Chilingar. It is noted that a pure dolomite deˆned by Chilingar has a (Mg/Ca)M of 1.0. The dolomite is expressed by Ca0.5 Mg0.5(CO3). In Fig. 9, this condition 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 test tubes and observed with an electron microscope. The microphotographs are shown in Photo 4. The indication by A, B and C are points where the electron beams were applied for the electron probe micro analysis (EPMA). The penetration depth of the beams was approximately 5 mm. The results of EPMA are indicated under the photos, respectively. It is noted that the measurement error by the EPMA is approximately one percent. Photograph 4(a) shows a calcite crystal precipitated using 1 M CaCl2. The EPMA shows that the Ca content is 95z. The crystals are typical of those of calcite. An addition of Mg2＋ changes the shape of the crystal of calcite (Folk, 1974). The calcite with a small amount of Mg can be called Mg-calcite. Photograph 4(b) is a good example of the transformation of the crystal in the presence of a small amount of Mg ions. The EPMA showed a relatively low content of Mg to Ca, though the applied Mg/Ca ratio was 0.25. This can be interpreted by a lack of the calcite stability under a relatively high Mg content (Chave, 1952). The minerals in Photo 4(b) can be characterized by Mg-calcite, basically calcite with Mg. Magnesium inhibits the crystal growth along the a and b crystal axes but allows growth along the c axis (Folk, 1974), as shown in Fig. 10. Under this situation, an extreme condition, this indicates that the crystal can grow in one direction and will produce needle/thorn type of crystals (Fig. 10(b)). In Photo 4(b), thorn-shaped precipitations are seen on the surfaces of calcites. In the photograph, it is seen that calcites stick together. This may be because the crystal growth of calcite was inhibited by Mg. Photographs 4(c) and (d) show that dolomite-like particles were produced for applied Mg/Ca molar ratios of 0.67 and 1.0. The EPMA showed a measured Mg/Ca weight ratio of approximately 0.14–0.33 (0.23–0.54 in molar ratio), while Fig. 9 indicated a measured Mg/Ca weight ratio of 0.3–0.5 (0.5–0.825 in molar ratio) for an applied Mg/Ca molar ratio between 0.67 and 1.0. The X-ray diŠraction pattern was observed on the

90

FUKUE ET AL.

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

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

precipitated carbonates within one week. The result obtained using a Mg/Ca ratio of 1 showed the presence of calcite, aragonite and possibly chlorartinite [Mg2(CO3) Cl(OH)･3(H2O)], but not dolomite. The ˆrst three minerals are likely to be produced. It is also considered that calcite and aragonite are primary products of dolomite in the presence of magnesium. Therefore, a long term experiment is required to conˆrm the ˆnal or ultimate products for engineering applications. When the Mg/Ca ratio is high, the products are spherical, as shown in Photos 4(e) and (f). In fact, the surfaces

Photo 5.

Fibrous crystal growth of MgCO3 without Ca

of the spherical magnesite are covered with ˆbrous crystals, as shown in Photo 5. As was mentioned, the crystal is possibly growing. It seems that from Fig. 9, the production of magnesite (MgCO3) is unstable with time and this is because of the hydration.

MICROBIAL CEMENTATION BY CARBONATES

Cementation of Sands due to Carbonates The sand layers in the acrylic pipes were cemented using the microbial process. The preparation of the specimens was described earlier. Specimen Nos. 1 to 4 were cemented with Mg and Ca (applied Mg/Ca＝1, but the measured Mg/Ca ratio＝0.5) and specimen Nos. 5 and 6 were cemented with 1 M calcite (without Mg). The cementation strength of the specimens was examined with the soft rock penetrometer (needle penetration shown in Photo 6). The needle was penetrated into the sand specimens after microbiologically cemented, and the penetration resistance (kPa) and penetration distance (mm) were measured. From the results, the unconˆned compressive strength of the sands was estimated using the correlation between unconˆned compressive strength and so called ``penetration gradient (N/mm)'' which can be determined penetration and penetration resistance of the needle, by log (qu)＝0.978x＋2.599

Photo 6.

Needle penetration device (Soft rock penetrometer)

Table 2. No.

Applied ions

natural soft rock samples and improved soils with cement. The results obtained in this study are a slightly complex because of the diŠerent speciˆc gravities for calcite (CaCO3) and Mg0.5Ca0.5(CO3) carbonate. The corrections and 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). The molecular 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 calcium ions in the pore space will produce 1 M CaCO3. The mass of carbonate can be obtained from the concentration, the molecular mass of CaCO3, the density of sand particles and pore volume. In this case, with sand and carbonate initially separated, it is convenient to use the following deˆnition of carbonate content modiˆed from (3) and evaluate the C value.

(12)

where x is the logarithm of ``the penetration gradient'', when the logarithm of qu is the unconˆned compressive strength. The relationship is presented as a calibration of the penetrometer. In the instrument manual, it is described that the relationship was conˆrmed by a correlation coe‹cient more than 0.9, for a su‹cient number of

91

C＝

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

(13)

c) Another way to obtain the carbonate content is to measure using the device shown in Fig. 4. For Mg–Ca carbonate, the carbonate content was ˆrst determined as calcite and it was multiplied by 0.77, i.e., dolomite/calcite mass ratio (77/100), where Mg/Ca ratio and carbonate ions were used for the calculation. The carbonate contents of the specimens are presented in Table 2. The measured carbonate content was obtained using the device shown in Fig. 4. The calculated carbonate content was determined using the concentration of the solution used. The measured one is a little lower than the calculated value for the ˆrst ‰ow (No. 1). The amount of precipitation was less at a lower pH than expected, because of the buŠer capacity of the sand. Though the initial pH of the solution was adjusted to 9.0 with the buŠer solution, it decreased to 6.7 the following day. The other data in Table 2 show that the measured Mg–Ca carbonate content is almost equal to the calculated value. For calcite the measured carbonate content was considerably higher than the calculated value.

Unconˆned compressive strength and conditions for experiments

Times of ‰ow

Conc.

Calculated carbonate content

Measured carbonate content

Penetration resistance

Penetration depth

Gradient of penetration

Uniaxial compressive 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/cm 3, void ratio＝0.86

92

FUKUE ET AL. Table 3. ing

Reduction of unconˆned compressive strength by acid wash-

Specimen Unconˆned compressive strength before washing (MPa) Unconˆned compressive strength after washing (MPa)

Mg-Ca carbonate (No.3)

Calcite (No.6)

2.05

2.4

1.35 1.42 0.97 0.40 0.53 0.90 1.45

Average strength (MPa)

1.25

0.819

Reduction due to dissolution (z)

39.0

65.9

Fig. 11. Comparison of unconˆned compressive strength for calcite and dolomite

In Fig. 9, it is likely that 0.5 M Mg and 0.5 M Ca reagents produce Mg–Ca carbonates (calcite, aragonite and possibly chlorartinite) with a Mg/Ca ratio of 0.5. Because of the crystal type, the calcite precipitated using CaCl2 solution has a lower cementation eŠect on sand particles. Therefore, the unconˆned compressive strength is higher for Mg–Ca carbonate than calcite for a similar carbonate content. At a carbonate content of about 8z, the strength of Mg–Ca carbonate (possibly dolomite) is approximately three times higher than calcite, as shown in Fig. 11.

Dissolution of Carbonates with Acid Acid rain or ground water with a low pH may result in the dissolution of carbonates in soils. Therefore, the eŠect of washing on the cemented sands was investigated using acetic acid. Specimens Nos. 3 and 6 were used for the experiment. The acrylic pipes of the specimens Nos. 3 and 6 were turned upside down and 70 mL of 0.5 M acetic acid solution was poured from the top and allowed to ‰ow into the sand. The solution was drained out from the bottom freely. When the acid solution was poured, gas bubbles were emitted from the sand. It took about 5 minutes to wash the sand with the acid. After washing with the acid, the specimens were washed with 70 mL of tap water and kept for one hour. The coarse sand layer shown in Fig. 6 was removed out from the pipes until the ˆne sand layer was exposed. The needle penetrometer shown in Photo 6 was used to evaluate the decrease in unconˆned compressive strength of the ˆne sand layers washed with acid. The needle was penetrated in the ˆne sand layer. The penetration resistance and penetration depth were measured. The gradient of penetration, deˆned as the penetration resistance divided by the penetration depth, was determined. The unconˆned compressive strength of the ˆne sand layers was estimated from the gradient of penetration using the relationship in Eq. (12). The results were presented in Table 3. The unconˆned compressive strengths of the specimens, Nos. 3 and 6, before and after washing with acid were evaluated from the

needle penetration tests. The average strengths after washing are also presented. The comparison between the unconˆned compressive strengths before and after washing was made to see the dissolution characteristics for Mg–Ca carbonates (No. 3) and calcite (No. 6). Specimen No. 3 shows about 40z reduction in unconˆned compressive strength, while No. 6 shows about 66z reduction. This indicates that the presence of Mg in carbonate decreased the dissolution of carbonate. Thus, it is consistent that from the observation of crystals produced, strength characteristics, and washing eŠect with acid, the addition of Mg can promote the cementation and inhibition of the dissolution of crystals by acid. It is possible that other metals also may have potential as an inhibitor or 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 in terms of microbial carbonate precipitation (MCP). The strain showed a very high urease activity at high concentrations of Ca and/or Mg ions. Their tolerance to Ca and Mg was su‹cient. To develop a feasible MCP for cementation of soils, various microbial minerals were produced using diŠerent Mg/Ca ratios. The results showed that the concentration of Mg in‰uenced the amount of carbonate precipitation and produced the diŠerent microbial minerals, which were possibly calcite, Mg-calcite, Ca-dolomite, dolomite Mg-dolomite and magnesite. The cementation of sand due to MCP was examined in terms of carbonate content and the types of minerals. The cementation due to the Mg/Ca ratio of 0.5 was stronger than calcite without magnesium. The resistance against acetic acid is stronger for the cementation with a Mg/Ca ratio of 0.5 than calcite without magnesium. The unconˆned compressive strength of the cemented sand with carbonate content of 8 percent was estimated to be 3.2 MPa at Mg/Ca of 0.5 and 1 MPa for calcite without Mg.

MICROBIAL 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 assistance from N. Uehara and Y. Takahashi. The authors would also like to thank and acknowledge Prof. C. N. Mulligan (Concordia University, Canada) for her useful comments.

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