MICROBIOLOGICALLY-ENHANCED CRACK REMEDIATION (MECR)

Sookie S. Bang∗and V. Ramakrishnan

South Dakota School of Mines and Technology
501 E. Saint Joseph Street
Rapid City, SD 57701

ABSTRACT

A novel approach of microbiologically-enhanced crack remediation (MECR) has been initiated

and evaluated in this report. Under the laboratory conditions, Bacillus pasteurii was used to induce
CaCO3 precipitation as the microbial urease hydrolyzes urea to produce ammonia and carbon dioxide.
The ammonia released in surroundings subsequently increases pH, leading to accumulation of insoluble
CaCO3. Scanning electron micrography (SEM) and x-ray diffraction (XRD) analyses evidenced the
direct involvement of microorganisms in CaCO3 precipitation. In biochemical studies, the primary roles
of microorganisms and microbial urease were defined. Furthermore, the role of urease in CaCO3
precipitation was characterized utilizing recombinant Escherichia coli that encoded B. pasteurii urease
genes in a plasmid. Microorganisms immobilized in polyurethane (PU) polymer were applied to
remediate concrete cracks. Although microbiologically- induced calcite precipitation enhanced neither
the tensile strength nor the modulus of elasticity of the PU polymer, cement mortar whose crack was
remediated with the cell-laden polymer showed a significant increase in compressive strength. Through
detailed investigation, MECR showed an excellent potential in cementing cracks in granite, concrete, and
beyond.

∗
Corresponding author. tel.: 1 605 3942426; fax: 1 605 3941232; e-mail: [email protected]

1
 INTRODUCTION

In natural environments, mineral precipitation processes constantly occur at a slow rate over
geologic time, plugging or selectively cementing permeable porous media (Hart et al., 1960; Kantzas et
al., 1992). Microbial metabolic activities often contribute to selective cementation by producing
relatively insoluble organic and inorganic compounds intra- and/or extracellularly, which remain in the
environment for a long time even after cell death. Some microorganisms produce glycocalyx and a
variety of organic polymers outside the cell wall (Lappin-Scott et al., 1988; MacLeod et al., 1988), while
others accumulate inorganic compounds such as phosphorites, carbonates, silicates, iron, and manganese
oxides in cytoplasm (Beveridge et al., 1983; Ghiorse, 1984; Knoll, 1985; Ruiz et al., 1988; Rivadeneya et
al., 1991). The importance of selective cementation has been widely recognized in Petroleum
Engineering, Geological Engineering and Civil Engineering. It has been documented that cracks in rock
formations surrounding oil reservoirs could be remediated by microorganisms to enhance the oil recovery
(Updegraff, 1982; Finnerty and Singer, 1983).

Cracks and fissures are a common problem in building structures, pavements, and historic
monuments. Methods currently used for crack remediation often use synthetic polymers that need to be
applied repeatedly. We have introduced a novel technique in fixing cracks with environmentally friendly
biological processes that is a continuous self-remediating process (Gollapudi et al., 1995). In the study,
Bacillus pasteurii that is abundant in soil has been used to induce CaCO3 precipitation. The long-term
goal of our research is to use microbiologically-induced CaCO3 in remediation of cracks and fissures in
natural and man-made structures. It is therefore imperative to understand the fundamentals of microbial
participation in crack remediation and establish the methodology of remediation prior to implementing
the microbiologically-enhanced crack remediation (MECR) process.

Microbiologically-induced CaCO3 precipitation results from a series of complex biochemical

reactions, including the concomitant participation of microorganisms, urease (urea amidohydrolase; EC
3.5.1.5), and high pH. As urease from B. pasteurii hydrolyzes urea to ammonia and carbon dioxide, the
ammonia increases the pH of the surrounding, which in turn induces CaCO3 precipitation. It is
hypothesized that urease is the main component that initiates the microbial CaCO3 precipitation. Urease
activity is found in a wide range of microorganisms and plants, some of which produce the enzyme in
large quantities (Mobley et al., 1995; Ciurli et al., 1996). In particular, B. pasteurii produces intracellular
urease constituting close to 1% of the cell dry weight. The urease from B. pasteurii consists of three
different subunits with two nickel atoms in individual active sites (Benini et al., 1999). Urease genes
from numerous microorganisms have been sequenced and expressed in the recombinant plasmids (Maeda
et al., 1993; Mordorf et al., 1994). To identify the role of urease in calcite precipitation, we have utilized
Escherichia coli HB101 that harbors a plasmid encoding B. pasteurii urease genes (You et al., 1995). B.
pasteurii and the recombinant E. coli were tested for the effects of acetohydroxamic acid (AHA) that
functions as a competitive inhibitor of urease on microbial calcite precipitation.

Use of microbial products as a long-term remediation tool has exhibited high potential for crack
cementation of various structural formations such as granite and concrete (Gollapudi et al., 1995; Stocks-
Fischer et al., 1999). However, the microbiological concrete remediation requires further consideration
before it can be used with confidence, mainly due to the fact that the pH of concrete remains extremely
high even after it is completely cured, i.e., above 12.5. Several reinforcement materials have been
considered for providing not only a protection from adverse environmental conditions in concrete but also
a higher bonding strength between the crack and the concrete. An immobilization technique for
remediation of cracks in concrete, where microbial cells are encapsulated in polymers, has been adopted
to enclose CaCO3 precipitation in the gap and to enhance the strength for selective cementation. The
immobilization technique offers several advantages for concrete remediation, in which encapsulated cells

2
retain high metabolic activities and are protected from adverse environmental conditions (O'Reilly and
Crawford, 1989).

Polyurethanes (PU) have been widely used as a vehicle for immobilization of enzymes and whole
cells because of its mechanically strong and biochemically inert characteristics (Fukushima et al., 1978;
Wang and Ruchenstein, 1993). PU makes open cell foam as a result of condensation of polycyanates
(R-CNO) and polyols (R-OH). Typically, porous matrices of PU not only increase the surface areas but
also minimize the diffusion limitation for substrates and products, which is a common disadvantage of
polymers currently used for encapsulation such as acrylamide, alginate, and carrageenan (Klein and
Kluge, 1981; Bang and Pazirandeh, 1999). Although metabolic activities of cells remain high in PU
matrices, it is however uncertain whether the rate of cell growth remains the same (O'Reilly and
Crawford, 1989; Sumino et al, 1992).

This paper summarizes our findings on the calcite precipitation induced by microorganisms and
the feasibility of using microbiologically-induced CaCO3 in concrete crack remediation. Data on the
mineral cementation by scanning electron microscopy (SEM) and x-ray diffraction (XRD) quantitative
analysis present physical evidence on microbial participation, which are detailed by the biochemical
studies of microbiological calcite precipitation. This report also includes the results of the molecular
investigation of the urease function in CaCO3 precipitation utilizing recombinant microorganisms and the
application of immobilization technology in concrete crack remediation.

MATERIALS AND METHODS

Microorganisms and Experimental Conditions

Microorganisms used for the study are Bacillus pasteurii ATCC 11859 (Bethesda, MD) and two
recombinant Escherichia coli HB101 (supE44 recA ara14 proA2 lacY1 galK2) strains containing
plasmids pBU11 and pBR322, respectively (You et al., 1995). The recombinant E. coli strains were
provided by S.D. Kim (Yeungnam University, Korea). Details of the growth conditions of
microorganisms and the experimental procedures for urease assay, CaCO3 precipitation, ammonia
production, XRD, and SEM are described elsewhere (Stocks-Fischer et al., 1999; Bang et al., 2001;
Ramachandran et al., 2001; Bachmeier et al., 2001). Water-based prepolymer of polyurethane (HYPOL
2000, 3000) was provided by the Hampshire Chemical Corp. (Boston, MA). All other essential chemicals
used were purchased from Aldrich Chemicals (St. Louis, MO).

RESULTS

Evidence of microbiologically-induced CaCO3 precipitation

Previously, we reported that the granite cracks remediated with a mixture of sand and B. pasteurii
cells showed a significant increase in compressive strength (Gollapudi, 1995; Zhong and Islam, 1995).
Table 1 summarizes the results of XRD quantitative analyses of sand samples that were tested under
different experimental conditions. The most abundant compound was clearly quartz, the main component
of sand. Microbiologically-induced CaCO3 crystals were identified as calcite, not aragonite that is more
common in seawater or magnesium-rich aqueous solutions (Berner, 1975; Rivadeneyra et al., 1991).
Calcite constituted 30.2% of the total weight of the sand samples plugged by bacteria, but none was
detected in the samples without live cells. In the sand samples treated with medium, urea was consumed
by growing bacteria (sample 4), while unmetabolized urea was detected in the samples with killed or no
bacteria. It is noteworthy that a small amount of Ca2+ (8.3%) was crystallized as a form of gehlenite in
the presence of killed bacteria. Details of the consolidated sand samples examined under SEM are
depicted in Fig. 1, where distinct calcite crystals embedded with microorganisms are found between and

3
on the surface of sand grains. Rod-shaped bacteria were prominent in all sediment samples and appeared
fossilized as intact bacilli within the calcite crystals. The presence of crystalline calcite associated with
bacteria suggests that bacteria served as nucleation sites during the mineralization process.

Table 1. XRD quantitative analysis of the final weight fractionsa of sand samplesb.
aNumbers represent an average of weight fraction values obtained from energy-dispersive XRD
quantitative analysis; ( ), variance errors;
Trace, <0.001; ND, not detected.
bSample 1, untreated sand; Sample 2, sand treated with medium; Sample 3, sand treated with killed B.
pasteurii and medium; Sample 4, sand treated with B. pasteurii and medium.

Quartz Calcite Gehlenite Mullite Hematite Goethite Urea

Sample SiO2 CaCO3 Ca2Al(Al,Si)2O7 Al6Si2O13 Fe2O3 α-FeO(OH) CH4N2O

1 0.960 ND ND 0.039 Trace ND ND

(0.010) (0.010)
2 0.954 ND ND 0.032 0.003 ND 0.011
(0.009) (0.008) (0.001) (0.003)
3 0.892 ND 0.083 0.012 Trace 0.003 0.030
(0.015) (0.011) (0.003) (0.001) (0.005)
4 0.683 0.302 ND 0.015 Trace ND ND
(0.065) (0.066) (0.004)

a b c

Figure 1. Scanning electron micrographs of microbiologically-induced calcite precipitation. a. Calcite

crystals have formed over the sand particles. Bar, 200 µm. b. Details of crystal formations shown in a.
At this magnification, the fine structure of the crystallites is easily discerned and evident. Bar, 20 µm. c.
Magnification of the area marked with an arrow in b shows B. pasteurii embedded in the crystals. Bar, 10
µm.

Biochemistry of microbiologically-induced CaCO3 precipitation

Fig. 2 presents the patterns of calcite precipitation, cell growth, ammonia production, and pH at
an initial concentration of 25.2 mM CaCl2 in the presence of live and killed cells (1 × 106 cells ml-1). The
microbiological CaCO3 precipitation began approximately at pH 8.3 and was completed at 9.2,
consolidating 98% of the initial concentration of Ca2+. Calcium carbonate precipitation appeared to be
correlated with the growth of B. pasteurii and was completed within 16 hours following inoculation.

4
However, ammonia was released continuously even during the stationary phase of cell growth. The pH of
the medium also increased slowly as the ammonia production increased. In the presence of killed cells,
there were no detectable changes in calcite precipitation, ammonia production, and pH.

900 10
30 800
700
20 108
600

500
Insoluble Ca (mM)

cell growth
10 ammonia in bacterial culture 9

NH4 (mM)
9
-1
2+

8 ammonia in control 400

Cells ml

pH
7 insoluble Ca2+ in bacterial culture

+
6 insoluble Ca2+ in control
5 107 pH in bacterial culture 300
pH in control
4

3
8
200
2

106
0 10 20 30 40 50 60

Time (hr)

Figure 2. Profiles of CaCO3 precipitation, NH4+ production, and pH changes in the presence of
live and killed B. pasteurii (inoculum size, 1.0 x 106 cells ml-1) in urea-CaCl2 medium.
Each point represents the average of duplicate assays.

Participation of urease in calcite precipitation

A urease inhibitor, acetohydroxamic acid (AHA), was used to identify the role of urease in calcite
precipitation. Fig. 3 shows the effects of AHA on the calcite precipitation induced by B. pasteurii and a
recombinant E. coli (pBU11) with the urease genes. E. coli (pBR322) encoding no urease genes served as
the negative control. In the absence of AHA, the recombinant E. coli induced calcite precipitation even
though its precipitation level was not as high as that by B. pasteurii, whereas there was no detectable
calcite precipitation by E. coli (pBR322). Similar trends were also noted for the ammonia production by
the same cells (data not included), corresponding to the patterns observed in calcite precipitation. In the
presence of AHA, B. pasteurii or E. coli (pBU11) induced little calcite precipitation. Subsequently, there
was neither pH increase nor ammonia release under the condition where AHA inhibited the urease
activity in the cell. However, the turbidity of both cultures containing AHA still increased, indicating that
the cell growth was not affected.

5
 25

20
B. pasteurii
Insoluble Ca2+ (mM)

B. pasteurii + AHA
E. coli HB101 (pBU11)
15 E. coli HB101 (pBU11) + AHA
E. coli HB101 (pBR322)

10

0
0 2 4 6 8 10 12 24 72

Time (hr)

Figure 3. Effects of acetohydroxamic acid (A H A ) on urease-induced calcite precipitation

by B. pasteurii and the recombinant E. coli H B101 strains.

Calcite precipitation induced by immobilized cells

Upon polymerization, PU foam was pliable and elastic with open-cell structure of matrices (Fig.
4a). Micrographs (Figs. 4b-4c) showing cell-laden PU matrices indicate that immobilization caused no
apparent morphological damage to the cells and microorganisms were entrapped throughout the polymer
matrices where cells were adhered or embedded with some clumping. As shown in Figs. 4d and 4e,
calcite precipitation occurred throughout the entire matrices, including the inside of pores as well as the
surface areas. It was also apparent that calcite crystals grew around the microorganisms and PU matrices
(Fig. 4f).

a b c

d e f

6
Figure 4. Scanning electron micrographs of calcite precipitation induced by B. pasteurii immobilized in
PU. a. Porous PU matrix without microbial cells showing open-cell structures. Bar, 1 mm. b.
Distribution of microorganisms on the PU surface. Bar, 1 µm. c. Microorganisms densely packed in a
pore of the PU matrix. Bar, 10 µm. d. Calcite crystals grown in the pore (shown in c) of the PU matrix.
Bar, 10 µm. e. Calcite crystals grown extensively over the PU polymer. Bar, 500 µm. f. Magnified
section pointed with an arrow in e shows crystals embedded with microorganisms. Bar, 20 µm.

Fig. 5 depicts the patterns of calcite precipitation and ammonia production by free and
immobilized cells. At 5 × 107 cells ml-1, calcite precipitation by both free and immobilized cells was
completed within 4 hours in medium, where 98% of the soluble Ca2+ became insoluble. There appear no
significant differences in the rate of calcite precipitation by free and immobilized microorganisms when
they are in the same concentration. As shown in the figure, controls including PU with no cells and killed
cells show neither calcite precipitation nor ammonium production. Viable cell counts in plates with
aliquots of the urea-CaCl2 medium containing PU-immobilized cells indicated that there was no apparent
cell leakage throughout the incubation.

25 700

600
20

500
Insoluble Ca2+ (mM)

NH4+ (mM)
15
400
insoluble Ca2+ by free cells
insoluble Ca2+ by immobilized cells
300
10 insoluble Ca2+ by cell-free PU
2+
insoluble Ca by immobilized dead cells
ammonia by free cells 200
ammonia by immobilized cells
5 ammonia by cell-free PU
ammonia by immobilized dead cells 100

0 0
0 5 10 15 20 25
Time (hr)

Figure 5. Patterns of calcite precipitation and ammonia production by free and immobilized
B. pasteurii (inoculum size, 5 x 107 cells ml-1).

Variations of the tensile strength and the stiffness of the PU foam with respect to the duration of
the incubation in urea-CaCl2 medium are shown in Figure 6. Immediately after immersion in medium,
there was a 42% increase in tensile strength of the foam with cells (21.18 psi) compared to that of the
foam without (14.94 psi). However, the tensile strength decreased with extended incubation period in
medium. Most of the decrease took place within the first ½ day, eventually approaching gradually to the
final decrease of 21% at the end of the 7-day incubation. On the contrary, there was no significant change
in the tensile strength of the foam without cells. At the end of the 7-day incubation, the overall tensile

7
strength of PU with cells was slightly higher than that of PU without cells. Results from the stiffness
(modulus of elasticity) tests of the foams are similar to those of tensile strength tests. Same trends were
also found in the modulus of elasticity of the foam except that the modulus of the PU with cells was
slightly less than that of PU without cells at the end of the 7-day incubation.

25 50

20
40

Modulus of Elasticity (psi)

Tensile Strength (psi)

15

tensile strength of PU without cells 30

tensile strength of PU with cells
10 elastic modulus of PU without cells
elastic modulus of PU with cells

20
5

0 10
0 6 12 18 160

Incubation Time (hr)

Figure 6. Effects of microbiologically-induced calcite precipitation on tensile strength

and modulus of elasticity of PU.

Application Immobilized Cells in Concrete Crack Remediation

In order to examine the effects of MECR, simulated cracks (d = 2.54 cm, w = 0.312 cm) in
cement mortar cubes (5.08 × 5.08 × 5.08 cm) were loaded with PU-encapsulated B. pasteurii and treated
with urea-CaCl2 medium for 7 and 28 days, respectively. Fig. 7 depicts the changes in compressive
strength of the concrete cubes treated with different concentrations of immobilized microorganisms when
compared to the control cubes treated with cell-free PU polymer. The highest compressive strength was
obtained with the cubes that were remediated with a concentration of 5 × 109 immobilized cells crack-1 for
7 days. With a longer incubation period (28 days), the increase in strength of all
microbiologically-remediated cubes was found to be marginal when compared to that with 7-day
incubation. It was also observed that the PU polymer with or without cells became less rigid when
incubated longer than 7 days in urea-CaCl2 medium. Nonetheless, there was no apparent change in
compressive strength of the control cubes even for a prolonged incubation period in medium.

8
 500

400
Compressive Strength (psi)

300
5 x 107 cells crack-1
5 x 108 cell crack-1
200 5 x 109 cells crack-1

100

0
7 28

Incubation Time (day)

Figure 7. Increase of compressive strengths of concrete with cracks remediated with

different concentrations of B. pasteurii encapsulated in PU.

DISCUSSION

Role of microorganisms in CaCO3 precipitation

The overall equilibrium reaction of calcite precipitation can be described below.

Ca2+ + CO32- CaCO3↓ (1)

The solubility of CaCO3 is a function of pH and affected by ionic strength in the aqueous medium
(Stumm and Morgan, 1981). In urea-CaCl2 medium that supports microbial growth, NH4+ and Cl- also
react with OH- and H+, respectively, at different pH, further interfering with chemically-induced CaCO3
precipitation. Microbiologically-induced CaCO3 precipitation occurs via far more complicated processes
than chemically-induced precipitation. The bacterial cell surface with a variety of ions could
nonspecifically induce mineral deposition by providing a nucleation site (Ferris et al., 1986, 1987).
Especially, Ca2+ is not likely utilized by microbial metabolic processes, rather accumulates outside the
cell (Silver et al., 1975). In medium, it is possible that individual microorganisms produce ammonia as a
result of enzymatic urea hydrolysis to create an alkaline micro-environment around the cell. The high pH
of these localized areas, without a significant increase in pH in the entire medium at the beginning,
apparently commences the growth of CaCO3 crystals around the cell. Possible biochemical reactions in
urea-CaCl2 medium to precipitate CaCO3 at the cell surface can be summarized as follows.

Ca2+ + Cell → Cell-Ca2+ (2)

Cl- + HCO3- + NH3 NH4Cl + CO32- (3)
2+ 2-
Cell-Ca + CO3 → Cell-CaCO3↓ (4)

9
 In addition, the results of kinetic studies render an explanation that the rate of CaCO3
precipitation correlates with the cell growth and urease exhibits higher enzymatic activities and stronger
affinity to urea at higher pH levels (pH 8-9) where calcite precipitation is favorable. Observations from
the XRD analysis and electron micrography have also added credence to our previous study results
(Gollapudi et al., 1995; Zhong and Islam, 1995), providing conclusive evidence of direct participation of
B. pasteurii in calcite formation. Undoubtedly, B. pasteurii not only provides a nucleation site for calcite
precipitation but also creates an alkaline environment inducing further precipitation of calcite. Findings
from this study provide additional supports to the notion that in-situ implementation of microbial mineral
plugging might provide a practical means for bioremediation of porous media.

Role of the microbial urease in calcite precipitation

The role of the microbial urease was defined from the data (Fig. 3) that the calcite precipitation
by B. pasteurii and E. coli (pBU11) expressing B. pasteurii urease was inhibited in the presence of a
urease inhibitor and a significant amount of calcite precipitation was induced by E. coli (pBU11), whereas
little was induced by E. coli (pBR322) lacking urease genes. These observations support our assumption
that the urease enzyme is a primary factor that initiates microbiologically-induced calcite precipitation. In
detail, reaction 1 produces calcium carbonate and protons in aqueous medium where CO32- primarily stays
as HCO3-.

Ca2+ + HCO3- → CaCO3 + H+ (5)

In biological systems, many calcareous organisms couple calcification to their metabolic assimilation
processes to scavenge protons (McConnaughey and Whelan, 1996). In urease-based reactions, NH3
released by the enzymatic hydrolysis of urea uses the protons generated from the calcite precipitation
(reaction 5) to produce NH4+.

NH3 + H+ → NH4+ (6)

The subsequent increase of pH in surrounding medium due to the presence of ammonia ions and the
additional release of CO2 from the enzymatic urea hydrolysis further accelerate the rate of the urease-
induced calcite precipitation. Thus, an active participation of urease is of essence in biochemical calcite
precipitation.

Concrete crack remediation

It was hypothesized at the beginning of the study that calcite precipitation in the polymer matrices
might influence the physical characteristics of PU, particularly enhancing the tensile strength and the
stiffness. However, microbiologically-induced calcite precipitation in PU matrices did not yield any
increase in tensile strength of the foam. Although the reason for this phenomenon is not yet clearly
identified, it appears that the precipitation by calcite results in no specific chemical bonding between
CaCO3 and the foam, rather in simple accumulation of CaCO3 in the matrices. In general, the increases in
tensile strength and stiffness depend on the bond between the precipitated calcium carbonate and the PU
foam. The initial increases in the strength and elastic modulus, however, indicate that there is a possible
nonspecific interaction between the precipitated calcite and the foam.

We observed previously that the use of a mixture of sand and microorganisms in concrete
remediation increases the compressive strength (Ramachandran et al., 2001). However, the
microbiological calcite precipitation occurred mainly close to the surface area of the crack where dense
growth of calcite crystals embedded with cells was observed. In this study, it is observed that PU-

10
immobilized B. pasteurii used for the remediation of concrete cracks induces calcite precipitation
throughout the matrices. The major advantage of using PU in B. pasteurii encapsulation is that its matrix
can provide the microorganisms with a means of protection from the extremely alkaline environment of
concrete, while serving as additional nucleation sites for calcite crystals. Microbiologically-induced
calcite remains intact in PU matrix mainly because of the high pH of concrete, where the solubility of
CaCO3 is extremely low.

In summary, the positive potential of MECR that has been demonstrated in our study offers an
interesting concept of the crack remediation technique in various applications. For instance, biological
metabolic products can be utilized not only in crack remediation but also in sealing the gaps in various
structures. The latter application of the technique can utilize the immobilized urease instead of the whole
cell. In practice, the urease-laden PU polymer is placed in cracks to which a subsequent application of the
substrate induces calcite precipitation. To explore possibilities of using the immobilized urease in crack
remediation, detailed biochemical studies are currently under investigation.

ACKNOWLEDGEMENTS

This research was funded by grants from the National Science Foundation (CMS-9412942; -
9802127; INT-0002608). Authors express sincere appreciation Dr. S. D. Kim for providing the
recombinant urease. Authors acknowledge the vital contribution of Dr. E. F. Duke of the Engineering and
Mining Experiment Station at the South Dakota School of Mines and Technology for his technical
assistance in SEM and XRD.

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