Isolation, Characterization and Application of Calcite Producing Bacteria from Urea Rich Soils

Abstract

Calcium carbonate is one of the most common minerals widespread on earth (4% by weight of the earth's crust). Bacteria are incredibly diverse and abundant and many bacterial species contribute to the precipitation of mineral carbonates in various natural environments. Alkaline pH is the primary means by which microbes promote calcite precipitation which results from the hydrolysis of urea. The study used selective enrichment culture technique to isolate urease-producing bacteria from local urea rich soil and others materials. All isolates were identified using conventional biochemical tests. In addition, all isolates were tested for their ability to enhance the consolidation of sand and compressive strength of mortar as well as absorption reduction properties. One isolate with promising results was selected and optimization of environmental and nutritional conditions was performed. The growth curve of the selected strain with optimized condition was investigated. Thirty three isolates were obtained from the enrichment culture technique. Among them 13 isolates showed increased consolidation of sand. The isolate that showed the highest performance was identified as Bacillus mycoides. The optimum pH of the isolate was shown to be 7.0 and an optimum temperature of 35 o C was found. The growth curve was constructed with a stationary phase starting after 10 hours. The test results indicated that inclusion of Bacillus mycoides isolate in cement mortar enhanced the compressive strength, with a maximum increase of 17% in compressive strength and 32% reduction in water absorption was observed with a 28-day mortar sample. In conclusion, locally isolated strain identified as Bacillus mycoides enhanced the properties of the cement mortar. It is recommended that a larger scale application of this isolate be implemented.

Full text

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
Isolation, Characterization and
Application of Calcite Producing Bacteria
from Urea Rich Soils
Abdelraouf A. Elmanama1,a and Mohammed T.
Alhour2,b
1Islamic University of Gaza, Medical laboratory Sciences
Department, Gaza, Palestine.
2Islamic University of Gaza, Department of biology and
Biotechnology, Gaza, Palestine.
aelmanama@mail.iugaza.edu.ps, bmhour@live.com
Article Info
Received: 10/1/2013
Accepted: 28/9 2013
Published online: 1/12/2013
ISSN 2231-8844
Abstract
Calcium carbonate is one of the most common minerals widespread on earth (4% by weight of the
earth's crust). Bacteria are incredibly diverse and abundant and many bacterial species contribute to
the precipitation of mineral carbonates in various natural environments. Alkaline pH is the primary
means by which microbes promote calcite precipitation which results from the hydrolysis of urea. The
study used selective enrichment culture technique to isolate urease-producing bacteria from local urea
rich soil and others materials. All isolates were identified using conventional biochemical tests. In
addition, all isolates were tested for their ability to enhance the consolidation of sand and compressive
strength of mortar as well as absorption reduction properties. One isolate with promising results was
selected and optimization of environmental and nutritional conditions was performed. The growth
curve of the selected strain with optimized condition was investigated. Thirty three isolates were
obtained from the enrichment culture technique. Among them 13 isolates showed increased
consolidation of sand. The isolate that showed the highest performance was identified as Bacillus
mycoides. The optimum pH of the isolate was shown to be 7.0 and an optimum temperature of 35 oC
was found. The growth curve was constructed with a stationary phase starting after 10 hours. The test
results indicated that inclusion of Bacillus mycoides isolate in cement mortar enhanced the
compressive strength, with a maximum increase of 17% in compressive strength and 32% reduction in
water absorption was observed with a 28-day mortar sample. In conclusion, locally isolated strain
identified as Bacillus mycoides enhanced the properties of the cement mortar. It is recommended that
a larger scale application of this isolate be implemented.
Keywords: Calcite precipitation; Urease, Bacillus mycoides; Biocementation; MCP; Palestine

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
389
1. Introduction
Calcium carbonate (CaCO3) is one of the most common minerals widespread on earth,
constituting 4% by weight of the earth's crust. It is naturally found in extensive sedimentary
rock masses, as limestone, marble and calcareous sandstone in marine, freshwater and
terrestrial environment (Ehrlich, 1998; Castanier et al., 1999; Hammes & Verstraete, 2002).
Numerous different bacterial species have previously been detected and assumed to be
associated with natural carbonate precipitates from diverse environments. The primary role of
bacteria in the precipitation process has subsequently been ascribed to their ability to create
an alkaline environment (high pH) through various physiological activities (Douglas &
Beveridge, 1998; Ehrlich, 1998; Castanier et al., 1999; Castanier et al., 2000; Fujita et al.,
2000).
Three main existing groups of organisms that can induce MCP through their metabolic
processes are; (i) photosynthetic organisms such as cyanobacteria and algae that remove
CO2, (ii) sulphate reducing bacteria that are responsible for the dissimilatory reduction of
sulphate and (iii) several organisms that are involved in the nitrogen cycle (Castanier et al.,
1999; Hammes & Verstraete, 2002; Ariyanti et al., 2012 ). Urease hydrolyses the substrate
urea generating ammonia and carbamate. Carbamate spontaneously decomposes to produce
another molecule of ammonia and carbonic acid (Mobley & Hausinger, 1989). The two
ammonia molecules and carbonic acid subsequently equilibrate in water with their
deprotonated and protonated forms, resulting in an increase in the pH (Mobley & Hausinger,
1989). Many organisms can use urea as a source of nitrogen by importing urea into the cell's
cytoplasm. One of the most robust ureolytic bacteria is Sporosarcina pasteurii (formerly
known as Bacillus pasteurii). S. pasteurii is an aerobic, spore forming, rod shaped bacterium.
It uses urea as an energy source and produce ammonia which increases the pH in the
environment and generate carbonate, causing Ca2+ and CO32- to be precipitated as CaCO3
(Kroll, 1990; Stocks-Fischer et al., 1999; Chahal et al., 2011).
Calcium carbonate precipitation is a rather straightforward chemical process governed
mainly by four key factors: (1) the calcium concentration, (2) the concentration of dissolved
inorganic carbon (DIC), (3) the pH and (4) the availability of nucleation sites (Hammes &
Verstraete, 2002).
The aim of this study is to isolate and characterize strains of urease-producing bacteria that
are capable of calcite precipitation and investigate the effect of the selected strain on
enhancing the strength of mortar and decreasing permeability.
2. Materials and methods
2.1 Selective enrichment
To screen for strains with high level of urease activity soil, sludge and freshly cut concrete
surface samples were collected from different locations in the middle zone of Gaza strip that
are likely to contain ureolytic bacteria. To enrich the samples for urease-producing bacteria, 1
g of each sample was inoculated into 50 ml of nutrient broth (250 ml shaking flasks, at 28oC,
for 36 hours). The enrichment media consist of 10 g.L-1 Yeast extract (YE), 1M urea, 152
mM ammonium sulphate and 100 mM sodium acetate. The bacterial isolates were tested for
their ability to grow on 5% urea contained on NA. The strains capable of growth at this

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
390
concentration of urea were selected and inoculated into higher concentration reaching to 10%
of urea concentration. Finally, the strains with the tolerance to the highest urea concentration
were selected and used in subsequent experiments. All isolates were introduce to sand
column to perform sand consolidation, the cementaion solution was used in consolidation
process was 1M of urea and 0.75M of CaCl2.2H2O. This work was performed the effect of
bacterial isolates on different parameters as a following:
2.2 Compressive strength test
All isolates were grown in NB media for 24 hour and suspended in saline buffer, and the
isolates which showed higher strength in sand consolidation (designated as TN1B, TN3B,
TN5A and TN3E) were suspended in phosphate buffer. Mortars cubes with each buffer
contain no cells were prepared and regarded as control 1 and control 2 by using cementation
solution and tap water respectively.
The cement to sand to water ratio was 1:3:0.5 (by weight). All components were thoroughly
mixed with bacterial inoculums with a buffer by using slandered motor mixer 65-L0005. The
mortar cubes was left in the hydraulic shrinkage of cement mortar 65-L0010/B for 24 hour.
Compressive strength test of saline buffer was performed interval with 3, 14 and 28 day and
phosphate buffer with interval 3, 21 and 28 days.
2.3 Water absorption
To determine the increase in resistance towards water penetration, all mortar specimens
(saline and phosphate mortar cubes) were cast and cured in tap water for 28 days, saturated
overnight in water and weighed. The bricks were then dried in an oven at 100 oC for 24 h,
cooled and weighed again. Water absorption was calculated by using following formula:
Where Wsaturation is the weight of bricks after saturation in water for 24 h, and WOven dried is
the weight of bricks after oven drying for 24 h (Sarda et al,. 2009).
2.4 Optimization of B. mycoides growth conditions
2.4.1 Temperature optimization
NB medium was prepared and distributed into several flasks. A 3 ml of an overnight culture
was used to inoculate 30 ml media in a 250 conical flask. 4 flasks were prepared and
incubated at the following temperature (20, 25, 30, 35) and were incubated for 20 hour.
Samples were collected after 20 hour of incubation to measure the optical density
spectrophotometrically at 660 nm. The experiment was done in triplicate and the average
absorbance was recorded.
2.4.2 Optimum pH
NB medium was prepared and distributed into several flasks. A 3 ml of an overnight culture
was used to inoculate 30 ml media in a 250 conical flask. The pH of the medium was
adjusted using 1N NaOH to obtain the following pH values (6, 6.5 7, 7.5, 8, 8.5, 9). An equal
volume of the inoculums was added to each flask and incubated at the 35oC for 18 hour.

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
391
Sample was collected after 18 hour of incubation to measure the optical density
spectrophotometrically at 660nm. The experiment was done in triplicate and the average
absorbance was recorded.
2.4.3 Selection of appropriate growth media
Four standard media (Yeast Extract (YE), Beef Extract, Brain Heart infusion Broth (BHB)
and Nutrient Broth (NB)) were used to determine the most effective medium for mass cell
production of the selected isolate. In addition, two cheap formulation (Rabbit feed and Corn
Steep Liquor (CSL) were tested as alternative media for growth. The experiment was done in
triplicate and the average absorbance was recorded.
2.5 Bacterial growth curve of B. mycoides
An experiment to determine the growth curve of the selected strain B. mycoides was carried
out using shake flask culture technique to set a growth comparison point at optimized
condition. A 3 ml of an overnight culture was used to inoculate 30 ml of Rabbit feed media in
a 250 conical flask. The culture was incubated at 35 oC by shaking for 28 hours at 180 rpm.
Inoculation time was considered as zero time. Samples were taken from the culture at
different time intervals and used for quantitative determination of growth which was
measured spectrophotometrically at 660 nm. Viable cell count was determined as ''colony
forming units/ml'' CFUs simultaneously. A growth curve was constructed by plotting the
absorbance at 660 nm against sampling time.
3. Results and discussion
This study was conducted with the aim of isolating locally urease producing bacteria that
could be potentially used in various biocementation processes. Urea agar was used for the
selection of urease producing microorganisms, producing a red-pink color due to the presence
of phenol red, a pH indicator. Based on the qualitative urease productions, 33 isolates were
obtained. A method to specifically enrich bacteria from most soil within a short cultivation
period (36-48 hours), ideally suitable for biocementation. Selection conditions (high pH,
presence of urea up to 1 M) have enriched for a superior Bacillus type bacteria that can
degrade urea, is highly tolerant to urea and ammonia at high pH and hence ideally suited the
biocementation process. From the enrichment cultures, different ureolytic bacterial strains
were isolated with high urease activity which is required for biocementation process as was
suggested by whiffin, (2004). The isolates were and biochemically identified by using ABIS
software (Costin and Ionut, 2007-2013). Although the differences in some bacterial behaviors
between the isolates were evident, the most effective isolates were closely related to one
another. Similar finding was shown in a previous study Hammes et al., (2003) on
identification of ureolytic strains isolated from various environmental locations. This close
relationship between the isolates might be due to the dominance presence of Bacillus species
as was confirmed by Fleske et al., (1998). Stock-Fischer et al., (1999) have stated that
Bacillus species are selected by the isolation and cultivation methods. The phenotypic and
biochemical properties of the bacteria isolates were resemble those of Bacillus species
reported previously (Stocks-Fischer et al., 1999).

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
392
B. mycoides (TN1B) was gram positive with opaque creamy appearance on agar plate, non-
motile, and catalase positive. B. mycoides was unable to hydrolyze starch, but hydrolyze
casein, and lecithin. Citrate was no utilized by B. mycoides. When subjected to salinity and
temperature tests, the selected strain was able to survive 0-10% NaCl when incubated for 72
hour.
The sand columns prepared with all bacterial isolates to get the most efficient isolates for
sand consolidation, 13 isolates were found to tightly pack through consolidation process
while the control sand column collapsed immediately after opening the plastic column. Table
1 show the results of isolates positive or negative to sand consolidation. The predominance of
calcite precipitation in upper most surface area of the sand column might be due to higher
growth of bacteria in the presence of oxygen which consequently induces active precipitation
of CaCO3 around the surface area (Achal et al., 2010b). Similar results were reported in sand
by Whiffin et al., (2007); Achal et al (2010a); Harkes et al., (2010) & Dhamia et al., (2012).
Table (1): Isolates were used in sand consolidation
Isolates
Status
Isolates
Status
Isolates
Status
TN1A
-
TN11A
+
TN7
-
TN1B
+
TN11B
-
TN8A
-
TN2A
-
TN13A
-
TN9A
-
TN2B
+
TN14A
+
TN13B
-
TN3A
-
TN15
-
TN14B
+
TN3B
-
TN2C
+
TN13C
-
TN5A
+
TN3C
+
TN11C
-
TN10A
+
TN3D
+
TN14C
-
TN10B
-
TN6
+
TN3E
+
TN5B
-
TN9B
-
Control1
-
TN8B
-
TN2D
-
Control 2
-
TN16
+
TN10C
-
Control 1: sand + cementation solution - Control 2: Sand + distilled water
The compressive strength of cube mortars with saline buffer was significantly increased for
some mortar cubes that contained microbial cells. Fig 1 show 28-day compressive strength
test results with saline buffer. The highest compressive strength was obtained with mortar
cubes TN3C (35.6 MPa) and TN3E (35.9 MPa) prepared with cementation solution that were
incubated for 28 days as compared to control 2 (32.8 MPa) prepared with tap water. TN3C
and TN3E mortar cubes improvement in the compressive strength were 8.5% and 9.4%
respectively compared to control 2.
The compressive strength had significantly increased for 2 mortar cubes that
contained microbial cells.

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
393
Fig 1 Compressive test results at 28 day for mortar specimens with salinebuffer.
Fig 2 show 28-day compressive strength test results with saline buffer. The highest
compressive strength was obtained with mortar cubes TN1B (39.23 MPa) and TN5A (38.71
MPa) prepared with cementation solution that were incubated for 28 days as compared to
control (33.4 MPa) prepared with tap water. TN1B and TN5A mortar cubes improvement in
the compressive strength were 17.3% and 15.89% respectively as compared to control.
Fig 2 Summarizes the 28 day compressive strength of differentcement mortar specimens with
phosphate buffer.
The greatest improvement in compressive strength occurred with B. mycoides isolate in
phosphate buffer, there was 17.3% improvement in the compressive strength compared to the
control at 28 days. Fig 1 show Portland cement mortar cubes prepared in saline have shown
slight decrease in compressive strength in the presence of the cells. The decrease in
compressive strength of the cubes containing saline may be due to the presence of chloride
ions in the solution, which is known to weaken the integrity of the cement matrix reported
previously by Berke et al., (1988). The compressive strength of mortar cubes prepared in the
phosphate buffer was consistently higher than the strength of saline-prepared cubes as
suggested by Berke et al., (1988).
This improvement in compressive strength is probably due to deposition of CaCO3 on the
bacterial cell surfaces and within the pores of cement–sand matrix, which plug the pores

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
394
within the mortar as suggested by Ramakrishnan et al., (1998); Ghosh et al., (2005); Achal et
al., (2009a); Achal et al., (2009b); Achal et al., (2009c); Achal et al., (2010); Achal et al.,
(2010a); Achal et al., (2011); Ramachandran et al., (2011) & Vempada et al., (2011).
There was a measurable increase in compressive strength of cement mortar cubes prepared
with S. pasteurii, supported by previous studies (Bang & Ramachandran, 2001 &
Ramachandran et al., 2001). Thus, it was concluded that the increase in compressive
strengths is mainly due to consolidation of the pores inside the cement mortar cubes with
microbiologically induced calcium carbonate precipitation.
The influence of bacteria on the water absorption of mortar cubes is given in table 2. The
water absorption test was conducted by using saline buffer to determine the increase in
resistance towards water penetration in mortars cubes. Mortar cubes treated with bacteria and
a calcium source showed significantly less water absorption compared to untreated specimens
(control). It can be seen from this table that with the inclusion of all bacterial isolates, water
absorption capacity of mortars cubes decreased with compared to control specimens.
Table (2): % Water absorption test with saline buffer
Mortar
samples
Weight
saturation
Weight
dried
% water
absorption
% reduction in
water absorption
TN1B
129.6
120.1
7.9
19.2
TN2B
133.4
123.1
8.3
14.6
TN5A
131.4
121.4
8.2
15.9
TN10A
116
106.9
8.4
13.5
TN11A
121.3
112.2
8.1
17.2
TN14A
126.3
116.8
8.1
17
TN2C
133.8
123.8
8.0
17.5
TN3C
106.5
98.6
7.9
19
TN3D
123.2
114.1
7.9
18.6
TN6
118
108.5
8.6
11.5
TN14B
124.8
115.3
8.2
15.9
TN3E
138.6
128.5
7.8
19.7
TN16
121.7
112.7
8
18.5
Control
131.1
119.4
9.79
---
The influence of bacteria on the water absorption of mortar cubes with phosphate buffer is
given in table 3. The water absorption test was conducted by using phosphate buffer to
determine the increase in resistance towards water penetration in mortars cubes. Mortar cubes
treated with bacteria and a calcium source showed significantly less water absorption
compared to untreated specimens (control). It can be seen from this table that with the
inclusion of bacterial isolates, water absorption capacity of mortars cubes decreased with
compared to control specimens. Maximum reduction in water absorption test was showed in
TN1B isolate.

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
395
Table (3): % Water absorption test with phosphate buffer
Mortar
samples
Saturated
weight
Dried weight
% water
absorption
% reduction in
water absorption
TN1B
126.7
118.5
6.9
32.2
TN3E
119.7
110.7
8.1
20.3
TN5A
125.2
116.7
7.2
28.6
TN3C
128.9
119.3
8
21.1
Control
127.4
115.6
10.2
-
Mortar cubes treated with bacteria and a calcium source showed significantly decrease of
the water uptake compared to control specimens. Maximum reduction in water absorption
was observed with B. mycodes isolate with phosphate buffer gave 32.21%. Similar
observations were made by previous reports by De Muynck et al., (2008) and Achal et al.,
(2010). Nemati & Voordouw, (2003) & Whiffin, (2004) noticed an additional decrease of the
permeability of sandstone cores after injecting CaCO3 forming reactants for a second time.
The deposition of a layer of calcium carbonate on the surface and inside pores of the mortar
specimens resulted in a decrease of water absorption and permeability. It is clear that the
presence of a layer of carbonate crystals on the surface by bacterial cells has the potential to
improve the resistance of cementitious materials towards degradation process as suggested by
Achal et al., (2010); Achal et al., (2011) & Chahal et al., (2012).
Through previous results we can say that the TN1B isolate which is identified as Bacillus
mycoides (B. mycoides) was the best strain capable to increase strength of the cement mortar
and has the important role to decrease the water penetration through the mortar compared to
the control.
Optimum growth of B. mycoides was at pH 7. Table 4 showed the optimization of pH by
using different pH values.
Table (4): Optimizing pH of B. mycoides isolate
pH degree
Spectrophotometer absorbance
Trial 1
Trial 2
Trial 3
Average
6
1.431
1.488
1.455
1.458
6.5
1.554
1.512
1.54
1.53
7
1.731
1.698
1.719
1.716
7.5
1.63
1.65
1.67
1.65
8
1.517
1.5
1.48
1.49
8.5
1.375
1.34
1.31
1.34
9
1.29
1.31
1.31
1.308
Table 5 summarizes the bacterial growth levels in different standard and cheap formulation
media and showed that the best growth of the bacteria was yeast extract (YE). The cheap
formulation "rabbit feed" was superior to nutrient broth which is a well-known favorable
media used to grow bacteria at liquid state.

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
396
Table 5: Optimization of growth media
Media type
Spectrophotometer absorbance
Trial 1
Trial 2
Trial 3
Average
YE
1.827
1.833
1.825
1.828
Beef Extract
1.746
1.721
1.734
1.733
BHB
1.708
1.719
1.727
1.718
Malt Extract
1.362
1.369
1.375
1.368
N.B
1.685
1.679
1.681
1.681
CSL
0.853
0.815
0.832
0.833
Rabbit feed
1.719
1.706
1.825
1.707
Optimum temperature required for growth B. mycoides was 35 oC, table 6 showed the
optimization of temperature by using different temperature degrees.
Table (6): Temperature optimization of the B. mycoides isolate
Temperature
Spectrophotometer absorbance
Trial 1
Trial 2
Trial 3
Average
25oC
1.281
1.286
1.276
1.281
30oC
1.509
1.493
1.498
1.5
35oC
1.781
1.815
1.793
1.79
40oC
0.864
0.743
0.724
0.777
Growth curve of the B. mycoides strain was done using the partially optimized
conditions Fig 3 shows hour by hour growth curves to B. mycoides isolates which is
considered as the selected isolate of this research project. Growth curve for B. mycoides
isolate was constructed by plotting the OD660nm on the Y axis and incubation time on the X
axis. The maximum OD was seen between 6 to 10 hours and referred as log phase.
Spectrophotometer reading showed that the cultures reached the stationary after 10 hour.
Figure 3: Hour by hour of growth curve of B. mycoides isolates
0.00
0.50
1.00
1.50
2.00
OD660nm
incubation time (min)
Absorbance growth curve of B. mycoides
average

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
397
Conclusions
Thirty three isolates of urease producing bacteria were isolated from sixteen soil samples.
Thirteen isolates were shown to increase strength and consolidation of sand samples. Among
them B. mycoides isolate showed the highest results in sand consolidation, compressive
strength and water absorption test. Rabbit feed was shown to be a good growth medium. It is
recommended to further optimize all growth conditions.
References
Achal, V., Mukherjee, A & Basu, P. (2009a). Lactose mother liquor as an alternative nutrient
source for microbial concrete production by Sporosarcina pasteurii. Journal of Industrial
Microbiology & Biotechnology 36(3): 433–438.
Achal, V., Mukherjee, A & Reddy, M. (2010). Microbial concrete: A way to enhance
durability of building structures. Second International Conference on Sustainable
Construction Materials and Technologies, Università Politecnica delle Marche, Ancona,
Italy.
Achal, V., Mukherjee, A & Reddy, M.S. (2010a). Biocalcification by Sporosarcina pasteurii
using Corn steep liquor as nutrient source. Industrial Biotechnology.6(3): 170-174.
Achal, V., Mukherjee, A & Reddy, M.S. (2010b). Effect of calcifying bacteria on permeation
properties of concrete structures. Journal of Industrial Microbiology & Biotechnology
38(9): 1229-1234.
Achal, V., Mukherjee, A., Basu, P & Reddy, M. (2009b). Strain improvement of
Sporosarcina pasteurii for enhanced urease and calcite production. Journal of Industrial
Microbiology and Biotechnology 36(7): 981-988.
Achal, V., Pan, X & Özyurt, N. (2011). Improved strength and durability of fly ash-amended
concrete by microbial calcite precipitation. Ecological Engineering 37(4): 554–559.
Achal, V., Siddique, R., Reddy, M & Mukherjee, A. (2009c). Improvement in the
compressive strength of cement mortar by the use of a microorganism – Bacillus
megaterium. Excellence in Concrete Construction through Innovation – Limbachiya &
Kew (eds).
Ariyanti, D., Handayani, N & Hadiyanto. (2012). Feasibility of using Microalgae for
biocement production through biocementation. Journal of Bioprocess Biotechniq
2(111): 1-4.
Bang, S., Galinat, J & Ramakrishnan, V. (2001). Calcite precipitation induced by
polyurethane-immobilized Bacillus pasteurii. Enzyme and Microbial Technology 28(4-
5): 404–409.
Berke, N., Pfeifer, D & Weil, T. (1988). Protection against chloride-induced corrosion.
Concrete international journal 10(12); 54-55.
Castanier, S., Le Métayer-Levrel, G., & Perthuisot, J. P. (1999). Carbonates precipitation and
limestone genesis–the microbiologist point of view. Sedimentary Geology, 126 (1-4), 9-
23.
Castanier, S., Le Metayer-Levrel, G., & Perthuisot, J. P. (2000). Bacterial roles in the
precipitation of carbonate minerals. Microbial Sediments, 32-39.
Chahal, N., Rajor, A & Siddique, R. (2011). Calcium carbonate precipitation by different
bacterial strains. African Journal of Biotechnology 10(42): 8359-8372.

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
398
Chahal, N., Siddique, R & Rajor, A. (2012). Influence of bacteria on the compressive
strength, water absorption and rapid chloride permeability of fly ash concrete.
Construction and Building Materials 28(1): 351–356.
De Muynck, W., Debrouwer, D., Belie N & Verstraete W. (2008). Bacterial carbonate
precipitation improves the durability of cementitious materials. Cement and Concrete
Research 38(7): 1005–1014.
Dhami, N., Reddy, M & Mukherjee, A. (2012). Improvement in strength properties of ash
bricks by bacterial calcite. Ecological Engineering 39: 31-35.
Douglas, S & Beveridge, T. (1998). Mineral formation by bacteria in natural microbial
communities. FEMS Microbiology Ecology 26(2): 79–88.
Ehrlich, H. L. (1998). Geomicrobiology: its significance for geology. Earth-Science
Reviews, 45(1), 45-60.
Fleske, A., Akkermans, A & De Vos, W. (1998). In situ detection of an uncultured
predominant Bacillus in Dutch grassland soils. Applied and Environmental
Microbiology 64: 4588-4590.
Fujita, Y., Ferris, F. G., Lawson, R. D., Colwell, F. S., & Smith, R. W. (2000). Calcium
carbonate precipitation by ureolytic subsurface bacteria. Geomicrobiology Journal, 17,
305- 318.
Ghosh, P., Mandal, S., Chattopadhyay, B. D & Pal, S. (2005). Use of microorganism to
improve the strength of cement mortar. Cement and Concrete Research, 35(10): 1980-
1983.
Hammes, F., & Verstraete, W. (2002). Key roles of pH and calcium metabolism in microbial
carbonate precipitation. Reviews in Environmental Science and Biotechnology, 1(1), 3-
7..
Hammes, F., Boon, N., Clement, G., de Villiers, J., Siciliano, S. D., & Verstraete, W. (2003).
Molecular, biochemical and ecological characterisation of a bio-catalytic calcification
reactor. Applied microbiology and biotechnology,62(2-3), 191-201.
Harkes, M., van Paassen, L. A., Booster, J. L., Whiffin, V. S., & van Loosdrecht, M. C. M.
(2010). Fixation and distribution of bacterial activity in sand to induce carbonate
precipitation for ground reinforcement. Ecological Engineering, 36, 112-117.
Kroll, R. (1990). Microbiology of extreme environments. McGraw-Hill. New York, p: 55-
92.
Mobley, H. L. T., & Hausinger, R. P. (1989). Microbial Ureases: Significance, regulation and
molecular characterisation. Microbiological Review, 53(1), 85-108.
Nemati, M., & Voordouw, G. (2003). Modification of porous media permeability, using
calcium carbonate produced enzymatically in situ. Enzyme and Microbial Technology,
33, 635-642.
Ramachandran, SK., Ramakrishnan, V & Bang, S.S. (2001). Remediation of concrete using
microorganisms. American Concrete Institute 98(1):3–9.
Ramachandran, SK., Ramakrishnan, V & Bang, S.S. (2001). Remediation of concrete using
microorganisms. American Concrete Institute 98(1):3–9.
Ramakrishnan, V., Bang, SS & Deo KS. (1998). A novel technique for repairing cracks in
high performance concrete using bacteria. In: Proceedings of international conference
on high performance high strength concrete. Perth, Australia, pp 597–618.

Journal of Advanced Science and Engineering Research Vol 3, No 4 December (2013) 388-399
399
Sarda, D., Choonia, H., Sarode, D & Lele, S. (2009). Biocalcification by Bacillus pasteurii
urease: a novel application. Journal of Industrial Microbiology and Biotechnology
36(8): 1111-1115.
Stocks-Fischer, S., Galinat, J. K., & Bang, S. S. (1999). Microbiological precipitation of
CaCO3. Soil Biology and Biochemistry, 31(11), 1563-1571.
Vempada S., Reddy, S.S., Rao, M.V & Sasikala C. (2011). Strength enhancement of cement
mortar using microorganisms-An Experimental Study. International Journal of Earth
Sciences and Engineering 4(6): 933-936.
Whiffin, V., van Paassen, L & Harkes, M. (2007). Microbial carbonate precipitation as a Soil
improvement Technique. Geomicrobiology Journal 24(5): 417-423.
Whiffin, V.S. (2004). Microbial CaCO3 precipitation for the production of biocement. Ph.D
Thesis. Murdoch University, Western Australia.