4 Simposio de Investigacin en Sistemas Constructivos, Computacionales y Arquitectnicos

Gmez Palacio Durango Mxico

23 y 24 de Noviembre de 2011
1
EXPERIMENTAL STUDY ON THE EFFECT OF UREOLYTIC BACTERIA IN
COMPRESSIVE STRENGTH OF CEMENT MORTAR
R. Narayanasamy
1
, S.O. Garca Prez
1
, A.E. Saavedra Martinez
1
, S.R. Hernandez
Martinez
2
, N. Balagurusamy
2
y

J. Betancourt Hernandez
1


1. Facultad de Ingeniera, Ciencias y Arquitectura de la Universidad Jurez del Estado de Durango, Calle
Universidad s/n, Fraccionamiento Filadelfia, Gmez Palacio, Durango, Mxico.
2. Escuela de Ciencias Biolgicas, Universidad Autnoma de Coahuila, Torren, Coahuila, Mxico.

RESUMEN

Nuevas tcnicas han emergiendo en el campo de la Ingeniera Civil como la tcnica de
precipitacin de carbonato inducida por microbios (MICP) para mejorar las propiedades fsicas
como la resistencia, rigidez y permeabilidad de la arena, cemento y piedra, para satisfacer las
necesidades. Se utilizaron en este estudio, los organismos que viven en el suelo de la regin de
La Comarca Lagunera que muestra la actividad de la ureasa y forma calcita. Veintids cepas de
bacterias fueron aisladas. De los cuales, slo seis cepas fueron seleccionados en base a
su actividad enzimtica. El estudio se inici con la preparacin de los cubos de mortero de
cemento con tres cepas (bacterias), junto con la muestra de control para comparar su resistencia a
la compresin de 28 das. Este estudio revel un aumento significativo en la resistencia a la
compresin debido a la adicin de las bacterias a una concentracin de 10
8
clulas por ml de agua
utilizada para la mezcla.

Palabras clave: Bacteria, Cemento, Resistencia a la compresin

ABSTRACT

Novel techniques are being emerged in the field of Civil Engineering using microbially induced
carbonate precipitation (MICP) to improve the physical properties such as strength, stiffness and
permeability of sand, cement & stone to satisfy their needs. Bacteria living in the soil of Laguna
region showing urease activity and having the ability to form calcite precipitates were used in this
study. Out of twenty two bacterial strains isolated, only six strains were selected based on their
enzyme activity. The study was carried out by casting cement mortar cubes with three strains
(bacteria) and compared with control not having bacteria for their 28 day compressive strength. A
significant increase in the compressive strength was observed due to the addition of bacteria at a
cell concentration of 10
8
cells per ml of water used for mixing.

Key words: Bacteria, Cement, Compressive strength
Narayanasamy
2
INTRODUCTION

One of the inherent weaknesses of the concrete is the developments of cracks due
to shrinkage and temperature changes resulting in weak tension of concrete. Normally
steel reinforcements are introduced to take care of the tensile forces. But when the micro
cracks are formed on the surface of the concrete, water and other salts seep through these
cracks and corrode the steel reinforcement, and thereby reduce the durability or life of
concrete structures. It is observed that the ingress of aggressive environment through the
pores of concrete leads to the corrosion of reinforcing steel and carbonation, affecting the
durability of concrete. Prevention of crack formation has not been achieved till date and
considerable expenses are incurred in maintenance work at regular intervals to safeguard
the structures.

Recent interest in the term sustainability involves use of environment friendly
green technology, which involves the use of an agent of biological origin. In the case of
civil engineering and construction field , there is a need to develop alternative sustainable
technologies since the production and use of conventional Portland cement is a significant
contributor to greenhouse gases and the resultant global warming. Less dependence on
fossil energy and the use of innovative materials are global challenges. Janine Benyus [1]
defined the term biomimicry as innovation inspired by nature; it is looking to the
natural world for developing sustainable technologies.

Existing biological principles and advances in knowledge on microbially induced
carbonate precipitation (MICP) offer opportunities to use natural stable systems to meet
these challenges. Microbial mineral precipitation involves various microorganisms,
pathways and environments. Carbonate precipitation is carried out by ureolytic bacteria
by the production of urease enzyme. This enzyme catalyzes the hydrolysis of urea to CO
2

and ammonia, resulting in an increase of the pH and carbonate concentration in the
bacterial environment [2, 3]. Recent studies reveal that the addition of bacteria like
Bacillus pasteurii promoted self healing of the cracks in concrete since they are capable of
carbonate precipitation [4]. Moreover, it is reported that the durability of the concrete
increased with the increase in the concentration of bacteria. Application of bacteria as an
integrated healing agent to the concrete mixture would save the environment and money.

Hence, the objective of this paper is to study the effect of ureolytic bacteria on the
compressive strength of cement mortar cubes.

EXPERIMENTAL INVESTIGATION

Materials used

a) Cement
Ordinary Portland cement available in local market was used in this study. The cement used
has been tested for various properties as per ASTM C187 - 98 and C191-08.
b) Fine Aggregate
Sand available in the local market was used in this work. The sand was graded (Fig. 1) to
meet the requirements ASTM C 778 and ASTM 136 06 specifications.
Narayanasamy
3





c) Water
Locally available potable water confirming to ACI 318 - 2008 was used.
d) Microorganisms
The bacterial strains ACRN 6, ACRN 5 & ACRN 4 which showed ureolytic activity (Fig. 2)
and which were isolated from the soil of Laguna Region were used. They were multiplied in a
medium containing urea, harvested after 48 h and used to prepare mortar cubes.





Compressive strength test of cement mortar cubes

A total of 12 mortar cubes of dimensions 50 x 50 x 50 mm were prepared (Fig. 3). Three
cubes for each bacterial strain and three cubes without bacteria (Control) were cast. Cement
and sand were mixed properly in the ratio of 1:4 and a water cement ratio of 0.4 was used.
Bacterial strains were added at a cell concentration of 10
8
per ml of water, which was used for
mixing to the cement sand mix. Control samples were prepared with only water to make
cement sand paste. The mortar cubes were cast and the molds were placed in the moist
curing cabinet for 24 2 hrs. After demolding, they were cured in water for 28 days. The
Fig. 1. Grading of sand (size and weight)
as specified in ASTM C 136 - 06
Fig. 2. Bacterial strains showing ureolytic activity
Narayanasamy
4
cubes were removed from the curing chamber and wiped out well before testing for
compressive strength. The test was done as per the requirements of ASTM C109 / C109M -
93 Standard Test Method for Compressive Strength of Hydraulic Cement Mortars (Using 2-
in. or [50-mm] Cube Specimens).







RESULTS
Compressive strength of Cement mortar cubes (28 days) prepared with different
bacterial strains at a cell concentration of 10
8
cells per ml of mixing water are presented in
Table 1. Results showed that there is an increase in the compressive strength of mortar cubes
due to the addition of bacterial strains ACRN 5 & ACRN 4 compared to the control
specimens without bacteria (Fig. 4). Bacterial strain ACRN4 showed a significant increase of
21.92%. Whereas ACRN 5 recorded a 7.48% increase in compressive strength. Although
there is reduction in the compressive strength two of the specimens with ACRN 6, the
average of 3 specimens too showed a slight increase of 2.3%.

Table 1. Effect of added bacteria strains on Compressive strength of Mortar cubes

Treatments 28 day Compressive Strength of Mortar in Kg/cm
2

Specimen #1 Specimen # 2 Specimen #3
Increase (%) Increase (%) Increase (%)
Control (without
bacteria)
187 - 184 - 185 -
With ACRN 6 219 17.11 169 -8.15 180 -2.78
With ACRN 5 201 7.48 191 3.8 196 5.95
With ACRN 4 228 21.92 201 9.24 203 9.73
Fig.3. Preparation of mortar cube and determination of compressive strength
Narayanasamy
5


Fig. 4. Effect of added bacterial strains on Compressive strength of Mortar cubes



Results of this study were similar to that of other studies reported earlier [4, 5, 6 & 7]
and found that they were similar. The increase in material strength is due to the partial
filling of the pores by the Calcite precipitates formed due to the addition of bacteria.


CONCLUSIONS

The following conclusions are drawn based on the experimental results.
a) Ureolytic bacteria that can form calcites are isolated from the soils of Laguna region.
b) Addition of bacteria improved the hydrated structure of cement mortar.
c) Addition of bacteria increased the compressive strength of mortar cubes by 21.92% in
comparison to the conventional mortar cube.

It can be concluded that addition of local strains of bacteria can improve the
characteristics and performance of concrete.


ACKNOWLEDGEMENTS

R.N. thanks IBQ Alejandra Alvarado for her help with microbiological analyses of
this work. Similarly, support from the Bioremediation laboratory, Escuela de Ciencias
Biologicas, UAdeC, Torreon and laboratory, FICA, UJED, Gomez Palacio are
acknowledged for providing space and facilities to carry out this study.



0
50
100
150
200
250
Specimen #1 Specimen #2 Specimen #3
Control
ACRN 6
ACRN 5
ACRN 4
Narayanasamy
6
BIBLIOGRAFIA

1. Benyus, J.M. 1998. Biomimicry: Innovation Inspired by Nature. Perennial (Harper
Collins), ISBN-13: 978-0688160999
2. Stocks-Fischer, S., Galinat, J.K. and Bang, S.S. 1999. Microbiological precipitation of
CaCO3, Journal of Soil Biology and Biochemistry, 31, 1563 1571.
3. Narayanasamy, R., Villegas Flores, N., Betancourt Silva, F, Betancourt Hernandez, J.,
Balagurusamy, N. 2010. Concrete under severe conditions Environment and Loading,
CRC Press, Taylor & Francis Group, London, UK, ISBN: 978-0415593168.
4. Ramachandran, S.K., Ramakrishnan, V., Bang, S.S., 2001. Remediation of concrete
using microorganisms. ACI Materials Journal, 98, 39.
5. Ghosh, P., Mandal S., Chattopadhyay B.D., Pal S., 2005. Use of microorganism to
improve the strength of cement mortar. Cement Concrete Research, 35, 1980-1983.
6. Jonkers, H.M., Thijssen, A., Muyzer, G., Copuroglu, O., Schlangen, E., 2010.
Application of bacteria as self-healing agent for the development of sustainable
concrete, Ecological Engineering, 36 (2): 230-235.
7. Biswas, M., Majumdar, S., Chowdhury, T., Chattopadhyay, B., Mandal, S., Halder,
U., Yamasaki, S.2010. Bioremediase a unique protein from a novel bacterium BKH1,
ushering a new hope in concrete technology. Enzyme and Microbial Technology, 46,
581- 587.









Evaluacin de las bacterias Ureoliticas a diferentes
concentraciones en la resistencia a la compresin del
mortero
Rajeswari Narayanasamy
1*
, Arturo Eduardo Saavedra Martinez
1
, Sixto Omar Garca Prez
1
,
Alejandra Alvarado
2
, Nagamani Balagurusamy
2
y

Jos Betancourt Hernandez
1
1. Facultad de Ingeniera, Ciencias y Arquitectura de la Universidad Jurez del Estado de Durango, Calle
Universidad s/n, Fraccionamiento Filadelfia, Gmez Palacio, Durango, Mxico. *naraya@ujed.mx
2. Escuela de Ciencias Biolgicas, Universidad Autnoma de Coahuila, Torren, Coahuila, Mxico.

Resumen
Recientes estudios han revelado que la adicin de bacterias con la capacidad de producir minerales permiten
promover la capacidad auto-curativa del concreto aumentando la durabilidad. Se utilizaron en este estudio, los organismos que
viven en el suelo de la regin de La Comarca Lagunera que muestra la actividad de la ureasa y forma calcita. Se aadieron las
bacterias ACRN 4 a la mezcla de cemento en diferentes concentraciones para definir la concentracin ptima que da el mximo
resistencia a la compresin en comparacin con el mismo de cubos de morteros convencionales (los de control). La resistencia a
la compresin de los cubos de morteros fueron analizados y se encontr que la concentracin de 10
5
dio la mxima resistencia a
compresin.
Desarrollo
Nuevas tcnicas han emergiendo en el campo
de la Ingeniera Civil como la tcnica de precipitacin de
carbonato inducida por microbios (MICP) para mejorar las
propiedades fsicas como la resistencia, rigidez y
permeabilidad de la arena, cemento y piedra, para
satisfacer las necesidades[1]. Veinticuatro cepas de
bacterias fueron aisladas del suelo de la regin Lagunera
[2]. De los cuales, slo seis cepas fueron seleccionados en
base a su actividad enzimtica. El estudio se inici con la
preparacin de los cubos de mortero de cemento con tres
cepas (bacterias), junto con la muestra de control para
comparar su resistencia a la compresin de 28 das. Este
estudio revel un aumento significativo en la resistencia a
la compresin debido a la adicin de las bacterias a una
concentracin de 108 clulas por ml de agua utilizada
para la mezcla. Las bacteria ACRN 4 dio mayor resultado
comparando con otros cepas bacterianas[3]. Con este
base, avanzo la investigacion para seguir con este bacteria
en diferentes concentraciones.
Materiales y Mtodos
Se utilizo cemento Portland. El agua utilizada en el
mezclado esta libre de cidos, grasas y aceites; de materia
vegetal y orgnica; potable y adecuada para la
elaboracin. Granulometra del Arena acuerdo con ASTM
C 136 06 y modificada. La bacteria ACRN 4 aislada y
cultivada despus de 48 horas.

Los morteros tienen una relacin de cemento y arena de
1:3 y agua cemento de 0.4. Se colaron 16 cubos de
morteros con bacteria de cada concentracin. Los
concentraciones hicieron son 10
4
, 10
5
, 10
6
, 10
7
y 10
8
y de
control (sin bacteria). Se probaron en una mquina
universal de las edades 7, 14, 21 y 28 das para todos los
especmenes de mortero.
Resultados
Estos resultados se coinciden con los resultados que
menciona en [4].


Conclusin
La concentracin 10
5
de la bacteria ACRN 4 dio la mxima resistencia a compresin comparando con las otras
concentraciones y de control (sin bacteria).
Bibliografa
1. Narayanasamy, R., Villegas Flores, N., Betancourt Silva, F, Betancourt Hernandez, J., Balagurusamy, N. 2010. Concrete under severe conditions Environment
and Loading, CRC Press, Taylor & Francis Group, London, UK, ISBN: 978-0415593168.
2. C. L. Obregn Calvillo, A. G. Alvarado Rodrguez, R. Narayanasamy, N. Balagurusamy, SISCCA 2010. Biorremediacin - una tcnica novedosa en la construccin.
3. R. Narayanasamy, S.O. Garca Prez, A.E. Saavedra Martnez, S.R. Hernndez Martnez, N. Balagurusamy

y

J. Betancourt Hernndez, SISCCA 2011.
Experimental study on the effect of ureolytic bacteria in compressive strength of cement mortar.
4. Ghosh, P., Mandal S., Chattopadhyay B.D., Pal S., 2005. Use of microorganism to improve the strength of cement mortar. Cement Concrete Research, 35,
1980-1983.






EVALUACION DE LAS BACTERIAS UREOLITICAS A DIFERENTES CONCENTRACIONES EN
LA RESISTENCIA A LA COMPRESIN DEL MORTERO


Rajeswari Narayanasamy
1
*, Jos Humberto Perales Villareal
1
, Arturo Eduardo Saavedra
Martinez
1
, Alejandra Alvarado
2
, Nagamani Balagurusamy
2
, Jos Betancourt Hernandez
1





RESUMEN

Nuevas tcnicas han emergido en el campo de la Ingeniera Civil como la tcnica de precipitacin de
carbonato inducida por microbios (MICP) para mejorar las propiedades fsicas de la arena, concreto y piedra.
Se utilizaron los organismos que viven en el suelo de la regin Comarca Lagunera que muestra la actividad de
ureasa y tener la capacidad para formar calcita. Se agregaron bacterianas ACRN 1 y ACRN 4 a la mezcla de
cemento y arena 1:3 en diferentes concentraciones para definir la concentracin ptima que da el mximo
resistencia comparando con cubos de morteros convencionales (control). Este estudio revel un aumento
significativo en la resistencia a la compresin con una concentracin de 10
5
debido a la adicin de las
bacterias.

ABSTRACT

New techniques are emerging in the field of Civil Engineering as the technique of Microbial induced
carbonate precipitation (MICP) to improve the physical properties of sand, cement and stone, to meet the
needs. In this study, the organisms that live in the soil of the region which show urease activity and form
calcite were utilized. Bacterial strains ACRN 1 and ACRN 4 were added to the cement mixture in various
concentrations to determine the optimal concentration that gives the maximum compressive strength in
comparison with the same conventional mortar cubes (the control). This study revealed a significant increase
in compressive strength with a concentration of 10
5
due to the addition of bacteria ACRN 1 and ACRN 4.

INTRODUCCIN

Una de las debilidades inherentes del concreto es la formacin de las grietas debido a los cambios de
contraccin y temperatura. Normalmente se introducen refuerzos de acero para cuidar de las fuerzas de
traccin. Pero cuando los micros fisuras se forman sobre la superficie del concreto, el agua y otras sales se
filtran a travs de estas grietas, corroen el acero de refuerzo y reducen la durabilidad o vida de estructuras de
concreto. La prevencin de la formacin de grietas no se ha logrado hasta la fecha y los gastos considerables
ocasionados por los trabajos de mantenimiento a intervalos regulares para proteger las estructuras.

El reciente inters en el termino Sustentabilidad involucra el uso de la tecnologa favorable al medio
ambiente, lo que implica la utilizacin, de un agente de origen biolgico. En el caso de la Ingeniera Civil y
sector de la construccin, hay una necesidad de desarrollar tecnologas alternativas sustentables como la
produccin y el uso de cemento Portland convencional ya que es un importante contribuyente a los gases de
efecto invernadero que resulta en el calentamiento global. Menos dependencia de energas fsiles y el uso de

1 Facultad de Ingeniera, Ciencias y Arquitectura de la Universidad Jurez del Estado de Durango,
Calle Universidad s/n, Fraccionamiento Filadelfia, Gmez Palacio, Durango. Tel: (871) 715 2017;
naraya@ujed.mx (
1
*Estudiante del programa de doctorado interinstitucional en ingeniera civil)
2 Escuela de Ciencias Biolgicas, Universidad Autnoma de Coahuila, Torren, Coahuila, Mxico.

VII Ctedra Nacional de Ingeniera Civil, 13-15 Junio 2012, Zacatecas
materiales innovadores son los desafos globales. Janine Benyus define el termino biomimetismo como la
innovacin inspirada por la naturaleza, que esta mirando hacia el mundo natural para el desarrollo de
tecnologas sustentables [Benyus, 1998].

Los principios biolgicos existentes y la diversidad de avances en el conocimiento de la precipitacin de
carbonato microbiana inducida (MICP) ofrece oportunidades para utilizar los sistemas naturales estables para
enfrentar estos desafos. La precipitacin de carbonato se lleva acabo por bacterias ureoliticas por la
produccin de la enzima ureasa. Esta enzima cataliza la hidrolisis de la urea en CO2 y amoniaco, resultando
en un aumento del pH y la concentracin de carbonato en el medio ambiente bacteriana [Stocks- Fischer, et
al., 1999& Narayanasamy, et al., 2010]. Estudios recientes revelan que la adicin de bacterias como Bacillus
pasteurii promueve la auto curacin de las grietas en el concreto [Ramachandran, et al., 2001]. La aplicacin
de las bacterias como un agente curativo integrado a la mezcla de concreto salva el medio ambiente y dinero.

Veinticuatro cepas de bacterias fueron aisladas del suelo de la regin Lagunera [Obregn Calvillo, et al.,
2010]. De los cuales, slo seis cepas fueron seleccionados en base a su actividad enzimtica. El estudio se
inici con la preparacin de cubos de mortero de cemento con ACRN 4, 5 y 6, la cepa bacteriana ACRN 4
mostro un aumento significativo de 21.92% en la resistencia a la compresin de 28 das con una
concentracin de 10
8
comparando

con el control [Narayanasamy, et al., 2011]. Con esta base, el estudio
sigui con dos cepas bacterianas ACRN 1 y 4, junto con la muestra de control para comparar su resistencia a
la compresin de 7, 14, 21 y 28 das.

El objectivo de este articulo es realizar un estudio experimental para evaluar el efecto de las bacterias
ureoliticas en diferentes concentraciones y obtener una concentracion optima en la resistencia a la
compresion de los cubos de mortero Cemento.

MTODOS Y MATERIALES

Los materiales Utilizados:

a) Cemento
Se utilizo Cemento Portland disponible en el Mercado local en este estudio. Se ha sido probado para diversas
propiedades segn la norma ASTM C 187 98 y C 191 08.

b) Agregado Fino
Se utilizo la arena que esta disponible en el mercado local para este trabajo. Se modifico la arena (Fig.1) para
cumplir con los requisitos que menciona en las especificaciones ASTM C 778 y ASTM 136 06.



Figura 1. La clasificacin de arena (Tamao y peso) como se especifica en la norma ASTM C 136-06.

c) Agua
Se utiliza Agua potable disponible a nivel local, confirmo a ACI 318 2008.

d) Microrganismos
Se agreg las cepas bacterianas ACRN 4 y ACRN 1 que mostraron actividad ureoliticas (Fig.2) fueron
aislados de la tierra de la Regin Lagunera. Ellos se multiplicaron en un medio que contiene urea,


cosechndose despus de 48 horas y utilizada para preparar cubos de mortero Cemento [Obregn Calvillo, et
al., 2010].


Figura 2. Las cepas bacterianas que muestran actividad Ureolitica

Prueba de Resistencia a la compresin de los cubos de mortero de cemento

Se elaboro un total de 16 cubos de mortero de dimensiones 50 x 50 x50 mm (Fig.3) para cada concentracin
de cepa bacteriana. Se aadi las cepas bacterianas con diferentes concentraciones de 10
4
, 10
5
, 10
6
, 10
7
y 10
8

por ml de agua con la mezcla de arena y cemento. Se mezclo adecuadamente el cemento y arena en una
proporcin de 1:3 y con una relacin de agua y cemento de 0.4. Las muestras de control (sin bacteria) fueron
preparadas solo con agua para hacer la pasta de cemento y arena. Despus de colado, los moldes se colocaron
en la cmara de curado hmedo de 24 2 horas. Se curo los cubos despus del desmolde, en agua durante 28
das. Se retiraron los cubos de la cmara de curado y se seco a cabo mucho antes de las pruebas de
resistencia a la compresin. Se probaron en una mquina universal de las edades 7, 14, 21 y 28 das para
todos los especmenes de mortero. Se realizo las pruebas segn los requisitos de la norma ASTM C 109 /
C 109 M 93, mtodo de prueba estndar para resistencia a la compresin de los morteros de cemento
hidrulico.



Figura3. Preparacin de cubo de mortero y la determinacin de la Resistencia a la compresin


Resultados

Se presenta las resistencias a la compresin de los cubos de mortero de cemento preparado con 2 cepas
bacterianas ACRN 1 y 4 a diferentes concentracin celular de 10
4
, 10
5
, 10
6
, 10
7
y 10
8
por ml de agua con la
mezcla de arena y cemento en la tabla 1 y 2. Los resultados mostraron que hay un aumento en la resistencia a
VII Ctedra Nacional de Ingeniera Civil, 13-15 Junio 2012, Zacatecas
la compresin de los cubos de mortero, debido a la adicin de cepas bacterianas ACRN 1 y 4 en comparacin
con las muestras de control sin bacteria.



Tabla 1: Efecto de la adicin de cepas de bacteria ACRN 1 en la resistencia a la compresin de
los cubos de mortero


Los morteros que preparo con las cepas bacteriana ACRN 1 con una concentracin de1x 10
5
por ml de agua
(ver Fig. 4) mostro un aumento significativo de 12.7, 15.7 y 22.9% en resistencia a compresin despus de 7,
14 y 28 das de vida comparando con otros concentraciones y el control (sin bacteria) como muestra en la
tabla 1.



Figura 4. La Resistencia a la compresin Vs Vida en das (ACRN 1)


Tambin los morteros que preparo con las cepas bacteriana ACRN 4 con una concentracin de1 x 10
5
por ml
de agua (ver Fig. 5) mostro un aumento significativo de 23.12%, 16.4% and 14.4% en resistencia a
compresin despus de 7, 14 y 28 das de vida respectivamente comparando con otros concentraciones y el
control (sin bacteria) como muestra en la tabla 2.





Concentraci
n de
bacteria por
ml de agua
ACRN 1
Resistencia promedio de los especmenes (kg/cm2)
7 das 14 das 21 das 28 das
Aumento
(%)
Aumento
(%)
Aumento
(%)
Aumento
(%)
Control 184.5 - 228.5 - 277 - 252.25 -
1 x 10
8
174 -5.7 186 -18.6 187 -32.5 235 -6.84
1 x 10
7
196 6.23 224 -1.97 264 -4.7 299 18.5
1 x 10
6
214 13.8 228 -0.22 258 -6.8 250 -0.89
1 x 10
5
208 12.7 271 15.7 249 -10.1 310 22.9


Tabla 2: Efecto de la adicin de cepas de bacteria ACRN 4 en la resistencia a la compresin de
los cubos de mortero

Concentraci
n de bacteria
por ml de
agua
ACRN 4
Resistencia promedio de los especmenes (kg/cm2)
7 das 14 das 21 das 28 das
Aumento
(%)
Aumento
(%)
Aumento
(%)
Aumento
(%)
Control 184.5 - 228.5 - 277 - 252.25 -
1 x 10
8
203 10.0 214.25 -6.2 214.75 -22.5 205.75 -18.4
1 x 10
7
185 0.3 230.25 1.75 245.5 -11.4 253 0.3
1 x 10
6
204 10.6 216.25 -5.36 233.5 -15.7 255.5 1.3
1 x 10
5
240 23.12 266 16.4 263.75 -4.8 288.75 14.4
1 x 10
4
196.5 6.5 207 -9.4 219.75 -26.05 243.75 -3.4




Figura 5. La Resistencia a la compresin Vs Vida en das (ACRN 4)

Se encontr los resultados de este estudio fueron similares a los de otros estudios que hicieron anteriormente
[Ramachandran, et al., 2001, Ghosh, et al., 2005, Jonkers, et al., 2010 y Biswas, et al., 2010]. El aumento de
la resistencia del mortero es debido al llenado parcial de los poros por la calcita precipitada formado debido a
la adicin de bacteria.

CONCLUSIONES

Las conclusiones de los resultados experimentales son
a) Las bacterias Ureoliticas que pueden producir calcitas son aislados de los suelos de la regin Laguna.
b) Adicin de bacteria mejora la estructura hidratada de mortero de cemento.
c) La concentracin 10
5
de la bacteria ACRN 1 y 4 dio a la mxima resistencia a compresin
comparando con las otras concentraciones y de control (sin bacteria).
d) Adicin de bacteria ACRN 1 aumento la resistencia a la compresin de los cubos de mortero por
12.7, 15.7 y 22.9% despus de 7, 14 y 28 das de vida comparando con otros concentraciones y el
control (sin bacteria).
e) Adicin de bacteria ACRN 4 aumento la resistencia a la compresin de los cubos de mortero por
23.12%, 16.4% and 14.4% despus de 7, 14 y 28 das de vida comparando con otros concentraciones
y el control (sin bacteria).
VII Ctedra Nacional de Ingeniera Civil, 13-15 Junio 2012, Zacatecas

Se puede concluir que la adicin de cepas locales de bacteria puede mejorar las caractersticas y rendimiento
del concreto.

AGRADECIMIENTOS

El apoyo de laboratorio de Biorremediacin, la Escuela de Ciencias Biolgicas, UA de C, Torren y
Laboratorio de materiales de FICA, UJED, Gmez Palacio son reconocidos por proporcionar el espacio y las
instalaciones para llevar a cabo este estudio.

BIBLIOGRAFIA

Benyus, J.M. 1998. Bio mimicry: Innovation Inspired by Nature. Perennial (Harper Collins), ISBN-13: 978-
0688160999

Biswas, M., Majumdar, S., Chowdhury, T., Chattopadhyay, B., Mandal, S., Halder, U., Yamasaki, S.2010.
Bioremediase a unique protein from a novel bacterium BKH1, ushering a new hope in concrete technology.
Enzyme and Microbial Technology, 46, 581- 587.

Ghosh, P., Mandal S., Chattopadhyay B.D., Pal S., 2005. Use of microorganism to improve the strength of
cement mortar. Cement Concrete Research, 35, 1980-1983.

Jonkers, H.M., Thijssen, A., Muyzer, G., Copuroglu, O., Schlangen, E., 2010. Application of bacteria as self-
healing agent for the development of sustainable concrete, Ecological Engineering, 36 (2): 230-235.

Narayanasamy, R., Villegas Flores, N., Betancourt Silva, F, Betancourt Hernandez, J., Balagurusamy, N.
2010. Concrete under severe conditions Environment and Loading, CRC Press, Taylor & Francis Group,
London, UK, ISBN: 978-0415593168.

Narayanasamy, R., Garcia Prez, S.O., Saavedra Martinez, A.E., Hernndez Martinez, S. R., Balagurusamy

,
N . y

Betancourt Hernndez, J. SISCCA 2011. Experimental study on the effect of ureolytic bacteria in
compressive strength of cement mortar.

Obregn Calvillo, C.L., Alvarado Rodrguez, A. G., Narayanasamy, R., Balagurusamy, N. SISCCA 2010.
Biorremediacin - una tcnica novedosa en la construccin.

Ramachandran, S.K., Ramakrishnan, V., Bang, S.S., 2001. Remediation of concrete using microorganisms.
ACI Materials Journal, 98, 39.

Stocks-Fischer, S., Galinat, J.K. and Bang, S.S. 1999. Microbiological precipitation of CaCO3, Journal of
Soil Biology and Biochemistry, 31, 1563 1571.













MICROBIAL BIOTECHNOLOGY: AN ECO-SUSTAINABLE APPROACH TO
INCREASE COMPRESSIVE STRENGTH OF CEMENT MORTAR

















Rajeswari Narayanasamy
1
, Sara R. Hernndez
2
, Alejandra Alvarado
2
, Jos H.
Perales
1
, Jose Betancourt
1
and Nagamani Balagurusamy
2*










1
Facultad de Ingeniera, Ciencias y Arquitectura, Universidad Jurez del Estado de
Durango, Gmez Palacio, Durango, Mxico.
2
Laboratorio de Biorremediacin. Escuela de Ciencias Biolgicas, Universidad Autnoma
de Coahuila, Carretera Torren-Matamoros km 7.5, Torren, Coahuila, Mxico.
* Tel & Fax: (871) 7571785; e-mail: naraya@ujed.mx; bnagamani@uadec.edu.mx








1. INTRODUCTION

Cracks and fracturing is perennial problem in concretes and various commercial
products, viz., structural epoxy, resins and epoxy mortar are available for quick
remedy [Neville, 1996]. Prevention of crack formation has not been achieved till
date and considerable expenses are incurred in maintenance work at regular
intervals to safeguard the structures. A novel strategy to restore corroded
structures and repair concrete cracks is microbiologically induced calcite
precipitation (MICP) [Bang et al., 2001, Ramachandran et al., 2001, Ramakrishnan
et al., 2005].
Recent interest in the term sustainability involves use of environment friendly
green technology, which involves the use of an agent of biological origin. In the
case of civil engineering and construction field , there is a need to develop
alternative sustainable technologies since the production and use of conventional
Portland cement is a significant contributor to greenhouse gases and the resultant
global warming. Less dependence on fossil energy and the use of innovative
materials are global challenges. The term bio mimicry was defined by Janine
Benyus as innovation inspired by nature; it is looking to the natural world for
developing sustainable technologies [Benyus, 1998].
Existing biological principles and advances in knowledge on microbial induced
carbonate precipitation (MICP) offer opportunities to use natural stable systems to
meet these challenges. Microbial mineral precipitation involves various
microorganisms, pathways and environments. Carbonate precipitation is carried
out by ureolytic bacteria by the production of urease enzyme. This enzyme
catalyzes the hydrolysis of urea to CO
2
and ammonia, resulting in an increase of
the pH and carbonate concentration in the bacterial environment [Bang et al.,
2001, Day et al., 2003]. Recent studies reveal that the addition of bacteria like
Bacillus pasteurii promoted self healing of the cracks in concrete since they are
capable of carbonate precipitation [Ramakrishnan et al., 2005]. Moreover, it is
reported that the durability of the concrete increased with the increase in the
concentration of bacteria. Application of bacteria as an integrated healing agent to
the concrete mixture would save the environment and money.
Table 1. Brief review work done earlier using Micro organisms

Bio-agent Application Reference
B. pasteurii Sand Consolidation Kantzas et al., 1992
B. pasteurii Sand Consolidation Gollapudi et al., 1995
B. pasteurii Microbial Plugging in sand and cracks Zhong et al., 1995
in granite
B. pasteurii Remediation of concrete Ramachandran et al., 2001
B. Sphaericus On mortar surfaces and on concrete Heirman et al., 2003
surfaces
B. pasteurii Concrete crack remediation Day et al., 2003
Myxococcus Consolidation and protection of porous Navarro et al., 2003
Xanthus carbonate stones used in sculptural sphaericus Bacillus sphaericus Bacillus
sph and architectural heritage
B. pasteurii Concrete crack remediation Ramakrishnan et al., 2005
Shewanella sp., Improvement of mortar compressive Ghosh et al., 2005
Escherichia coli strength
Shewanella sp., Crack filling of concrete Mandal & Chattopadhyay, 2006
B. pseudofirmus Self healing concrete Henk et al., 2009
& B. Cohnii
B. Subtilis Improvement of strength of Concrete Sunil et al., 2010
B. Sphaericus Improvement of stength of Concrete Gavimath et al., 2012

The Importance of microbial mineral precipitation has been widely recognized in
Petroleum, Geological and Civil Engineering based on the investigation reports of
the remediation of cracks in rock formations, especially in oil reservoirs, sand
consolidation, ornamental stone repair, etc. The applications made so far are given
in chronological order (Table 1). After analyzing the behavior of microorganisms in
plugging the pores of rock by adhering to the available surfaces through
extracellular organic compounds, the research has been initiated in remediation of
cracks in man-made structures such as concrete. So far, the research groups from
United States of America (USA), Spain, Belgium, India, United Kingdom (UK) and
Netherlands are concentrating to solve this macro problem by using
microorganisms.
The first life-size experiments were carried out on the south-east tower of the
Saint Medard church (limestone) built during 12
th
century in Thouars, Deux Sevres,
France [Heirman et al., 2003]. The biomineralisation treatment was evaluated after
6 months and 1 year of application and found that the calcite layer behaves like the
natural calcin and it ages in the same way as the stone.
This study is aimed at isolation and selection of urease producing bacterial
strains from the soils of Comarca Lagunera of North-East Mexico. Twenty four
strains were tested, only six were selected due to their highest activity during the
scanning tests. Such strains were identified as ACRN1 (L2), ACRN2 (L1), ACRN3
(L2), ACRN4 (L2), ACRN5 (L1) and ACRN6 (L2). In this paper, the behavior of
bacterial strains ACRN 5 which showed ureolytic activity was studied and their
potential in increasing the compressive strength of cement mortar under different
cell concentrations were evaluated.

2. MATERIALS USED & METHODS
2.1. Cement
Ordinary Portland cement available in local market was used in this study. The
cement used has been tested for various properties as per ASTM C187 - 98 and
C191-08.
2.2. Fine Aggregate
Sand available in the local market was used in this work. The sand was graded
(Fig. 1) to meet the requirements ASTM C 778 and ASTM 136 06 specifications.

Figure 1. Granulometry of Sand
2.3. Water
Locally available potable water confirming to ACI 318 - 2008 was used.
2.4. Microorganisms y Isolation of urease producing bacteria & assay of
enzyme activity
Bacterial strains ACRN 5 which showed ureolytic activity were isolated and
multiplied in a medium containing urea, harvested after 48 h and used to prepare
mortar cubes. Urease producing bacteria were isolated from Comarca Lagunera
soils by using a selective medium containing (g/l) NaHCO
3,
2.12; urea, 20; peptone,
0.5; meat extract, 1.5; NH
4
Cl, 2.12; CaCl
2
2H
2
O, 30 mM; agar, 20. One unit of
urease activity is defined as the release of one mol of ammonia per min at 37 C
[Obregon et al., 2010].
2.5. Cement Mortar cube preparation and resistance test
Cement and sand were mixed properly at the ratio of 1:3 and a water cement
ratio of 0.4 was used. A total of 16 mortar cubes of dimensions 50x50x50 mm were
prepared by adding bacteria at different cell concentrations (10
4
, 10
5
, 10
6
, 10
7
y 10
8

per ml of water) to the water used for preparing the cement mortar. Control
samples were prepared with water only. Mortar cubes were cast and the molds
were placed in water in the moist curing cabinet. After 7, 14, 21 and 28 days, the
cubes were removed; wiped clear of water and the compressive strength were
determined by following the protocols mentioned in ASTM C109/C109M-93
Standard Test Method for Compressive Strength of Hydraulic Cement Mortars.
Scanning electron micrograph of the mortar prepared was also studied.

3. Results and Discussion
3.1 Growth of bacteria and enzyme activity
All strains reached their exponential phase after 24 hr, and dead phase after
three days. ACRN4 showed the highest biomass generation, while ACRN6 did not
grow at the same rate as the others (Figure 2).
According to the results of extracellular protein (Figure 3) strains ACRN2,
ACRN6, ACRN5 y ACRN3 increased protein production at 24 hr. of incubation at
35C, suggesting a rapid activity. In the case of strain ACRN1 extracellular protein
production was induced after two days, while in ACRN4 this happened after four

Figure 2. Cellular growth of the isolated ureolytic bacteria
days. In the case of cellular protein, ACRN6 and ACRN1 had the highest initial
concentration of 0,168 mg/ml both of them. ACRN1 strain reduced the amount of
protein in a 41.66% while the strain ACRN6 reduced 23.8%.
Figure 3. Extracellular protein (lines) and cellular protein (bars) of the ureolytic
strains isolated from soil of the Comarca Lagunera
Results of enzymatic activity show that all strains except ACRN5 reached their
maximum activity after 24 hours (Table 2 & Figure 4). ACRN6 displayed the
highest activity of 37.35 mol/ml, followed by ACRN3.
Table 2. Enzymatic activity of selected strains




Figure. 4. Enzyme activity (mol/ml).
After 72 hr. of incubation, cellular activity of ACRN1, ACRN4, and ACRN6 ACRN5
was measured. Unlike results in extracellular protein, ACRN4 showed better
activity, followed by ACRN1, ACRN5 and finally by ACRN6, which showed the best
extracellular activity. All strains resulted to be positive in the Gram test, and most of
them are rod-shaped bacteria.

Hour 0 24 48 72
ACRN 1 0 7.28 0.83 0.37
ACRN 3 0 18.65 2.89 0.15
ACRN 4 0 37.35 1.22 1.72
ACRN 5 0 0 2.86 1.32
ACRN 6 0 0 0.28 0.25
2.5. Compressive strength of mortar cubes

The results were noted in Table. 3 and comparative study has been made with
the control ones and also with the different concentrations of bacteria such as 10
8
,
10
7
, 10
6
, 10
5
and 10
4
. It was observed that the compressive strength of mortar

Table 3. Average Compressive strength of the specimens with various
concentrations of bacteria per ml of water.


Fig . 5. Compressive strength of mortar cubes in relation to bacterial population
0
50
100
150
200
250
300
350
400
0 7 14 21 28
CONTROL
1X10^8
1X10^7
1X10^6
1X10^5
1X10^4
Days
ACRN 5
ACRN -5
cubes increased due to the addition of ACRN 5 strain at a concentration of 10
5

cells per ml of water after 7, 21 & 28 days of curing (Fig. 5). SEM photomicrograph
of cement mortar prepared with bacteria showing calcite formation is shown in
Fig.6.

Fig. 6. Scanning Electron Micrograph of cement mortar prepared with bacteria

4. Conclusions
Addition of ureolytic bacteria isolated from the soils of Laguna region
improved the hydrated structure of cement mortar and increased the material
strength by the calcite precipitates formed, which partially filled the pores. From the
results it can be concluded that MICP is an efficient method for self-healing of
concrete. Addition of ACRN 5 strain at a concentration of 10
5
cells per ml of water
resulted in higher compressive strength of 37.7, 35.29 and 29.46% after 7, 21 and
28 days of curing compared with the other concentrations and the control
specimens. But, further research on growth phase of bacteria, their survival in
cement mortar and their self-healing property is warranted for authentication of this
technology.
5. REFERENCES
Bang, S.S., Galinat, J.K., Ramakrishnan, V., 2001. Calcite precipitation induced by
polyurethane-immobilized Bacillus pasteurii. Enzyme Microb. Technol. 28, 404
409.
Benyus, J.M. 1998. Bio mimicry: Innovation Inspired by Nature. Perennial (Harper
Collins), ISBN-13: 978-0688160999
Day, J.L., Ramakrishnan, V., and Bang, S.S., "Microbiologically induced sealant for
concrete crack remediation," Proceedings of the 16th Engineering Mechanics
Conference, Seattle, WA, 2003.
Gavimath. C.C., Mali B.M., Hooli V.R., Mallpur J.D., Patil A.B., Gaddi D.P.,
Ternikar C.R. and Ravishankera B.E. (2012). Potential application of bacteria to
improve the strength of cement concrete, International Journal of Advanced
Biotechnology and Research, 3(1), 541- 544.
Gollapudi, U.K., Knutson, C.L., Bang, S.S., Islam, M.R., 1995. A new method for
controlling leaching through permeable channels. Chemosphere 30, 695705.
Ghosh, P., Mandal S., Chattopadhyay B.D., Pal S., 2005. Use of microorganism to
improve the strength of cement mortar. Cement Concrete Res 35(10):1980-
1983.
Heirman, G., Herremans.T., Vangheel.T., Van Gemert. D., 2003. Biological repair
of damaged concrete and mortar surfaces: Biomineralisation. 6th Int. Conf. on
Materials Science and Restoration (MSR VI). Karlsruhe, 16-18 September
2003, 501-508.
Henk M. J., Arjan T., Gerard M., Oguzhan C., Erik S., 2008. Application of bacteria

as self-healing agent for the development of sustainable concrete, Ecological
Engineering, 2009.
Kantzas, A., Ferris, G.F., Jha, K.N., Mourits, F.M., 1992. A novel method of Sand
Consolidation through Bacteriogenic Mineral Plugging, paper presented at
the CIM Annual Technical Conference, Calgary, June 8-10.
Mandal, S., Chattopadhyay, B.D., 2006. Big Patents India, A process for preparing
modified bioconcrete, Application 263/KOL/2006 published 2006-04-28, filed
2006-03-27.
Neville. A.M., 1996. Properties of concrete, 4
th
edition, Pearson Higher Education,
Prentice Hall, NJ.
Obregn Calvillo, C.L., Alvarado Rodrguez, A. G., Narayanasamy, R.,
Balagurusamy, N. SISCCA 2010. Biorremediacin - una tcnica novedosa
en la construccin.
Ramachandran, S.K., Ramakrishnan, V., Bang, S.S., 2001. Remediation of
concrete using microorganisms. ACI Materials Journal, 98, 39.
Ramakrishnan, V., Panchalan, R.K., and Bang, S.S. 2005. Improvement of
Concrete durability by bacterial mineral precipitation. Proceedings of the 11
th

International conference on Fracture, Turin, Italy.
Rodrguez- Navarro, C., Rodrguez-Gallego, M., Ben Chekroun, K., and Gonzlez-
Muoz, M.T., 2003. Conservation of ornamental Stone by Myxococcus
Xanthus- Induced Carbonate biomineralization, Appl. Environ. Microb. 69,
2182-2193.
Sunil Pratap Reddy S., Sheshagiri Rao M.V., Aparna P., Sasikala C.H. (2010).
Performance of standard grade bacterial concrete, Asian Journal of Civil
Engineering, 11(1), 43-55.
Zhong, L. and Islam, M.R. (1995). A new microbial plugging process and its impact
on fracture remediation (SPE 30519). Proceedings of the 70
th
Annual Technical
Conference and Exhibition of the Society of Petroleum Engineers, Dallas,
Texas, 703-715.









Ghent University Magnel Laboratory for Concrete Research Technologiepark-Zwijnaarde 904 B-9052 Ghent (Belgium)
Tel : +32 9 264 55 18 Fax : +32 9 264 58 45 ICSHM2013@UGent.be www.ICSHM2013.be
Conference Chairs
Professor Nele DE BELIE
Ghent University, Belgium

Professor Sybrand VAN DER ZWAAG
Delft University of Technology, the Netherlands

Symposium Secretariat
Marijke Reunes
e-mail: ICSHM2013@UGent.be
website: www.ICSHM2013.be


BELGIUM Ghent
16 20 June 2013


Prof. M. Ing. Rajeswari Narayanasamy
Universidad Juarez del Estado de Durango
RFC : UJE 570321 HBO
Constitucion No. 404 Sur,
Col. Centro, Durango, Durango, Mexico 34000



Ghent, 2013-04-04


Letter of invitation



Dear Prof.,

It is my pleasure to invite you to the ICSHM2013 Fourth International Conference on Self-
Healing Materials in Ghent (Belgium) on 16-20 June 2013.
It is very important for us that you come to Ghent to present your paper (ID no. CM-56
Potential of soil bacteria from the Comarca Lagunera, North-East Mexico for bioconcrete
development and to share your experiences on self-healing materials and to make the
conference more meaningful.

The Conference fee will be 340 euro.

We are looking forward to welcoming you in Ghent in June 2013.

Sincerely,




Prof. Dr. Ir. Nele DE BELIE
Conference Chair
Dear author,

We are pleased to inform you that your submission (Previous ID no. : BIM-07; New
ID no. : CM-56) has been accepted for an oral presentation at the International
Conference on Self-Healing Materials which will take place in Ghent (Belgium)
from 16 till 20 June 2013. Possibly, the abstract ID No of your contribution has
changed, based on the comments of the reviewers.

The comments of the reviewers are inserted below.
REVIEWER #1: No comments

REVIEWER #2: No comments

Please revise your abstract in correspondence with these comments
and prepare your four-page manuscript (including the abstract) according to
the instructions given on the website (http://www.icshm2013.be).

The four-page manuscript and the revised abstract can be e-mailed as an
attachment to the symposium secretariat at ICSHM2013@UGent.be before 15
March 2013 (word and pdf document). Please indicate in the subject of your e-mail
your abstract ID No (as given above by the symposium secretariat).

Please do not forget to revise your abstract (max. 300 words), taking into account
the remarks of the reviewers. Only the 300-words abstracts will be published in an
abstract book. The manuscripts will be distributed to the participants via USB.

The early registration deadline at reduced fee is 15 March 2013. Please do not
forget that at least the presenting author has to be registered before 15 March
2013 in order to guarantee that the contribution will be part of the conference
program and proceedings.

Please also communicate this notification of acceptance to the co-authors of your
publication.

Please do not hesitate to contact the symposium secretariat
at ICSHM2013@UGent.be in case of any questions.

Kind regards,
Reunes Marijke
Secretariat
___________________________________________________________

The 4
th
International Conference on Self-Healing Materials
will be held in the city of Ghent from 16 to 20 June 2013.
For more information about this event, we refer to
the conference website www.ICSHM2013.be.
Potential of soil bacteria from the Comarca Lagunera, North-East
Mexico for bioconcrete development

R. Narayanasamy
1
, A. Alvarado
2
, J. Sanchez Medrano
1
, J. Betancourt Hernandez
1

and N. Balagurusamy
2

1
Facultad de Ingeniera, Ciencias y Arquitectura, Universidad Jurez del Estado de Durango,
Av. Universidad S/N Fracc. Filadelfia, C.P. 35010, Apdo. Postal 36 B, Gmez Palacio,
Durango, Mxico. e-mail: naraya@ujed.mx; jesus_ocl@hotmail.com; jbetancourth@ujed.mx
2
Laboratorio de Biorremediacin, Escuela de Ciencias Biolgicas, Universidad Autnoma de
Coahuila, Carretera Torren- Matamoros km 7.5, C.P.27000, Torren, Coahuila, Mxico.
e-mail: ale.alv89@gmail.com; bnagamani@uadec.edu.mx


Keywords: concrete, cement mortar, bacteria, self-healing, compressive strength

Abstract No: Previous ID no. : BIM-07; New ID no. : CM-56

Sustainability or environment friendly green technology is based on the use of agents
of biological origin that can mimic nature in their process applications. The production
and use of conventional Portland cement is significant contributor to emission of
greenhouse gases and the resultant global warming. Microbial induced carbonate
precipitation (MICP) is an emerging technology to minimize the environmental
problems, to improve the concrete qualities, and more importantly as a self-healing
agent.

This study was aimed at isolation, selection and evaluation of urease producing
bacterial strains from the soils of Comarca Lagunera of North-East Mexico.
Carbonate precipitation is achieved by urease enzyme, which catalyzes the
hydrolysis of urea to CO
2
and ammonia, resulting in an increase of the pH and
carbonate precipitation.

Out of twenty four bacterial strains isolated, six were selected based on their urease
activity and were denoted as ACRN1 to ACRN6. All strains recorded their maximum
growth after 24 h and death phase after three days. ACRN4 showed the highest
biomass production at 35C, while ACRN6 recorded the least growth rate. Initially the
ACRN4, ACRN5 and ACRN6 were evaluated for their potential in increasing the
compressive strength of cement mortar by varying the cell concentrations and were
observed that addition of bacteria at 10
5
cells significantly increased the compressive
strength around 35%.

However, concrete specimens were prepared with ACRN4 at same concentration, in
the presence and absence of water reducing additives showed an increase of 4%
and a decrease of 6.64% in their compressive strength on 14th day, and a decrease
of 6% and 7.6% on 28th day, with and without additive respectively. Scanning
electron microscopic and X-ray diffraction studies are in progress to understand the
phenomenon observed.


1. INTRODUCTION

Recent interest in the term sustainability involves use of environment friendly green
technology, which involves the use of an agent of biological origin.Existing biological
principles and advances in knowledge on microbial induced carbonate precipitation
(MICP) offer opportunities to use natural stable systems to meet these challenges.
Recent studies reveal that the addition of bacteria like Bacillus pasteurii, Bacillus
sphaericus, Shewanella sp., Bacillus pseudofirmus, B. Cohnii and Bacillus Subtilis
promoted self healing of the cracks in concrete since they are capable of carbonate
precipitation [1]. This study was focused to evaluate the behavior of bacterial strains
on the bio concrete.

2. MATERIALS & METHODS

Ordinary Portland cement, gravel and sand available in the local market were used
for this study. Distilled water was used for the isolation of bacterial strains. Locally
available potable water (tap water) was used for the preparation of mortar cubes and
concrete cylinders. Out of 24 bacterial strains from the soils of Comarca Laguna of
North-East Mexico which has the ability to produce Urease were isolated, evaluated
and the best six (ACRN1 to ACRN6) were selected based on their urease activity for
the mortar and concrete preparation. Bacterial strains were multiplied in the urea
medium and harvested after 48 hrs. were used to prepare mortar cubes and concrete
cylinders.
Cement Mortar Cubes preparation and Compressive Strength Testing
a) Three cubes of dimensions 50 x 50 x 50 mm for each bacterial strain and three
cubes without bacteria (Control) were cast. Bacterial strains ACRN 4, ACRN 5 and
ACRN 6 were added at a cell concentration of 10
8
per ml of water to prepare the
cement mortar. The compressive strength was determined after 28 days of curing.
b) Mortar cubes were prepared by adding bacteria ACRN 4 (higher compressive
strength compared to other strains) at different cell concentrations (10
4
, 10
5
, 10
6
, 10
7

y 10
8
per ml of water) to the water used for preparing the cement mortar. The tap
water was used for the dilutions of the bacteria cells and was used directly for the
preparation of mortar samples. After 24 hrs. of casting, all the specimens with or
without cells were demolded and were cured under water in the moist curing cabinet.
The compressive strength was determined after 7, 14, 21 and 28 days.
Concrete cylinder preparation and Compressive Strength Testing
Cement, coarse aggregate and fine aggregate were mixed properly to obtain a
concrete of strength, fc 200 kg/cm
2
. A total of 60 concrete cylinders of dimensions
150 mm diameter with 300 mm height were prepared by adding bacteria ACRN 4 at
its optimum concentration (identified from the mortar cube samples) of 10
5
per ml of
water to the water used for preparing the concrete. After 24 hrs. of casting, all the
specimens with or without bacteria were demolded and were cured under water in
the moist curing cabinet. The compressive strength of the cylinders was determined
after 3, 7, 14 and 28 days.
SEM analysis of Concrete Cylinder specimens
The broken concrete cylinder samples (with / without bacteria) collected after
compressive strength testing were thinned down to small pieces and were coated
with carbon prior to SEM examination. The prepared samples were examined in
ESEM (Fei Quanta 600 ESEM
TM
) with Tungsten Filament (W) with high vacuum
mode to take the microphotographs.

3. RESULTS & DISCUSSION

Compared to control, the mortar cubes prepared with bacterial strain ACRN 4 with
10
8
cells per ml of water showed higher compressive strength of 21.92 % after 28
days (Table 1). From the compressive strength results of the mortar cubes prepared
with 10
8
, 10
7
, 10
6
, 10
5
and 10
4
cells per ml of mixing water, it was found that the
optimum cell concentration of the bacterial strain ACRN 4 was 10
5
cells per ml
(Figure 1) with an increase of 18.83% compared to the control after 28 days, which is
almost similar to the results reported earlier [2].

Table 1. Effect of added bacteria strains on Compressive strength of Mortar cubes

Treatments 28 day Compressive Strength of Mortar (Kg/cm
2
)
Specimen #1 Specimen # 2 Specimen #3
Increase (%) Increase (%) Increase (%)
Control(without
bacteria)
187 - 184 - 185 -
With ACRN 6 219 17.11 169 -8.15 180 -2.78
With ACRN 5 201 7.48 191 3.8 196 5.95
With ACRN 4 228 21.92 201 9.24 203 9.73


Figure 1: Compressive strength of Cement mortar cubes in relation to different cell
concentrations of bacterial strain ACRN 4.

The addition of ACRN 4 strain at a concentration of 10
5
cells per ml of water in
concrete cylinders resulted in higher compressive strength (4.11% increase) after 14
days of curing compared with the control specimens. The cylinders prepared with
bacteria added with admixture showed lower compressive strength (7.60 %
decrease) after 28 days of curing period. But the cylinders prepared with admixture
only showed higher compressive strength (8.57% increase) after 28 days of curing
period (Figure 2). The prepared broken samples of concrete cylinders, control one
and with bacteria and admixture were examined (Figure 3). Figure 3(b) revealed the
formation of calcite crystals with well-formed rhombohedrical shape as reported [3].
SEM image (Figure 3(c)) showed the presence of microorganisms on the surface of
the concrete cylinder sample prepared with bacteria and admixture.

0
100
200
300
400
0 7 14 21 28
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ACRN-4
CONTROL
1X10^8
1X10^7
1X10^6
1X10^5
1X10^4
Days


Figure 2: Compressive strength of Concrete Cylinders in relation to bacteria



Figure 3: SEM images of Concrete Cylinder Samples (a) control (b) & (c) with
bacteria and additive

4. CONCLUSION

Ureolytic bacteria that can form calcites are isolated from the soils of Laguna region.
Cell density (ACRN 4) of 10
5
per ml of water was found to be optimum concentration,
which increased the compressive strength of concrete cylinders. SEM studies
confirmed the formation of calcite crystals and the presence of bacteria on the
surface of the broken samples of concrete cylinder.

ACKNOWLEDGEMENTS
Authors gratefully acknowledge the help of M.C. Juan Fernando de la Rosa, CIDT,
Peoles, Torreon in SEM analysis.

REFERENCES
[1] W. De Muynck. N. De Belie, W. Verstraete, Microbial carbonate precipitation in
construction materials: A review, Ecological Engineering 36 (2009)118 136.
[2] M. Biswas, S. Majumdar, T. Chowdhury, B. Chattopadhyay, S. Mandal, U. Halder,
S. Yamasaki, Bioremediase a unique protein from a novel bacterium BKH1, ushering
a new hope in concrete technology, Enzyme and Microbial Technology 46 (2010)
581- 587.
[3] J. Dick, W. de Windt, B. de Graef, H. Saveyn, P. Van der Meeren, N. de Belie &
W. Verstraete, Bio- deposition of a calcium carbonate layer on degraded limestone
by Bacillus species, Biodegradation (2006) 17: 357-367.
0
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Concrete Specimens
CONTROL
with bacteria
control with
additive
with bacteria
& additive
Days
(a)
(b) (c)