Accepted Manuscript

Title: Isolation and identification of bacteria to improve the

strength of concrete

Author: S. Krishnapriya D.L. Venkatesh Babu G. Prince

Arulraj

PII: S0944-5013(15)00050-6
DOI: http://dx.doi.org/doi:10.1016/j.micres.2015.03.009
Reference: MICRES 25763

To appear in:

Received date: 2-11-2014

Revised date: 12-3-2015
Accepted date: 16-3-2015

Please cite this article as: Krishnapriya S, Venkatesh Babu DL, Arulraj GP, Isolation and
identification of bacteria to improve the strength of concrete, Microbiological Research
(2015), http://dx.doi.org/10.1016/j.micres.2015.03.009

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Isolation and identification of bacteria to improve the strength of concrete

S.Krishnapriyaa , D.L.Venkatesh Babub, G.Prince Arulrajc

a

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Department of Civil Engineering, SNS College of Technology, Coimbatore ,India,

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E mail:kpriyasivakumar@gmail.com

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b
Department of Civil Engineering, JSS Academy of Technical Education,

Bangalore ,India,

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E mail:drdlvbabu@gmail.com

an
c
Department of Civil Engineering, SNS College of Technology, Coimbatore ,India,

E mail:princearulraj@yahoo.com
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Corresponding Author: Mrs.S.Krishnapriya, Department of Civil Engineering,

SNS College of Technology, Coimbatore - 641035, India.

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E-mail:kpriyasivakumar@gmail.com,pnskumar01@gmail.com
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Phone: +91 9688218146

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Fax:+914222665138
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ABSTRACT

The objective of this research work is to isolate and identify calcite precipitating
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bacteria and to check the suitability of these bacteria for use in concrete to improve

its strength. Bacteria to be incorporated in concrete should be alkali resistant to

endure the high pH of concrete and endospore forming to withstand the mechanical

stresses induced in concrete during mixing. They must exhibit high urease activity

to precipitate calcium carbonate in the form of calcite. Bacterial strains were

Page 1 of 37
isolated from alkaline soil samples of a cement factory and were tested for urease

activity, potential to form endospores and precipitation of calcium carbonate. Based

on these results, three isolates were selected and identified by 16S rRNA gene

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ip
sequencing. They were identified as Bacillus megaterium BSKAU, Bacillus

licheniformis BSKNAU and Bacillus flexus BSKNAU. The results were compared

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with Bacillus megaterium MTCC 1684 obtained from Microbial Type Culture

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Collection and Gene Bank , Chandigarh, India. Experimental work was carried out

to assess the influence of bacteria on the compressive strength and tests revealed

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that bacterial concrete specimens showed enhancement in compressive strength.

The efficiency of bacteria towards crack healing was also tested. Substantial
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increase in strength and complete healing of cracks was observed in concrete
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specimens cast with Bacillus megaterium BSKAU, Bacillus licheniformis

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BSKNAU and Bacillus megaterium MTCC 1684.This indicates the suitability of

these bacterial strains for use in concrete. The enhancement of strength and healing
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of cracks can be attributed to the filling of cracks in concrete by calcite which was
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visualized by Scanning Electron Microscope.

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Keywords: Bacteria. Concrete. Suitability tests. Compressive strength. Crack

healing. SEM

1. Introduction

Concrete is the second most consumed material on earth, next only to water. But

it is susceptible to micro crack formation and has pores in it. Micro cracks and pores

in concrete are highly undesirable because they provide an open pathway for the

Page 2 of 37
ingress of water and other deleterious substances. This leads to corrosion of

reinforcement and reduces the strength and durability of concrete. Large costs are

incurred all over the globe to repair cracks in concrete. For repairing cracks, a

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variety of techniques are available but majority of traditional repair systems are

chemical based, expensive and lead to environmental and health hazards. Recently,

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microbiologically induced calcite precipitation has been proposed as an effective

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alternative repair technique for plugging of micro cracks and pores in concrete. This

bacterial remediation technique surpasses other techniques as it is bio based, eco

friendly, cost effective and durable.

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Urease positive bacteria have been found to influence the precipitation of
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calcium carbonate (calcite) by the production of an urease enzyme. This enzyme
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catalyzes the hydrolysis of urea to CO2 and ammonia, resulting in an increase of the
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pH and calcite precipitation in the bacterial environment (Stocks-Fischer et al.

1999).
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This innovative environmental friendly method was first used for the repair
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of cracks to prevent leaching in channels (Gollapudi et al.1995). It can also be used

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for enhanced oil recovery, prevention of acid mine drainage, for remediation of

granite, mortar, limestone and concrete (Ramakrishnan et al. 2001;Zhong and Islam,

1995).

The calcite precipitation induced by Bacillus pasteruii and

Bacillus sphaericus was found to be effective in remediating cracks of concrete and

increased its compressive strength (Ramakrishnan et al. 2001;

Page 3 of 37
Van Tittelboom et al.2010).The durability of concrete specimens treated with

Bacillus pasteruii exposed to alkaline, sulphate and freeze-thaw environments was

also reported to increase (Ramakrishnan et al.2005).

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Considerable research work on concrete incorporating bacterial species Bacillus

pasteruii and Bacillus sphaericus has been reported in the literature. But limited

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research has been done on other species of bacteria. The present study deals with the

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isolation and identification of indigenous calcite precipitating bacteria and checking

the suitability of these bacteria for use in concrete. The influence of bacteria on the

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compressive strength and healing of cracks in concrete has also been studied. The

calcite precipitates were visually examined by SEM. Bacillus megaterium MTCC

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1684 obtained from Microbial Type Culture Collection and Gene Bank,
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Chandigarh, India was used as the standard culture. This study also uses wheat bran
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as an alternative substrate for growth of bacteria which will result in reduction of

cost of bacterial treatment.

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2. Materials and methods

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2.1 Bacterial strains and wheat bran as alternative substrate

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Five pure alkali resistant bacterial strains were isolated from the alkaline soil

samples of a cement factory at Coimbatore, Tamil Nadu, India. They were

maintained constantly on nutrient agar slants. Contamination from other bacteria

was checked periodically by streaking on nutrient agar plates.

Wheat bran samples were obtained from local market and were used as an

alternative substrate for cultivating bacterial cultures to reduce the cost of substrate.

Page 4 of 37
Whenever required, few colonies of the pure culture were inoculated into nutrient

broth of 25ml in 100ml conical flask and the growth condition was maintained at

37°C temperature and placed in 125 rpm orbital shaker. Media composition used for

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the growth of culture was Yeast Extract 5 g/l, Beef Extract 5 g/l and Wheat Bran 20

g/l. pH was maintained alkaline at 8.

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2.2 Quantitative urease assay by electric conductivity

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Conductivity method for urease activity assay was conducted as per Ibtisam et

al. (2013) and Marien et al. (2010).The urease reaction involved the hydrolysis of

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non-ionic substrate urea to ionic products thus generating a proportionate increase in

conductivity under standard conditions. For enzyme assay, 1.0 ml of bacterial broth
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culture (Nutrient Broth -Urea) was added to 9.0 ml of 1.11 M urea solution. Final
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conductivity record was taken after 5 minutes of incubation at 20oC by electric

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conductivity meter. Urease activity is presented by the rate of conductivity increase

as mS/min. Table 1 gives the electric conductivity [EC (mS/m)] of urease assay at
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different time intervals.

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Based on the high urease activity, three bacterial isolates (BI 1, BI 2 and BI 5)
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and Bacillus megaterium MTCC 1684 were selected for use in concrete. Bacillus

megaterium MTCC 1684 was used as the standard culture and was also used in

concrete for comparing the results with the bacterial isolates.

Page 5 of 37
2.3 Endospore Staining and CaCo3 (calcium carbonate) precipitation in broth

state

The selected bacterial isolates and Bacillus megaterium MTCC 1684 were

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tested for their ability to form endospores by Schaeffer –Fulton endospore

staining procedure as per Geeta and Mehrotra (2009).The smears of bacterial

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isolates and Bacillus megaterium MTCC 1684 were prepared and heat fixed. The

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smears were covered with a piece of absorbent paper cut to fit the slide and the

slides were placed on wire gauze on a ring stand. The paper was saturated with

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malachite green and the slide was heated until steam could be seen rising from

the surface. The slide was removed from heat and reheated to keep the slide
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steaming for about three minutes. As the paper began to dry, a drop of malachite
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green was added to keep it moist. The paper was removed with tweezers and the
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slide was rinsed thoroughly with tap water. The slide was drained and

counterstained for 45 seconds with 0.5% safranin. It was then washed, blotted
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and examined under a research compound microscope (100 x). The vegetative
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cells will appear red or pink and the endospores will appear green.
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The selected isolates were further tested for CaCO3 precipitation in broth

state as per Ibtisam et al. (2013).For measurement of CaCO3 precipitation in

broth, nutrient broth supplemented with 2% urea and calcium chloride (NB-

U/Ca) was used.30ml of NB-U/Ca was inoculated with 2% inoculum then

incubated under shaking condition (at 130 rpm) at 30ºC for 7 days. Three

Page 6 of 37
 replicates were tested. Precipitated CaCO3 was filtered through filter paper

(Whatman filter paper), which was dried in 60°C oven for 3 hours and then

weighed. CaCO3 precipitant weight (Wc) was determined from the equation:

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Wc = Wfc - Wf

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where (Wfc) is the weight of filter paper containing precipitant; and (Wf) is the
weight of empty filter paper.

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2.4 Molecular identification

Further identification of the isolate was performed using 16S rRNA gene

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sequencing. The DNA was isolated and the analysis of DNA sequences was

performed by using the Blastx software (BLAST), National center for

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biotechnology information.

2.5 Concrete
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Ordinary portland cement of 53 grade available in the market was used. The
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physical and chemical properties of cement are given in Table 2. It satisfies the
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requirements as per IS: 12269 (1987). Locally available sand passing through 4.75
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mm sieve and conforming to zone II of IS: 383 (1987) was used. Crushed stones of

maximum size 20 mm conforming to IS: 383 (1987) were used as coarse aggregate.
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The specific gravity of coarse aggregate and fine aggregate were found as 3.0 and

2.69 respectively.

Page 7 of 37
 2.6 Preparation of test concrete specimens and compressive strength test

Control concrete mixture was designed as per IS: 10262 (2009) to develop 28-

day compressive strength of 25 MPa and to have a slump of 25 mm to 50 mm.

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Concrete cube specimens of size 150 mm x 150 mm x 150mm were cast. The cell

concentration of 105 cells/ml of mixing water was added during mixing of bacterial

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concrete cube test specimens for BI 1, BI 2, BI 5 and Bacillus megaterium MTCC

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1684.

Control concrete cube specimens (M1) were cast using potable water

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conforming to IS: 456 (2000).Concrete mixture proportions are given in
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Table 3.The materials required for the M 25 concrete mix were weighed. The test

specimens were cast immediately after mixing. The specimens were removed from
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moulds after 24 hours and cured in water for 28 days. After completion of 28 days
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of curing, the specimens were removed from the curing tank and subjected to

compressive strength testing using compression testing machine as per IS:516

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(1959).The specimens were tested immediately on removal from the water and
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while they were still in the wet condition. Each specimen to be tested was centrally
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placed in the compression testing machine and subjected to loading. The load was

applied without any shock and increased continuously at the rate of approximately

140 kg/cm2/minute until the resistance of the specimen to the increasing load breaks

down and no greater load can be sustained. The maximum load was recorded and

the compressive strength of the specimen was calculated by dividing the maximum

Page 8 of 37
load by the cross-sectional area of the specimen. The compressive strength test was

conducted on three specimens and the average value was taken.

Cube specimens were also cast using media alone without inoculating bacteria

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to assess whether there was increase in compressive strength due to media.

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2.7 Preparation of cracks in concrete beam specimens and quantification of

crack healing with bacteria

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Concrete beam specimens of size 500 mm x 100 mm x 100mm were cast. The

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bacterial concrete beam specimens were cast with the bacterial cell concentration of

105 cells/ml of mixing water with BI 1, BI 2, BI 5 and Bacillus megaterium MTCC

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1684.

Control concrete beam specimen was cast using potable water conforming to
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IS: 456 (2000).All the beam specimens were cast with the concrete mixture
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proportions given in Table 3.The materials required for the M 25 concrete mix were
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weighed. The test specimens were cast immediately after mixing. Cracks were
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induced in the beam specimens by introducing a thin copper plate of thickness

0.3 mm up to a depth of 10 mm in the fresh concrete. The plates were removed

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before final setting of concrete such that a crack was clearly visible in the beam

specimens. The specimens were removed from moulds after 24 hours and cured in

water. Photographs were taken to visualise the cracks in the control and bacterial

beam specimens. Every week, the beam specimens were removed from water and

Page 9 of 37
the cracks were inspected for the presence of any white precipitates and for the

closure or healing of cracks.

2.8 Characterization of calcite precipitates by SEM (Scanning Electron

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Microscope)

The calcite precipitation by bacterial isolates in the micro cracks and pores in

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concrete specimens were analyzed using SEM micrographs. These micrographs

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were obtained using Jeol JSM – 6390 apparatus at an accelerating voltage of 0.5 to

30 kV. The broken pieces of cube specimens obtained from compressive strength

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test were collected. All the samples were dried at 1000C in oven for 3 days. These

samples were gold coated with a sputter coating and subjected to SEM analysis.
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3. Results
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3.1 Endospore Staining

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The endospores designated by green rods within red or pink cells were clearly
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visible in the results obtained from endospore staining test. The endospores in
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green color are visible as dark black colored rods in Fig.1a, Fig.1b, Fig.1c and

Fig.1d .The vegetative cells are visible as light colored rods in these figures. This
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indicates that all the selected bacterial isolates are capable of forming endospores.

The Fig.1c which shows the endospore staining picture of BI 5 (Bacillus flexus

BSKNAU), the amount of endospores were limited when compared to the other

bacterial isolates and B.megaterium MTCC 1684.

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Page 10 of 37
 3.2 CaCO3 (calcium carbonate) precipitation in broth state

Directly after inoculation of NB-U/Ca, a white powder appeared in the media

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and its density increased with incubation. After 7 days of incubation, CaCO3

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precipitants were collected and weighed.

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All the three bacterial isolates precipitated calcite. The highest amount of

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calcite (1.08g) was precipitated by B. megaterium MTCC 1684 followed by

BI 1.The bacterial isolates BI 1,BI 2 and BI 5 precipitated 0.84g, 0.82g and 0.76 g

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of calcium carbonate respectively (Fig.2).

3.3 Molecular identification

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The nucleotide sequences were compared with known sequences using the
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Blastx software (BLAST), National center for biotechnology information.

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The bacterial isolate BI 1 was identified as Bacillus megaterium having 96%

similarity with Bacillus megaterium J-65 and Bacillus megaterium BAB-2443,

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bacterial isolate BI 2 was identified as Bacillus licheniformis having 99% similarity

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with Bacillus licheniformis AS-4 and bacterial isolate BI 5 was identified as

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Bacillus flexus having 100% similarity with Bacillus flexus ME BHU10 and

Bacillus flexus M2.We named our strains as Bacillus megaterium BSKAU , Bacillus

licheniformis BSKNAU and Bacillus flexus BSKNAU and have been submitted to

Genbank with submission IDs BankIt1779541, BankIt1779544 and BankIt1779548

respectively.

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Page 11 of 37
 3.4 Compressive strength

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From the compressive strength results given in Table 4, it is clear that the

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concrete specimens with bacterial isolates and Bacillus megaterium MTCC 1684

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(M2, M3, M4 and M5) performed better than the control concrete specimens

(M1).The maximum increase in compressive strength (38.3 MPa) was obtained for

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concrete specimens cast with Bacillus megaterium MTCC 1684 (M5).This is

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16.1% more than control concrete specimens. Bacterial concrete specimens cast

with Bacillus megaterium BSKAU (M2), Bacillus licheniformis BSKAU (M3) and
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Bacillus flexus BSKAU (M4) have yielded compressive strengths of 37 MPa,

36.5 MPa and 35 MPa respectively which amount to 12.1%, 10.6 % and 6.1%,
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compared to control concrete specimens. Incorporation of bacteria has increased

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the compressive strength of concrete.

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The compressive strength of specimens cast using media alone without

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inoculating bacteria did not show any significant improvement (± 1%) compared

to control concrete specimens.

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3.5 Quantification of crack healing by bacteria

Fig.4 shows the photographs of cracks from control beam specimens and

bacterial beam specimens before and after healing of cracks. After 70 days

(10 weeks), white precipitates were observed in the cracks of bacterial concrete

specimens. At the end of 81 days, complete healing of cracks was observed in

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Page 12 of 37
bacterial beam specimens cast with Bacillus megaterium BSKAU, Bacillus

licheniformis BSKNAUand Bacillus megaterium MTCC 1684 (Fig.4e, 4f and 4j).

On the other hand, the crack in the bacterial beam specimen cast with Bacillus

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flexus BSKNAU was only partially sealed as observed in Fig.4i. The crack in the

control beam specimen cast with water was also partially sealed as seen in Fig.4b.

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3.6 Characterization of calcite precipitates by SEM (Scanning Electron

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Microscope)

The presence of individual crystals can be observed in bacterial concrete

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specimens (Fig.3b, 3c, 3d and 3e).On the other hand, the matrix of the control

concrete (Fig.3a) shows no such crystal growth.

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4. Discussion
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All the three bacterial isolates namely Bacillus megaterium BSKAU,

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Bacillus licheniformis BSKNAU, Bacillus flexus BSKNAU and Bacillus

megaterium MTCC 1684 were capable of forming endospores. Endospores are

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special resistant dormant structures formed within a cell that is capable of

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surviving in adverse environmental or hostile conditions. They are extremely

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resistant to heat, desiccation, chemicals and radiation. Bacteria can form

endospores in approximately 6 to 8 hours after being exposed to adverse

conditions. Spores are metabolically inactive and dehydrated. When spores are

exposed to favorable conditions, they can germinate into a vegetative cell within

90 minutes (Geeta and Mehrotra, 2009; Micheal et al. 1998;

http://www.austincc.edu/microbugz/endospore_stain.php). By forming

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Page 13 of 37
endospores, bacteria can withstand large mechanical stresses and chemically

induced stresses during mixing of concrete (Sagripanti and Bonifacino, 1996) and

can remain viable for periods up to 200 years (Schlegel, 1993).When water and air

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enters through micro cracks, endospores can germinate into vegetative (active)

cells within concrete and start precipitating calcite to heal micro cracks in

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concrete. Bacillus flexus BSKNAU (Fig.1c) could produce only limited number of

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endospores when compared with Bacillus megaterium BSKAU (Fig.1a) , Bacillus

licheniformis BSKNAU (Fig.1b) and Bacillus megaterium MTCC 1684 (Fig.1d).

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Hence the efficiency of Bacillus flexus BSKNAU in increasing strength and in

healing of cracks in concrete will be low as lesser endospores will be produced

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after mixing in concrete.
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The formation of CaCO3 precipitates by all the four bacterial strains is due to
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the hydrolysis of urea which results in the production of ammonia and carbonate.

Ammonia release act to raise the pH of the medium which is a favorable

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condition for the precipitation of calcium carbonate. Carbonate binds calcium

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ions present in medium resulting in the formation of calcium carbonate crystals

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which was deposited in agar as well as in broth media (Stocks-Fischer et al.

1999). Similar results were reported for Sporosarcina pasteurii by Ibtisam et al.

(2013).All the three bacterial isolates namely Bacillus megaterium BSKAU,

Bacillus licheniformis BSKNAU, Bacillus flexus BSKNAU and Bacillus

megaterium MTCC 1684 are capable of precipitating calcite which will plug the

micro cracks and pores in concrete. The lowest amount of calcite has been

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Page 14 of 37
 produced by Bacillus flexus BSKNAU and will be less effective in increasing

strength and in healing of cracks in concrete. The amount of calcite precipitated

by Bacillus megaterium BSKAU and Bacillus licheniformis BSKNAU are

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analogous to that of Bacillus megaterium MTCC 1684. Hence Bacillus

megaterium BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium

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MTCC 1684 are suitable for use in concrete even though there are differences in

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the amount of CaCO3 precipitated.

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The inclusion of bacteria has increased the compressive strength of concrete.

The insignificant improvement in the compressive strength of cube specimens cast

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using media alone without inoculating bacteria shows that the cause for strength

improvement is due to calcite precipitation by bacteria and not due to media.

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It has been observed from the results in Table 4 that the strength properties
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of all the bacterial concrete specimens (M2, M3, M4 and M5) have increased with
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respect to the control concrete specimens (M1). The improvement in compressive

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strength is due to the plugging of micro cracks and pores in concrete with calcite

precipitated by bacteria.
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The improvement in compressive strength by inclusion of bacteria is

probably due to deposition of calcite on the microorganism cell surfaces and

within the pores of cement sand matrix, which plug the pores (Ramachandran et

al.2001; Ramakrishnan et al.1998; Ramakrishnan et al.1999). Ghosh et al. (2006)

reported that the improvement in compressive strength may be due to the

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Page 15 of 37
deposition of some minute filler material produced by the bacteria that reduce the

pore size and modify the microstructure of concrete. Navneet Chahal et al. (2012)

attributed that the increase in compressive strength is mainly due to filling of the

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pores inside the cement concrete cubes with microbiologically induced calcium

carbonate precipitation. The greatest improvement in compressive strength was

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obtained for concrete specimens with Bacillus megaterium MTCC 1684.The

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lowest compressive strength has been reported for Bacillus flexus BSKNAU which

has produced lesser endospores and less amount of calcite.When compared to

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bacterial concrete specimens cast with Bacillus megaterium MTCC 1684, the

difference in compressive strength of bacterial concrete specimens cast with

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Bacillus megaterium BSKAU is only 3.5%.This difference is not high and hence it
d

can be interpreted that Bacillus megaterium BSKAU has performed well in

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increasing the compressive strength of concrete specimens. Minimum difference in

compressive strength of 4.9% was also observed in bacterial concrete specimens

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cast with Bacillus megaterium MTCC 1684 and Bacillus licheniformis BSKNAU.
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The compressive strength test results of concrete specimens with Bacillus

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megaterium BSKAU and Bacillus licheniformis BSKNAU are comparable to that

of Bacillus megaterium MTCC 1684.Hence it can be concluded that Bacillus

megaterium BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium

MTCC 1684 are suitable for use in concrete.

In Fig.4a, 4c, 4d, 4g and 4h, the cracks can be clearly seen. These figures

show the cracks in the specimens before healing and correspond to control beam

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Page 16 of 37
specimens and bacterial beam specimens cast with Bacillus megaterium BSKAU,

Bacillus licheniformis BSKNAU, Bacillus flexus BSKNAU and Bacillus

megaterium MTCC 1684 respectively. Similarly, the figures, Fig.4b, 4e, 4f, 4i and

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4 j show the difference in cracks after healing of cracks at the end of 81 days. In

Fig.4e, 4f and 4j, complete healing of cracks can be observed. This sealing of

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cracks is due to the precipitation of calcite by the bacterial isolates Bacillus

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megaterium BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium

MTCC 1684.Fig.4b and 4i show the crack which was partially healed. The cracks

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have not been completely healed and the crack is visible at some points. This

indicates that in the beam specimens cast with Bacillus flexus BSKNAU (Fig.4i),
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the bacteria was able to precipitate calcite, but the calcite precipitation was low
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and not sufficient to completely seal the cracks. In Fig.4b, partial healing of crack
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in control beam specimen can be observed. This indicates that the self healing of

cracks has also taken place in control concrete to certain extent.

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The Fig. 3a shows the SEM micrograph of control concrete specimens. In

Fig. 3a, no crystals can be visualized. This indicates the absence of calcite
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precipitating bacteria which is the reason for lower strength of control concrete

specimens. Fig.3b, 3c, 3d and 3e show the SEM micrographs of bacterial concrete

specimens with Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAU,

Bacillus flexus BSKNAU and Bacillus megaterium MTCC 1684 respectively. The

SEM micrographs of Fig.3b, 3c, 3d and 3e reveal individual crystals and

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Page 17 of 37
individual crystals can be noticed. This was the reason for enhancement in strength

properties of concrete (Table 4) and for the healing of cracks in concrete. This

difference in texture of control specimens (Fig.3a) and bacterial specimens

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(Fig.3b – 3e) as visualized in SEM micrographs may due to precipitation of calcite

crystals in the micro cracks and pores of concrete by bacterial isolates. These

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results are in agreement with the earlier results which also reported the contrasting

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textures of matrices of bacterial concrete specimens and control concrete

specimens. The matrix of untreated ones (control concrete specimens) appeared to

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be amorphous, showing no conspicuous crystal growth. On the other hand,

bacterial concrete specimens showed crystalline matrix where individual crystals

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could be recognized (Ghosh et al. 2009).Navneet Chahal et al. (2012) also reported
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that distinct calcite crystals could be seen in the SEM analysis of concrete with
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S.pasteurii.
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Conclusions
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All the three newly isolated bacterial strains and Bacillus megaterium MTCC

1684 exhibited high urease activity. They formed endospores and precipitated
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calcium carbonate. The newly isolated bacterial strains were identified by

16S rRNA gene sequencing as Bacillus megaterium BSKAU,

Bacillus licheniformis BSKNAU and Bacillus flexus BSKNAU. The urease

activity, endospore formation and calcium carbonate precipitation of

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Page 18 of 37
Bacillus megaterium BSKAU and Bacillus licheniformis BSKNAU is close to

Bacillus megaterium MTCC 1684.

The compressive strength of bacterial concrete specimens with Bacillus

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megaterium BSKAU, Bacillus licheniformis BSKNAU, Bacillus flexus

BSKNAU and Bacillus megaterium MTCC 1684 has increased when compared to

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control concrete specimens. Even though the maximum increase in strength was

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found for bacterial concrete specimens with Bacillus megaterium MTCC 1684, the

increase in strength of bacterial concrete specimens with Bacillus megaterium

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BSKAU and Bacillus licheniformis BSKNAU are equally good. Complete healing

of cracks was observed in concrete specimens cast with Bacillus megaterium

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BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium MTCC 1684.
d

The presence of distinct calcite crystals was seen in the SEM analysis of bacterial
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concrete specimens which has increased the strength of concrete specimens. It can

be concluded that Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAU

p

and Bacillus megaterium MTCC 1684 are suitable for use in concrete as they
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have resulted in increased strength and complete healing of cracks in concrete

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specimens. The inclusion of these bacteria in concrete will result in high strength,

crack free and durable concrete structures in the future. Bacillus flexus BSKNAU

is less effective in increasing strength and in healing of cracks and hence is not a

better choice for use in concrete.

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Page 19 of 37
 The use of wheat bran as an alternative substrate for growth of bacteria will

result in cost effectiveness of bacterial concrete specimens when compared to

control concrete specimens.

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References

Geeta Sumbali, Mehrotra RS.Principles of Microbiology. 1st ed. New Delhi: Tata

cr
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Ghosh P, Mandal S. Development of bioconcrete material using an enrichment

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culture of novel thermophilic anaerobic bacteria. Indian J Exp Biol2006;44:336-

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Ghosh S, Biswas M, Chattopadhyay BD. Microbial activity on the microstructure

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leaching through permeable channels.Chemosphere 1995;30(4):695-705.

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CM van Loosdrecht. Fixation and distribution of bacterial activity in sand to

induce carbonate precipitation for ground reinforcement.Ecol Eng 2010; 36:112-

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Navneet Chahal, Rafat Siddique, Anita Rajor. Influence of bacteria on the

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on High Performance. High Strength Concrete;1998; Perth, Australia.1998. p. 597-

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 Ramakrishnan V, Panchalan RK , Bang SS.Improvement of concrete durability by

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Web reference

http://www.austincc.edu/microbugz/endospore_stain.php last accessed on

02/11/2014.

23

Page 23 of 37
Figure captions

Fig.1. Endospore staining pictures of the bacterial isolates (a) BI 1. (b) BI 2.

t
(c) BI 5 and (d) B.megaterium MTCC 1684.

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Fig.2. Comparison of CaCo3 precipitation by different bacterial isolates and Bacillus

cr
megaterium MTCC 1684 (B.mega MTCC 1684).

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Fig.3. Scanning electron micrographs of (a) control concrete specimen with no

an
calcite crystals. (b) concrete specimen with Bacillus megaterium BSKAU

indicating the presence of calcite crystals. (c) concrete specimen with

M
Bacillus licheniformis BSKAU indicating the presence of calcite crystals.(d)

concrete specimen with Bacillus flexus BSKAU indicating the presence of

d

calcite crystals and (e) concrete specimen with Bacillus megaterium MTCC
te

1684 indicating the presence of calcite crystals.

p

Fig.4. Photographs of crack healing (a) in control concrete beam specimen before
ce

healing. (b) in control concrete beam specimen after 81 days of healing. (c)

in concrete beam specimen with Bacillus megaterium BSKAU before

Ac

healing. (d) in concrete beam specimen with Bacillus licheniformis

BSKNAU before healing. (e) in concrete beam specimen with Bacillus

megaterium BSKAU after 81 days of healing. (f) in concrete beam specimen

with Bacillus licheniformis BSKNAU after 81 days of healing. (g) in

concrete beam specimen with Bacillus flexus BSKNAU before healing. (h)

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Page 24 of 37
 in concrete beam specimen with Bacillus megaterium MTCC 1684 before

healing. (i) in concrete beam specimen with Bacillus flexus BSKNAU after

81 days of healing and (j) in concrete beam specimen with Bacillus

t
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megaterium MTCC 1684 after 81 days of healing.

cr
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M
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p te
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Table 1

Table 1
Electric Conductivity [EC (mS / m)] of Urease Assay Mixture at Different Time
Intervals

t
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Bacillus
BI 1 BI 2 BI 3 BI 4 BI 5 megaterium
MTCC 1684

cr
EC EC EC EC EC EC
Time Time Time Time Time Time
(mS / (mS / (mS / (mS / (mS / (mS
(s) (s) (s) (s) (s) (s)

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m) m) m) m) m) /m)

0 135.6 0 121.9 0 122.7 0 118.7 0 128.1 0 240

300 138.7 300 136.5 300 123.2

an300 119.8 300 131.9 300 251

M
3960 142.1 3960 139.2 3960 125.0 3960 121.8 3960 141.2 3960 282

6360 161.2 6360 140.6 6360 129.2 6360 126.2 6360 145.9 6360 296
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Page 26 of 37
Table 2

Table 2
Physical and Chemical Properties of Cement
Physical property

Colour Grey
Specific Gravity 3.15
Chemical constituent (%)
SiO2 21

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CaO 62

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Al2O3 5.04
Fe2O3 3.16

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MgO 4.56
Na2O 0.08
Loss on Ignition 1.29

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Page 27 of 37
Table 3

Table 3
Concrete Mix Proportions

Concrete Mixture No.

M11 M2 M3 M4 M5
Cement (kg/m3) 413 413 413 413 413

Natural Sand 680 680 680 680 680

(kg/m3)

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Bacteria used (105

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- B.megaterium
cells/ml of mixing BI 1 BI 2 BI 5 MTCC 1684
water)

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Coarse aggregate 1290 1290 1290 1290 1290
(kg/m3)

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Water Cement Ratio 0.55 0.55 0.55 0.55 0.55

Water (kg/m3) 186 (tap 186 186 186 186

water)
*M stands for Concrete Mixture
1
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control concrete cast with no bacterial cells
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Page 28 of 37
Table 4

Table 4

Compressive Strength Values of Concrete Mixtures [Values are Mean ± Standard Deviation]

Bacteria Bacterial Concrete Mixture Compressive strength at

cells / ml of 28 days in MPa

t
mixing

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water
No bacterial
0 M1 (Control) 33.00 ± 0.33
cells

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105 M2 37.00 ± 1.06

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105 M3 36.50 ± 0.44
Live cells
105 M4 35.00 ± 0.19

105 M5
an 38.30 ± 0.53
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Page 29 of 37
Figure 1

(a)

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(b)
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Page 30 of 37
(c)

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(d)
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Fig.1

Page 31 of 37
Figure 2

1.2
Weight of CaCo3 precipitation
1
0.8
0.6
in g/l

0.4
0.2
0
BI 1 BI 2 BI 5 B.mega

t
MTCC 1684

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Bacterial isolates

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Fig. 2.

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pt
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Page 32 of 37
Figure 3

(a)

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(b)
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Page 33 of 37
(c)

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(d)
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Page 34 of 37
(e)

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Fig.3.
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Page 35 of 37
Figure4

(a)

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Visible crack

(b)
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Visible Crack
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Partial healing of cracks

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Page 36 of 37
(c) (d)

t
ip
Visible crack Visible crack

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(e) (f)

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`

Complete healing of cracks

an Complete healing of cracks

M
(g) (h)
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Visible crack
Visible crack
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(i) (j)
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`
Partial healing of cracks

Visible crack Complete healing of cracks

Fig.4.

Page 37 of 37