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: DOI: Reference:
S0944-5013(15)00050-6 http://dx.doi.org/doi:10.1016/j.micres.2015.03.009 MICRES 25763
To appear in: Received date: Revised date: Accepted date:
2-11-2014 12-3-2015 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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Isolation and identification of bacteria to improve the strength of concrete
a
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S.Krishnapriyaa , D.L.Venkatesh Babub, G.Prince Arulrajc Department of Civil Engineering, SNS College of Technology, Coimbatore ,India,
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E mail:[email protected]
Department of Civil Engineering, JSS Academy of Technical Education,
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Bangalore ,India,
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E mail:[email protected]
Department of Civil Engineering, SNS College of Technology, Coimbatore ,India,
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E mail:[email protected]
Corresponding Author: Mrs.S.Krishnapriya, Department of Civil Engineering,
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SNS College of Technology, Coimbatore - 641035, India.
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E-mail:[email protected],[email protected]
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Phone: +91 9688218146 Fax:+914222665138 ABSTRACT
The objective of this research work is to isolate and identify calcite precipitating 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
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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
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on these results, three isolates were selected and identified by 16S rRNA gene sequencing. They were identified as Bacillus megaterium BSKAU, Bacillus
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licheniformis BSKNAU and Bacillus flexus BSKNAU. The results were compared
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with Bacillus megaterium MTCC 1684 obtained from Microbial Type Culture Collection and Gene Bank , Chandigarh, India. Experimental work was carried out
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to assess the influence of bacteria on the compressive strength and tests revealed that bacterial concrete specimens showed enhancement in compressive strength.
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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 BSKNAU and Bacillus megaterium MTCC 1684.This indicates the suitability of
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these bacterial strains for use in concrete. The enhancement of strength and healing of cracks can be attributed to the filling of cracks in concrete by calcite which was visualized by Scanning Electron Microscope. 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
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ingress of water and other deleterious substances. This leads to corrosion of reinforcement and reduces the strength and durability of concrete. Large costs are
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incurred all over the globe to repair cracks in concrete. For repairing cracks, a
variety of techniques are available but majority of traditional repair systems are
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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 alternative repair technique for plugging of micro cracks and pores in concrete. This
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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 pH and calcite precipitation in the bacterial environment (Stocks-Fischer et al.
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1999).
This innovative environmental friendly method was first used for the repair
of cracks to prevent leaching in channels (Gollapudi et al.1995). It can also be used 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;
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Van Tittelboom et al.2010).The durability of concrete specimens treated with Bacillus pasteruii exposed to alkaline, sulphate and freeze-thaw environments was
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also reported to increase (Ramakrishnan et al.2005).
Considerable research work on concrete incorporating bacterial species Bacillus
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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 isolation and identification of indigenous calcite precipitating bacteria and checking
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the suitability of these bacteria for use in concrete. The influence of bacteria on the compressive strength and healing of cracks in concrete has also been studied. The
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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 as an alternative substrate for growth of bacteria which will result in reduction of
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cost of bacterial treatment.
2. Materials and methods 2.1 Bacterial strains and wheat bran as alternative substrate 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.
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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
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37°C temperature and placed in 125 rpm orbital shaker. Media composition used for
g/l. pH was maintained alkaline at 8.
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2.2 Quantitative urease assay by electric conductivity
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the growth of culture was Yeast Extract 5 g/l, Beef Extract 5 g/l and Wheat Bran 20
Conductivity method for urease activity assay was conducted as per Ibtisam et
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al. (2013) and Marien et al. (2010).The urease reaction involved the hydrolysis of non-ionic substrate urea to ionic products thus generating a proportionate increase in
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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 conductivity meter. Urease activity is presented by the rate of conductivity increase
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as mS/min. Table 1 gives the electric conductivity [EC (mS/m)] of urease assay at different time intervals.
Based on the high urease activity, three bacterial isolates (BI 1, BI 2 and BI 5)
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.
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2.3 Endospore Staining and CaCo3 (calcium carbonate) precipitation in broth state
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The selected bacterial isolates and Bacillus megaterium MTCC 1684 were tested for their ability to form endospores by Schaeffer –Fulton endospore
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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 smears were covered with a piece of absorbent paper cut to fit the slide and the
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slides were placed on wire gauze on a ring stand. The paper was saturated with malachite green and the slide was heated until steam could be seen rising from
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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 slide was rinsed thoroughly with tap water. The slide was drained and
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counterstained for 45 seconds with 0.5% safranin. It was then washed, blotted and examined under a research compound microscope (100 x). The vegetative cells will appear red or pink and the endospores will appear green.
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 (NBU/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
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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
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weighed. CaCO3 precipitant weight (Wc) was determined from the equation:
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Wc = Wfc - Wf
where (Wfc) is the weight of filter paper containing precipitant; and (Wf) is the
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weight of empty filter paper. 2.4 Molecular identification
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Further identification of the isolate was performed using 16S rRNA gene sequencing. The DNA was isolated and the analysis of DNA sequences was
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performed by using the Blastx software (BLAST), National center for
2.5 Concrete
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biotechnology information.
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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 requirements as per IS: 12269 (1987). Locally available sand passing through 4.75 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. The specific gravity of coarse aggregate and fine aggregate were found as 3.0 and 2.69 respectively.
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2.6 Preparation of test concrete specimens and compressive strength test Control concrete mixture was designed as per IS: 10262 (2009) to develop 28-
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day compressive strength of 25 MPa and to have a slump of 25 mm to 50 mm.
Concrete cube specimens of size 150 mm x 150 mm x 150mm were cast. The cell
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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 1684.
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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 Table 3.The materials required for the M 25 concrete mix were weighed. The test
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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 of curing, the specimens were removed from the curing tank and subjected to
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compressive strength testing using compression testing machine as per IS:516 (1959).The specimens were tested immediately on removal from the water and while they were still in the wet condition. Each specimen to be tested was centrally 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
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load by the cross-sectional area of the specimen. The compressive strength test was conducted on three specimens and the average value was taken.
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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.
2.7 Preparation of cracks in concrete beam specimens and quantification of
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crack healing with bacteria
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Concrete beam specimens of size 500 mm x 100 mm x 100mm were cast. The bacterial concrete beam specimens were cast with the bacterial cell concentration of
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105 cells/ml of mixing water with BI 1, BI 2, BI 5 and Bacillus megaterium MTCC 1684.
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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 weighed. The test specimens were cast immediately after mixing. Cracks were 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 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
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the cracks were inspected for the presence of any white precipitates and for the closure or healing of cracks.
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2.8 Characterization of calcite precipitates by SEM (Scanning Electron Microscope)
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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 were obtained using Jeol JSM – 6390 apparatus at an accelerating voltage of 0.5 to
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30 kV. The broken pieces of cube specimens obtained from compressive strength test were collected. All the samples were dried at 1000C in oven for 3 days. These
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samples were gold coated with a sputter coating and subjected to SEM analysis.
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3.1 Endospore Staining
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3. Results
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The endospores designated by green rods within red or pink cells were clearly visible in the results obtained from endospore staining test. The endospores in 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 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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3.2 CaCO3 (calcium carbonate) precipitation in broth state
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Directly after inoculation of NB-U/Ca, a white powder appeared in the media 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 calcite (1.08g) was precipitated by B. megaterium MTCC 1684 followed by
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BI 1.The bacterial isolates BI 1,BI 2 and BI 5 precipitated 0.84g, 0.82g and 0.76 g
3.3 Molecular identification
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of calcium carbonate respectively (Fig.2).
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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. The bacterial isolate BI 1 was identified as Bacillus megaterium having 96%
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similarity with Bacillus megaterium J-65 and Bacillus megaterium BAB-2443, bacterial isolate BI 2 was identified as Bacillus licheniformis having 99% similarity with Bacillus licheniformis AS-4 and bacterial isolate BI 5 was identified as 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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3.4 Compressive strength 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 (M2, M3, M4 and M5) performed better than the control concrete specimens
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(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 16.1% more than control concrete specimens. Bacterial concrete specimens cast
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with Bacillus megaterium BSKAU (M2), Bacillus licheniformis BSKAU (M3) and Bacillus flexus BSKAU (M4) have yielded compressive strengths of 37 MPa,
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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. The compressive strength of specimens cast using media alone without
inoculating bacteria did not show any significant improvement (± 1%) compared to control concrete specimens. 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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bacterial beam specimens cast with Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAUand Bacillus megaterium MTCC 1684 (Fig.4e, 4f and 4j).
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On the other hand, the crack in the bacterial beam specimen cast with Bacillus
flexus BSKNAU was only partially sealed as observed in Fig.4i. The crack in the
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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 Microscope)
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The presence of individual crystals can be observed in bacterial concrete specimens (Fig.3b, 3c, 3d and 3e).On the other hand, the matrix of the control
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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, Bacillus licheniformis BSKNAU, Bacillus flexus BSKNAU and Bacillus
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megaterium MTCC 1684 were capable of forming endospores. Endospores are special resistant dormant structures formed within a cell that is capable of surviving in adverse environmental or hostile conditions. They are extremely 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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endospores, bacteria can withstand large mechanical stresses and chemically induced stresses during mixing of concrete (Sagripanti and Bonifacino, 1996) and
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can remain viable for periods up to 200 years (Schlegel, 1993).When water and air enters through micro cracks, endospores can germinate into vegetative (active)
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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 endospores when compared with Bacillus megaterium BSKAU (Fig.1a) , Bacillus
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licheniformis BSKNAU (Fig.1b) and Bacillus megaterium MTCC 1684 (Fig.1d). Hence the efficiency of Bacillus flexus BSKNAU in increasing strength and in
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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 the hydrolysis of urea which results in the production of ammonia and carbonate.
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Ammonia release act to raise the pH of the medium which is a favorable condition for the precipitation of calcium carbonate. Carbonate binds calcium ions present in medium resulting in the formation of calcium carbonate crystals 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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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
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by Bacillus megaterium BSKAU and Bacillus licheniformis BSKNAU are analogous to that of Bacillus megaterium MTCC 1684. Hence Bacillus
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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.
The inclusion of bacteria has increased the compressive strength of concrete.
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The insignificant improvement in the compressive strength of cube specimens cast using media alone without inoculating bacteria shows that the cause for strength
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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 respect to the control concrete specimens (M1). The improvement in compressive strength is due to the plugging of micro cracks and pores in concrete with calcite precipitated by bacteria.
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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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)
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attributed that the increase in compressive strength is mainly due to filling of the pores inside the cement concrete cubes with microbiologically induced calcium
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carbonate precipitation. The greatest improvement in compressive strength was
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obtained for concrete specimens with Bacillus megaterium MTCC 1684.The lowest compressive strength has been reported for Bacillus flexus BSKNAU which
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has produced lesser endospores and less amount of calcite.When compared to bacterial concrete specimens cast with Bacillus megaterium MTCC 1684, the
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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
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can be interpreted that Bacillus megaterium BSKAU has performed well in increasing the compressive strength of concrete specimens. Minimum difference in
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compressive strength of 4.9% was also observed in bacterial concrete specimens cast with Bacillus megaterium MTCC 1684 and Bacillus licheniformis BSKNAU. The compressive strength test results of concrete specimens with Bacillus 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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specimens and bacterial beam specimens cast with Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAU, Bacillus flexus BSKNAU and Bacillus
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megaterium MTCC 1684 respectively. Similarly, the figures, Fig.4b, 4e, 4f, 4i and 4 j show the difference in cracks after healing of cracks at the end of 81 days. In
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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 megaterium BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium
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MTCC 1684.Fig.4b and 4i show the crack which was partially healed. The cracks have not been completely healed and the crack is visible at some points. This
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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 in control beam specimen can be observed. This indicates that the self healing of
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cracks has also taken place in control concrete to certain extent. 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 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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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
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difference in texture of control specimens (Fig.3a) and bacterial specimens
(Fig.3b – 3e) as visualized in SEM micrographs may due to precipitation of calcite
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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 textures of matrices of bacterial concrete specimens and control concrete
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specimens. The matrix of untreated ones (control concrete specimens) appeared to be amorphous, showing no conspicuous crystal growth. On the other hand,
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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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S.pasteurii.
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that distinct calcite crystals could be seen in the SEM analysis of concrete with
Conclusions
All the three newly isolated bacterial strains and Bacillus megaterium MTCC
1684 exhibited high urease activity. They formed endospores and precipitated 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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Bacillus megaterium BSKAU and Bacillus licheniformis BSKNAU is close to Bacillus megaterium MTCC 1684.
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The compressive strength of bacterial concrete specimens with Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAU, Bacillus flexus
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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 found for bacterial concrete specimens with Bacillus megaterium MTCC 1684, the
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increase in strength of bacterial concrete specimens with Bacillus megaterium BSKAU and Bacillus licheniformis BSKNAU are equally good. Complete healing
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of cracks was observed in concrete specimens cast with Bacillus megaterium
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BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium MTCC 1684.
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The presence of distinct calcite crystals was seen in the SEM analysis of bacterial concrete specimens which has increased the strength of concrete specimens. It can
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be concluded that Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium MTCC 1684 are suitable for use in concrete as they have resulted in increased strength and complete healing of cracks in concrete 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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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
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Geeta Sumbali, Mehrotra RS.Principles of Microbiology. 1st ed. New Delhi: Tata
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Ghosh P, Mandal S. Development of bioconcrete material using an enrichment 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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Ibtisam A Hammad, Fatma N Talkhan, Ahmed E Zoheir. Urease activity and induction of calcium carbonate precipitation by Sporosarcina pasteurii NCIMB 8841. J Appl Sci Res 2013;9(3):1525-1533.
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IS:516 (1959).Methods for test for strength of concrete. Amendment No.2,Reprint
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concrete. 8th reprint October 1991;Bureau of Indian Standards.New Delhi,India.
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IS:12269 (1987).Specification for 53 grade ordinary portland cement. Bureau of
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IS:456 (2000).Plain and reinforced concrete - Code of Practice. 4th revision;
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Micheal J Pelczar Jr, Chan ECS, Noel R Krieg. Microbiology.5th ed. New
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Delhi:Tata McGraw Hill Education Private Limited; 1998. Navneet Chahal, Rafat Siddique, Anita Rajor. Influence of bacteria on the
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compressive strength, water absorption and rapid chloride permeability of fly ash concrete. Constr Build Mater2012;28:351-356.doi:10.1016/j.conbuildmat.
us
2011.07.042.
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Ramachandran SK, Ramakrishnan V, Bang SS. Remediation of concrete using
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microorganisms.ACI Mater J2001;98:3-9.
Ramakrishnan V, Bang SS, Deo KS.A novel technique for repairing cracks in high
d
performance concrete using bacteria.Proceeding of the International Conference
Ac ce p
617.
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on High Performance. High Strength Concrete;1998; Perth, Australia.1998. p. 597-
Ramakrishnan V, Deo KS, Duke EF, Bang SS.SEM investigation of microbial calcite precipitation in cement. Proceeding of the International Conference on Cement Microscopy;1999; Las Vegas, Nevada. 1999. p. 406-414. Ramakrishnan V, Panchalan RK , Bang SS. Bacterial concrete. Proceedings of SPIE- International Society of Optical Engineering, Smart Materials;2001; Australia. 2001;4234: p. 168-176 . doi:10.1117/12.424404.
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Ramakrishnan V, Panchalan RK , Bang SS.Improvement of concrete durability by bacterial mineral precipitation.Proceedings of the 11th International Conference on
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Fracture; 2005; Turin, Italy. 2005.
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Sagripanti JL, Bonifacino A.Comparative sporicidal effects of liquid chemical
us
agents.Appl Environ Microbiol1996;62 (2):545.
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SchlegelHG.General microbiology. Cambridge:Cambridge University Press; 1993. Stocks-Fischer S,Galinat JK,Bang SS. Microbiological precipitation of CaCo3.Soil
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Biol Biochem 1999;31(11):1563-1571.
d
Van Tittelboom K, De Belie N, De Muynck W, Verstraete W.Use of bacteria to
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repair cracks in concrete.Cem Concr Res2010;40:157-
Ac ce p
166,doi:10.1016/j.cemconres.2009.08.025. ZhongL,Islam MR.A new microbial process and its impact on fracture remediation.Paper presented at the 70th Annual Technical Conference and Exhibition of the Society of Petroleum Engineers,1995; Dallas, Texas. 1995.doi:10.2118/30519-MS.
Web reference
http://www.austincc.edu/microbugz/endospore_stain.php last accessed on 02/11/2014.
23 Page 23 of 37
Figure captions
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Fig.1. Endospore staining pictures of the bacterial isolates (a) BI 1. (b) BI 2. (c) BI 5 and (d) B.megaterium MTCC 1684.
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megaterium MTCC 1684 (B.mega MTCC 1684).
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Fig.2. Comparison of CaCo3 precipitation by different bacterial isolates and Bacillus
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Fig.3. Scanning electron micrographs of (a) control concrete specimen with no calcite crystals. (b) concrete specimen with Bacillus megaterium BSKAU
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indicating the presence of calcite crystals. (c) concrete specimen with Bacillus licheniformis BSKAU indicating the presence of calcite crystals.(d)
d
concrete specimen with Bacillus flexus BSKAU indicating the presence of
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calcite crystals and (e) concrete specimen with Bacillus megaterium MTCC
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1684 indicating the presence of calcite crystals. Fig.4. Photographs of crack healing (a) in control concrete beam specimen before healing. (b) in control concrete beam specimen after 81 days of healing. (c) in concrete beam specimen with Bacillus megaterium BSKAU before 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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in concrete beam specimen with Bacillus megaterium MTCC 1684 before healing. (i) in concrete beam specimen with Bacillus flexus BSKNAU after
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te
d
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an
us
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megaterium MTCC 1684 after 81 days of healing.
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81 days of healing and (j) in concrete beam specimen with Bacillus
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Table 1
BI 2
BI 3
BI 4
BI 5
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BI 1
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Table 1 Electric Conductivity [EC (mS / m)] of Urease Assay Mixture at Different Time Intervals
Bacillus megaterium MTCC 1684
EC (mS / m)
Time (s)
EC (mS / m)
Time (s)
EC (mS / m)
Time (s)
EC (mS / m)
Time (s)
EC (mS / m)
Time (s)
EC (mS /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
3960
142.1
3960
139.2
3960
6360
161.2
6360
140.6
6360
an
us
Time (s)
119.8
300
131.9
300
251
125.0
3960
121.8
3960
141.2
3960
282
129.2
6360
126.2
6360
145.9
6360
296
Ac ce p
te
d
M
300
1 Page 26 of 37
Table 2
Table 2 Physical and Chemical Properties of Cement Physical property Colour Specific Gravity
Grey 3.15
cr
21 62 5.04 3.16 4.56 0.08 1.29
Ac
ce pt
ed
M
an
us
SiO2 CaO Al2O3 Fe2O3 MgO Na2O Loss on Ignition
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Chemical constituent (%)
Page 27 of 37
Table 3
Table 3 Concrete Mix Proportions Concrete Mixture No. M2 413
M3 413
M4 413
M5 413
Natural Sand (kg/m3) Bacteria used (105 cells/ml of mixing water)
680
680
680
680
680
Coarse aggregate (kg/m3) Water Cement Ratio
B.megaterium MTCC 1684
BI 1
BI 2
BI 5
1290
1290
1290
cr
-
0.55
0.55
0.55
0.55
0.55
186
186
186
Water (kg/m3)
1290
Ac
ce pt
ed
M
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186 (tap 186 water) *M stands for Concrete Mixture 1 control concrete cast with no bacterial cells
1290
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Cement (kg/m3)
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M11 413
Page 28 of 37
Table 4
Table 4 Compressive Strength Values of Concrete Mixtures [Values are Mean ± Standard Deviation]
Compressive strength at 28 days in MPa
0
M1 (Control)
33.00 ± 0.33
105
M2
37.00 ± 1.06
105
M3
105
M4
105
M5
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Concrete Mixture
cr
No bacterial cells
Bacterial cells / ml of mixing water
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Bacteria
36.50 ± 0.44
Live cells
38.30 ± 0.53
Ac
ce pt
ed
M
an
35.00 ± 0.19
Page 29 of 37
Figure 1
te Ac ce p
(b)
d
M
an
us
cr
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(a)
Page 30 of 37
Ac ce p
te
(d)
d
M
an
us
cr
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(c)
Fig.1
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1.2 1 0.8 0.6 0.4 0.2 0 BI 1
BI 2
BI 5
B.mega MTCC 1684
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Bacterial isolates
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Weight of CaCo3 precipitation in g/l
Figure 2
Ac
ce pt
ed
M
an
us
Fig. 2.
Page 32 of 37
Figure 3
an
us
cr
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(a)
Ac
ce pt
ed
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(b)
Page 33 of 37
an
us
cr
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(c)
Ac
ce pt
ed
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(d)
Page 34 of 37
M
an
us
cr
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(e)
Ac
ce pt
ed
Fig.3.
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Figure4
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cr
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(a)
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Visible crack
Ac ce p
te
d
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(b)
Visible Crack
Partial healing of cracks
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(d)
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(c)
Visible crack (f)
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(e)
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Visible crack
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`
Complete healing of cracks
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Complete healing of cracks
(h)
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te
d
(g)
Visible crack
(i)
Visible crack
(j) `
Partial healing of cracks
Visible crack
Complete healing of cracks
Fig.4.
Page 37 of 37
S0944-5013(15)00050-6 http://dx.doi.org/doi:10.1016/j.micres.2015.03.009 MICRES 25763
To appear in: Received date: Revised date: Accepted date:
2-11-2014 12-3-2015 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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Isolation and identification of bacteria to improve the strength of concrete
a
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S.Krishnapriyaa , D.L.Venkatesh Babub, G.Prince Arulrajc Department of Civil Engineering, SNS College of Technology, Coimbatore ,India,
b
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E mail:[email protected]
Department of Civil Engineering, JSS Academy of Technical Education,
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Bangalore ,India,
c
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E mail:[email protected]
Department of Civil Engineering, SNS College of Technology, Coimbatore ,India,
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E mail:[email protected]
Corresponding Author: Mrs.S.Krishnapriya, Department of Civil Engineering,
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SNS College of Technology, Coimbatore - 641035, India.
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E-mail:[email protected],[email protected]
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Phone: +91 9688218146 Fax:+914222665138 ABSTRACT
The objective of this research work is to isolate and identify calcite precipitating 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
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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
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on these results, three isolates were selected and identified by 16S rRNA gene sequencing. They were identified as Bacillus megaterium BSKAU, Bacillus
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licheniformis BSKNAU and Bacillus flexus BSKNAU. The results were compared
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with Bacillus megaterium MTCC 1684 obtained from Microbial Type Culture Collection and Gene Bank , Chandigarh, India. Experimental work was carried out
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to assess the influence of bacteria on the compressive strength and tests revealed that bacterial concrete specimens showed enhancement in compressive strength.
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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 BSKNAU and Bacillus megaterium MTCC 1684.This indicates the suitability of
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these bacterial strains for use in concrete. The enhancement of strength and healing of cracks can be attributed to the filling of cracks in concrete by calcite which was visualized by Scanning Electron Microscope. 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
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ingress of water and other deleterious substances. This leads to corrosion of reinforcement and reduces the strength and durability of concrete. Large costs are
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incurred all over the globe to repair cracks in concrete. For repairing cracks, a
variety of techniques are available but majority of traditional repair systems are
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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 alternative repair technique for plugging of micro cracks and pores in concrete. This
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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 pH and calcite precipitation in the bacterial environment (Stocks-Fischer et al.
Ac ce p
1999).
This innovative environmental friendly method was first used for the repair
of cracks to prevent leaching in channels (Gollapudi et al.1995). It can also be used 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;
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Van Tittelboom et al.2010).The durability of concrete specimens treated with Bacillus pasteruii exposed to alkaline, sulphate and freeze-thaw environments was
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also reported to increase (Ramakrishnan et al.2005).
Considerable research work on concrete incorporating bacterial species Bacillus
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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 isolation and identification of indigenous calcite precipitating bacteria and checking
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the suitability of these bacteria for use in concrete. The influence of bacteria on the compressive strength and healing of cracks in concrete has also been studied. The
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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 as an alternative substrate for growth of bacteria which will result in reduction of
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cost of bacterial treatment.
2. Materials and methods 2.1 Bacterial strains and wheat bran as alternative substrate 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.
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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
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37°C temperature and placed in 125 rpm orbital shaker. Media composition used for
g/l. pH was maintained alkaline at 8.
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2.2 Quantitative urease assay by electric conductivity
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the growth of culture was Yeast Extract 5 g/l, Beef Extract 5 g/l and Wheat Bran 20
Conductivity method for urease activity assay was conducted as per Ibtisam et
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al. (2013) and Marien et al. (2010).The urease reaction involved the hydrolysis of non-ionic substrate urea to ionic products thus generating a proportionate increase in
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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 conductivity meter. Urease activity is presented by the rate of conductivity increase
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as mS/min. Table 1 gives the electric conductivity [EC (mS/m)] of urease assay at different time intervals.
Based on the high urease activity, three bacterial isolates (BI 1, BI 2 and BI 5)
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.
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2.3 Endospore Staining and CaCo3 (calcium carbonate) precipitation in broth state
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The selected bacterial isolates and Bacillus megaterium MTCC 1684 were tested for their ability to form endospores by Schaeffer –Fulton endospore
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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 smears were covered with a piece of absorbent paper cut to fit the slide and the
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slides were placed on wire gauze on a ring stand. The paper was saturated with malachite green and the slide was heated until steam could be seen rising from
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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 slide was rinsed thoroughly with tap water. The slide was drained and
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counterstained for 45 seconds with 0.5% safranin. It was then washed, blotted and examined under a research compound microscope (100 x). The vegetative cells will appear red or pink and the endospores will appear green.
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 (NBU/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
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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
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weighed. CaCO3 precipitant weight (Wc) was determined from the equation:
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Wc = Wfc - Wf
where (Wfc) is the weight of filter paper containing precipitant; and (Wf) is the
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weight of empty filter paper. 2.4 Molecular identification
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Further identification of the isolate was performed using 16S rRNA gene sequencing. The DNA was isolated and the analysis of DNA sequences was
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performed by using the Blastx software (BLAST), National center for
2.5 Concrete
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biotechnology information.
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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 requirements as per IS: 12269 (1987). Locally available sand passing through 4.75 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. The specific gravity of coarse aggregate and fine aggregate were found as 3.0 and 2.69 respectively.
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2.6 Preparation of test concrete specimens and compressive strength test Control concrete mixture was designed as per IS: 10262 (2009) to develop 28-
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day compressive strength of 25 MPa and to have a slump of 25 mm to 50 mm.
Concrete cube specimens of size 150 mm x 150 mm x 150mm were cast. The cell
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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 1684.
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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 Table 3.The materials required for the M 25 concrete mix were weighed. The test
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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 of curing, the specimens were removed from the curing tank and subjected to
Ac ce p
compressive strength testing using compression testing machine as per IS:516 (1959).The specimens were tested immediately on removal from the water and while they were still in the wet condition. Each specimen to be tested was centrally 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
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load by the cross-sectional area of the specimen. The compressive strength test was conducted on three specimens and the average value was taken.
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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.
2.7 Preparation of cracks in concrete beam specimens and quantification of
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crack healing with bacteria
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Concrete beam specimens of size 500 mm x 100 mm x 100mm were cast. The bacterial concrete beam specimens were cast with the bacterial cell concentration of
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105 cells/ml of mixing water with BI 1, BI 2, BI 5 and Bacillus megaterium MTCC 1684.
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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 weighed. The test specimens were cast immediately after mixing. Cracks were 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 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
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the cracks were inspected for the presence of any white precipitates and for the closure or healing of cracks.
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2.8 Characterization of calcite precipitates by SEM (Scanning Electron Microscope)
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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 were obtained using Jeol JSM – 6390 apparatus at an accelerating voltage of 0.5 to
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30 kV. The broken pieces of cube specimens obtained from compressive strength test were collected. All the samples were dried at 1000C in oven for 3 days. These
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samples were gold coated with a sputter coating and subjected to SEM analysis.
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3.1 Endospore Staining
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3. Results
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The endospores designated by green rods within red or pink cells were clearly visible in the results obtained from endospore staining test. The endospores in 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 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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3.2 CaCO3 (calcium carbonate) precipitation in broth state
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Directly after inoculation of NB-U/Ca, a white powder appeared in the media 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 calcite (1.08g) was precipitated by B. megaterium MTCC 1684 followed by
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BI 1.The bacterial isolates BI 1,BI 2 and BI 5 precipitated 0.84g, 0.82g and 0.76 g
3.3 Molecular identification
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of calcium carbonate respectively (Fig.2).
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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. The bacterial isolate BI 1 was identified as Bacillus megaterium having 96%
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similarity with Bacillus megaterium J-65 and Bacillus megaterium BAB-2443, bacterial isolate BI 2 was identified as Bacillus licheniformis having 99% similarity with Bacillus licheniformis AS-4 and bacterial isolate BI 5 was identified as 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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3.4 Compressive strength 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 (M2, M3, M4 and M5) performed better than the control concrete specimens
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(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 16.1% more than control concrete specimens. Bacterial concrete specimens cast
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with Bacillus megaterium BSKAU (M2), Bacillus licheniformis BSKAU (M3) and Bacillus flexus BSKAU (M4) have yielded compressive strengths of 37 MPa,
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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. The compressive strength of specimens cast using media alone without
inoculating bacteria did not show any significant improvement (± 1%) compared to control concrete specimens. 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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bacterial beam specimens cast with Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAUand Bacillus megaterium MTCC 1684 (Fig.4e, 4f and 4j).
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On the other hand, the crack in the bacterial beam specimen cast with Bacillus
flexus BSKNAU was only partially sealed as observed in Fig.4i. The crack in the
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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 Microscope)
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The presence of individual crystals can be observed in bacterial concrete specimens (Fig.3b, 3c, 3d and 3e).On the other hand, the matrix of the control
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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, Bacillus licheniformis BSKNAU, Bacillus flexus BSKNAU and Bacillus
Ac ce p
megaterium MTCC 1684 were capable of forming endospores. Endospores are special resistant dormant structures formed within a cell that is capable of surviving in adverse environmental or hostile conditions. They are extremely 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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endospores, bacteria can withstand large mechanical stresses and chemically induced stresses during mixing of concrete (Sagripanti and Bonifacino, 1996) and
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can remain viable for periods up to 200 years (Schlegel, 1993).When water and air enters through micro cracks, endospores can germinate into vegetative (active)
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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 endospores when compared with Bacillus megaterium BSKAU (Fig.1a) , Bacillus
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licheniformis BSKNAU (Fig.1b) and Bacillus megaterium MTCC 1684 (Fig.1d). Hence the efficiency of Bacillus flexus BSKNAU in increasing strength and in
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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 the hydrolysis of urea which results in the production of ammonia and carbonate.
Ac ce p
Ammonia release act to raise the pH of the medium which is a favorable condition for the precipitation of calcium carbonate. Carbonate binds calcium ions present in medium resulting in the formation of calcium carbonate crystals 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
14 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
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by Bacillus megaterium BSKAU and Bacillus licheniformis BSKNAU are analogous to that of Bacillus megaterium MTCC 1684. Hence Bacillus
cr
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
an
the amount of CaCO3 precipitated.
The inclusion of bacteria has increased the compressive strength of concrete.
M
The insignificant improvement in the compressive strength of cube specimens cast using media alone without inoculating bacteria shows that the cause for strength
d
improvement is due to calcite precipitation by bacteria and not due to media.
te
It has been observed from the results in Table 4 that the strength properties
Ac ce p
of all the bacterial concrete specimens (M2, M3, M4 and M5) have increased with respect to the control concrete specimens (M1). The improvement in compressive strength is due to the plugging of micro cracks and pores in concrete with calcite precipitated by bacteria.
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
15 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)
ip t
attributed that the increase in compressive strength is mainly due to filling of the pores inside the cement concrete cubes with microbiologically induced calcium
cr
carbonate precipitation. The greatest improvement in compressive strength was
us
obtained for concrete specimens with Bacillus megaterium MTCC 1684.The lowest compressive strength has been reported for Bacillus flexus BSKNAU which
an
has produced lesser endospores and less amount of calcite.When compared to bacterial concrete specimens cast with Bacillus megaterium MTCC 1684, the
M
difference in compressive strength of bacterial concrete specimens cast with
d
Bacillus megaterium BSKAU is only 3.5%.This difference is not high and hence it
te
can be interpreted that Bacillus megaterium BSKAU has performed well in increasing the compressive strength of concrete specimens. Minimum difference in
Ac ce p
compressive strength of 4.9% was also observed in bacterial concrete specimens cast with Bacillus megaterium MTCC 1684 and Bacillus licheniformis BSKNAU. The compressive strength test results of concrete specimens with Bacillus 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
16 Page 16 of 37
specimens and bacterial beam specimens cast with Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAU, Bacillus flexus BSKNAU and Bacillus
ip t
megaterium MTCC 1684 respectively. Similarly, the figures, Fig.4b, 4e, 4f, 4i and 4 j show the difference in cracks after healing of cracks at the end of 81 days. In
cr
Fig.4e, 4f and 4j, complete healing of cracks can be observed. This sealing of
us
cracks is due to the precipitation of calcite by the bacterial isolates Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium
an
MTCC 1684.Fig.4b and 4i show the crack which was partially healed. The cracks have not been completely healed and the crack is visible at some points. This
M
indicates that in the beam specimens cast with Bacillus flexus BSKNAU (Fig.4i),
d
the bacteria was able to precipitate calcite, but the calcite precipitation was low
te
and not sufficient to completely seal the cracks. In Fig.4b, partial healing of crack in control beam specimen can be observed. This indicates that the self healing of
Ac ce p
cracks has also taken place in control concrete to certain extent. 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 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
17 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
ip t
difference in texture of control specimens (Fig.3a) and bacterial specimens
(Fig.3b – 3e) as visualized in SEM micrographs may due to precipitation of calcite
cr
crystals in the micro cracks and pores of concrete by bacterial isolates. These
us
results are in agreement with the earlier results which also reported the contrasting textures of matrices of bacterial concrete specimens and control concrete
an
specimens. The matrix of untreated ones (control concrete specimens) appeared to be amorphous, showing no conspicuous crystal growth. On the other hand,
M
bacterial concrete specimens showed crystalline matrix where individual crystals
d
could be recognized (Ghosh et al. 2009).Navneet Chahal et al. (2012) also reported
Ac ce p
S.pasteurii.
te
that distinct calcite crystals could be seen in the SEM analysis of concrete with
Conclusions
All the three newly isolated bacterial strains and Bacillus megaterium MTCC
1684 exhibited high urease activity. They formed endospores and precipitated 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
18 Page 18 of 37
Bacillus megaterium BSKAU and Bacillus licheniformis BSKNAU is close to Bacillus megaterium MTCC 1684.
ip t
The compressive strength of bacterial concrete specimens with Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAU, Bacillus flexus
cr
BSKNAU and Bacillus megaterium MTCC 1684 has increased when compared to
us
control concrete specimens. Even though the maximum increase in strength was found for bacterial concrete specimens with Bacillus megaterium MTCC 1684, the
an
increase in strength of bacterial concrete specimens with Bacillus megaterium BSKAU and Bacillus licheniformis BSKNAU are equally good. Complete healing
M
of cracks was observed in concrete specimens cast with Bacillus megaterium
d
BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium MTCC 1684.
te
The presence of distinct calcite crystals was seen in the SEM analysis of bacterial concrete specimens which has increased the strength of concrete specimens. It can
Ac ce p
be concluded that Bacillus megaterium BSKAU, Bacillus licheniformis BSKNAU and Bacillus megaterium MTCC 1684 are suitable for use in concrete as they have resulted in increased strength and complete healing of cracks in concrete 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.
19 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
ip t
control concrete specimens. References
cr
Geeta Sumbali, Mehrotra RS.Principles of Microbiology. 1st ed. New Delhi: Tata
us
McGraw Hill Education Private Limited;2009.
an
Ghosh P, Mandal S. Development of bioconcrete material using an enrichment culture of novel thermophilic anaerobic bacteria. Indian J Exp Biol2006;44:336-
d
M
339.
te
Ghosh S, Biswas M, Chattopadhyay BD. Microbial activity on the microstructure
Ac ce p
of bacteria modified mortar. Cem Concr Compos 2009;31:93-98. doi:10.1016/j.cemconcomp.2009.01.001.
Gollapudi UK, Knutson CL,Bang SS,Islam MR. A new method for controlling leaching through permeable channels.Chemosphere 1995;30(4):695-705.
Ibtisam A Hammad, Fatma N Talkhan, Ahmed E Zoheir. Urease activity and induction of calcium carbonate precipitation by Sporosarcina pasteurii NCIMB 8841. J Appl Sci Res 2013;9(3):1525-1533.
20 Page 20 of 37
1993;Bureau of Indian Standards.New Delhi,India.
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IS:516 (1959).Methods for test for strength of concrete. Amendment No.2,Reprint
cr
IS:383 (1987).Specification for coarse and fine aggregate from natural sources for
us
concrete. 8th reprint October 1991;Bureau of Indian Standards.New Delhi,India.
an
IS:12269 (1987).Specification for 53 grade ordinary portland cement. Bureau of
M
Indian Standards.New Delhi,India.
d
IS:456 (2000).Plain and reinforced concrete - Code of Practice. 4th revision;
te
Bureau of Indian Standards.New Delhi,India.
Ac ce p
IS:10262 (2009).Guidelines for concrete mix proportioning. Bureau of Indian Standards.New Delhi,India.
Marien PHarkes, Leon A van Paassen,Jacco L Booster,Victoria S Whiffin,Mark CM van Loosdrecht. Fixation and distribution of bacterial activity in sand to induce carbonate precipitation for ground reinforcement.Ecol Eng 2010; 36:112117.doi:10.1016/j.ecoleng.2009.01.004
21 Page 21 of 37
Micheal J Pelczar Jr, Chan ECS, Noel R Krieg. Microbiology.5th ed. New
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Delhi:Tata McGraw Hill Education Private Limited; 1998. Navneet Chahal, Rafat Siddique, Anita Rajor. Influence of bacteria on the
cr
compressive strength, water absorption and rapid chloride permeability of fly ash concrete. Constr Build Mater2012;28:351-356.doi:10.1016/j.conbuildmat.
us
2011.07.042.
an
Ramachandran SK, Ramakrishnan V, Bang SS. Remediation of concrete using
M
microorganisms.ACI Mater J2001;98:3-9.
Ramakrishnan V, Bang SS, Deo KS.A novel technique for repairing cracks in high
d
performance concrete using bacteria.Proceeding of the International Conference
Ac ce p
617.
te
on High Performance. High Strength Concrete;1998; Perth, Australia.1998. p. 597-
Ramakrishnan V, Deo KS, Duke EF, Bang SS.SEM investigation of microbial calcite precipitation in cement. Proceeding of the International Conference on Cement Microscopy;1999; Las Vegas, Nevada. 1999. p. 406-414. Ramakrishnan V, Panchalan RK , Bang SS. Bacterial concrete. Proceedings of SPIE- International Society of Optical Engineering, Smart Materials;2001; Australia. 2001;4234: p. 168-176 . doi:10.1117/12.424404.
22 Page 22 of 37
Ramakrishnan V, Panchalan RK , Bang SS.Improvement of concrete durability by bacterial mineral precipitation.Proceedings of the 11th International Conference on
ip t
Fracture; 2005; Turin, Italy. 2005.
cr
Sagripanti JL, Bonifacino A.Comparative sporicidal effects of liquid chemical
us
agents.Appl Environ Microbiol1996;62 (2):545.
an
SchlegelHG.General microbiology. Cambridge:Cambridge University Press; 1993. Stocks-Fischer S,Galinat JK,Bang SS. Microbiological precipitation of CaCo3.Soil
M
Biol Biochem 1999;31(11):1563-1571.
d
Van Tittelboom K, De Belie N, De Muynck W, Verstraete W.Use of bacteria to
te
repair cracks in concrete.Cem Concr Res2010;40:157-
Ac ce p
166,doi:10.1016/j.cemconres.2009.08.025. ZhongL,Islam MR.A new microbial process and its impact on fracture remediation.Paper presented at the 70th Annual Technical Conference and Exhibition of the Society of Petroleum Engineers,1995; Dallas, Texas. 1995.doi:10.2118/30519-MS.
Web reference
http://www.austincc.edu/microbugz/endospore_stain.php last accessed on 02/11/2014.
23 Page 23 of 37
Figure captions
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Fig.1. Endospore staining pictures of the bacterial isolates (a) BI 1. (b) BI 2. (c) BI 5 and (d) B.megaterium MTCC 1684.
us
megaterium MTCC 1684 (B.mega MTCC 1684).
cr
Fig.2. Comparison of CaCo3 precipitation by different bacterial isolates and Bacillus
an
Fig.3. Scanning electron micrographs of (a) control concrete specimen with no calcite crystals. (b) concrete specimen with Bacillus megaterium BSKAU
M
indicating the presence of calcite crystals. (c) concrete specimen with Bacillus licheniformis BSKAU indicating the presence of calcite crystals.(d)
d
concrete specimen with Bacillus flexus BSKAU indicating the presence of
te
calcite crystals and (e) concrete specimen with Bacillus megaterium MTCC
Ac ce p
1684 indicating the presence of calcite crystals. Fig.4. Photographs of crack healing (a) in control concrete beam specimen before healing. (b) in control concrete beam specimen after 81 days of healing. (c) in concrete beam specimen with Bacillus megaterium BSKAU before 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)
24 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
Ac ce p
te
d
M
an
us
cr
megaterium MTCC 1684 after 81 days of healing.
ip t
81 days of healing and (j) in concrete beam specimen with Bacillus
25 Page 25 of 37
Table 1
BI 2
BI 3
BI 4
BI 5
cr
BI 1
ip t
Table 1 Electric Conductivity [EC (mS / m)] of Urease Assay Mixture at Different Time Intervals
Bacillus megaterium MTCC 1684
EC (mS / m)
Time (s)
EC (mS / m)
Time (s)
EC (mS / m)
Time (s)
EC (mS / m)
Time (s)
EC (mS / m)
Time (s)
EC (mS /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
3960
142.1
3960
139.2
3960
6360
161.2
6360
140.6
6360
an
us
Time (s)
119.8
300
131.9
300
251
125.0
3960
121.8
3960
141.2
3960
282
129.2
6360
126.2
6360
145.9
6360
296
Ac ce p
te
d
M
300
1 Page 26 of 37
Table 2
Table 2 Physical and Chemical Properties of Cement Physical property Colour Specific Gravity
Grey 3.15
cr
21 62 5.04 3.16 4.56 0.08 1.29
Ac
ce pt
ed
M
an
us
SiO2 CaO Al2O3 Fe2O3 MgO Na2O Loss on Ignition
ip t
Chemical constituent (%)
Page 27 of 37
Table 3
Table 3 Concrete Mix Proportions Concrete Mixture No. M2 413
M3 413
M4 413
M5 413
Natural Sand (kg/m3) Bacteria used (105 cells/ml of mixing water)
680
680
680
680
680
Coarse aggregate (kg/m3) Water Cement Ratio
B.megaterium MTCC 1684
BI 1
BI 2
BI 5
1290
1290
1290
cr
-
0.55
0.55
0.55
0.55
0.55
186
186
186
Water (kg/m3)
1290
Ac
ce pt
ed
M
an
186 (tap 186 water) *M stands for Concrete Mixture 1 control concrete cast with no bacterial cells
1290
us
Cement (kg/m3)
ip t
M11 413
Page 28 of 37
Table 4
Table 4 Compressive Strength Values of Concrete Mixtures [Values are Mean ± Standard Deviation]
Compressive strength at 28 days in MPa
0
M1 (Control)
33.00 ± 0.33
105
M2
37.00 ± 1.06
105
M3
105
M4
105
M5
ip t
Concrete Mixture
cr
No bacterial cells
Bacterial cells / ml of mixing water
us
Bacteria
36.50 ± 0.44
Live cells
38.30 ± 0.53
Ac
ce pt
ed
M
an
35.00 ± 0.19
Page 29 of 37
Figure 1
te Ac ce p
(b)
d
M
an
us
cr
ip t
(a)
Page 30 of 37
Ac ce p
te
(d)
d
M
an
us
cr
ip t
(c)
Fig.1
Page 31 of 37
1.2 1 0.8 0.6 0.4 0.2 0 BI 1
BI 2
BI 5
B.mega MTCC 1684
cr
Bacterial isolates
ip t
Weight of CaCo3 precipitation in g/l
Figure 2
Ac
ce pt
ed
M
an
us
Fig. 2.
Page 32 of 37
Figure 3
an
us
cr
ip t
(a)
Ac
ce pt
ed
M
(b)
Page 33 of 37
an
us
cr
ip t
(c)
Ac
ce pt
ed
M
(d)
Page 34 of 37
M
an
us
cr
ip t
(e)
Ac
ce pt
ed
Fig.3.
Page 35 of 37
Figure4
us
cr
ip t
(a)
an
Visible crack
Ac ce p
te
d
M
(b)
Visible Crack
Partial healing of cracks
Page 36 of 37
(d)
ip t
(c)
Visible crack (f)
us
(e)
cr
Visible crack
an
`
Complete healing of cracks
M
Complete healing of cracks
(h)
Ac ce p
te
d
(g)
Visible crack
(i)
Visible crack
(j) `
Partial healing of cracks
Visible crack
Complete healing of cracks
Fig.4.
Page 37 of 37
