Int. J. Life. Sci. Scienti. Res., 4(5): 2025-2036, September 2018

 

Isolation, Characterization, and Application of Calcite Producing Bacteria for Self-Healing Concrete Preparation

 

Diksha H. Jokyani¹, Dharmvir A. Chouhan²*

 

¹Student, P. G Department of Microbiology, Dhote Bandhu Science College, Gondia, India

²Assistant Professor and HOD, P. G Department of Microbiology, Dhote Bandhu Science College, Gondia, India

 

^*Address for Correspondence: Dr. D.A. Chouhan, Head and Assistant Professor, P.G Department of Microbiology, Dhote Bandhu Science College, Kudwa Road Gondia- 441614, Maharashtra, India

 

ABSTRACT- Concrete being the foremost building material broadly used in the construction sector is subjected to crack formation due to low tensile strength, durability, and ductility. So this issue is of great curiosity to the researchers in pursuit for the concrete production with better properties. Micro-cracks are the main reason for structural failure occurs, when the load applied exceeds its limits. This causes the seepage of water and other salts. In order to overcome this, the carbonate precipitating, non-pathogenic, spore-forming, alkaline resistant strain of Bacillus subtilis has been explored as a bio-cementing material. Cracks in M20 grade concrete blocks are injected by direct means with screened bacterial strain Bacillus subtilis (BH3) at the cell concentration of 10⁴, 10⁵, 10⁶ CFU/ml with silica gel as an immobilizing agent and calcium lactate as a food source.  The cracks were allowed to heal for appropriate time duration at specific pH, temperature, and urea concentration. These findings suggest the potential of Bacillus subtilis in an autogenously healing process.

Keywords: Bacillus subtilis, BH3, Carbonate precipitation, Crack, Healing and Bio-cementation

 

INTRODUCTION- A large amount of non-renewable resource is consumed by the construction engineering sector, most of which contribute to the highest proportion of global CO₂ emission at their production or application stage. The concrete production process is an energy-intensive process if in case of mining, transportation and processing are considered. Its production level lies at about 2.35 billion metric tonnes per year and contributes an astonishing 10% of CO₂ emission in the atmosphere ^[1]. Another issue concerns is the huge maintenance costs for structure built in past. About 10% of the bridges in the USA are considered structurally deficient and 10% of it is considered functionally obsolete ^[2]. Apart from this the factors like freeze-thaw reactions, shrinkage, results in the cracking of concrete structures during its hardening and this ultimately leads to structural deformation. Latter the ingress of some hazardous chemicals and moisture into the concrete may lead to decrement in its serviceability whereas; the penetration of sulphates and chlorides in cracks causes the durability to be affected. Hence the more concern should be given to this so as to prevent the expansion of cracks by a sustainable means that might involve the natural microbial mechanism of bio-cementation which is promising.

Autonomously healed concrete is nothing but the biologically produced limestone by which the cracks can be healed effectively. The screened bacterial strain of genus “Bacillus” along with the calcium based nutrient like calcium lactate and an immobilizing agent like silica gel is mixed in appropriate proportion and injected into the preformed cracks by direct means. In case if it is added initially to the concrete mixture then the self-healing agent can lie in the dormant stage within the concrete for up to about 200 years. Due to any of the damage the water starts to percolate deep into the structure and the dormant bacterial form if get the growth favourable conditions to acquire its vegetative form and starts to feed on calcium lactate consuming oxygen and thereby converting soluble calcium lactate to insoluble calcium carbonate better called limestone. Here the consumption of oxygen by the bacterial strain is advantageous as oxygen is responsible for steel corrosion thereby enhancing the durability of steel structures ^[3], Since 1980’s several researchers had been working on this concept and had used the different bacterial strains such as Jonker et al. ^[4] used Bacillus cohnii bacteria to precipitate CaCO₃ while Santhosh et al. ^[5] and Bang et al. ^[6] used Bacillus pasteurii.

As the bacterial cell wall is negatively charged, it will draw the cations from the environment, Ca²⁺ and deposit it on their cell surface serving as a nucleation site. ^[6]

Ca²⁺ + Cell     ----------------------à   Cell- Ca²⁺                                                                            1

This bacterial genus is known to have specific urease activity that is utilized for the hydrolysis of urea into 1 mole of ammonia and 1 mole of carbonic acid ^[7].

H₂N-CO-NH₂ + H₂O    ----------------------à         NH₃ + H₂N-CO-OH                                     2

                      

H₂N-CO-OH + H₂O          ----------------------à        NH₃ + H₂CO₃                                                              3   

These two products will subsequently form 2 moles of ammonium and hydroxide ion as,

 H₂CO₃         ----------------------à        HCO^3- + H⁺                                                                           4

2NH₃ + 2H₂O     ----------------------à    2NH₄⁺ + 2OH^-                                                                     5

Reaction 4 and 5 in turn will results in pH increase and shifts the equilibrium, resulting in the carbonate ion formation as,

HCO^3- + H⁺ + 2NH₄⁺ + 2OH^-   ----------------------à     CO₃^2- + 2NH₄⁺ + 2H₂O                             6

The carbonate formed in reaction 6 will subsequently react with CO₃^2- ions, leading to CaCO₃ precipitation.

 Cell- Ca²⁺ + CO₃^2-     ----------------------à            Cell- CaCO₃                                                    7    

 

MATERIAL AND METHODS

Materials and Chemical used- Soil samples used for isolation of potent strain were collected from 12 different construction sites across the Gondia district, India. All the required chemicals in this work were purchased from Hi-media laboratories, India. The research work was carried out in the P.G Department of Microbiology, Dhote Bandhu Science College, Gondia, India from August 2017 to March 2018.

Preparation and artificial cracking of concrete blocks- The concrete blocks of M20 grade were first prepared with the mould size of 100 mm x 60 mm and left for 28 days curing process. Further, the blocks were subjected to indigenous cracking by plastic shrinkage method (plastic shrinkage cracks usually ranges between 1 - 2 mm depth) ^[8].

Determining Size of cracks- Size of the cracks in concrete block was measured using scale ^[9].

Isolation of new ureolytic bacteria:  Soil samples pasteurized at 80⁰C for 15 minutes then subjected to tenfold dilution. The 0.2 ml of pasteurised diluted sample was then plated on sterile urea agar plates with 5% urea concentration. Plates were incubated at 37⁰C for 24 - 48 hrs and observed the isolated colonies. Pink colour colonies were selected and further screened based on the capability to degrade urea in Christensen’s agar medium and to tolerate high alkaline condition based on pH optimization studies. The single selected potent organism was in turn identified based on its morphological and biochemical characteristics. Latter on the identified isolate was pure cultured on urea agar slants and preserved at low temperature (4⁰C) ^[10].

 

Study on effect of environmental and nutritional condition on urease production a capability of BH3 strains: Bacillus subtilis strain with promising result was selected and the effect of environmental condition like temperature, and nutritional condition such as urea concentration were characterized. For temperature optimization, 1 ml of overnight growth culture was inoculated in 3 tubes containing urease broth except the control tube and incubated at 3 different temperature ranges as 25⁰C, 35⁰C, and 45⁰C then after 24 hours the tubes were observed for change in colour intensity and biomass was determined nephlometrically. For urea concentration, optimization purpose 1 ml of overnight growth culture was inoculated in 3 tubes containing urea in concentration range of 2%, 5% and 10% except the control tube and incubated at 35⁰C. After 24 hours the tubes were observed for change in colour intensity and biomass was determined nephlometrically. ^[11]

 

Growth Curve of Bacillus subtilis strains:  The growth curve of screened isolate was determined to set a growth comparison point at optimum condition. Three ml of overnight growth culture was used to inoculate in 30 ml of urea broth in 250 ml conical flask then the culture was incubated for 27 hours at 35⁰C, pH 8, and 5% urea concentration. Inoculation time was considered as zero time and the quantitative determination of growth was carried out by spectrophotometer at 600 nm.

Simultaneously the viable cell count was determined as a colony forming units/ml (CFUs) ^[12]. The desired isolate was purified and mixed with silica gel in 1: 100 dilution factors with the cell count 10⁴,10⁵,10⁶ cells so as to immobilize the bacterial cells and hence it can remain embedded for the long time in the concrete matrix. Along with it, calcium lactate is also added to it with molar mass 218 g/mole that serve as food source for bacterial cells.  As it gets the growth favourable conditions it will soon start to precipitate CaCO₃ and the bacterial cells will be in turn coated with it resulting in the autogenous healing ^[13].

Water Permeability Test of Concrete- To determine the water absorption ability of bacterial concrete, this test was carried out in a comparative manner. For this, the control and bacteria treated concrete blocks were kept overnight saturated in the saline buffer and weighed. Then both blocks were dried in the oven at 100⁰C for 24 hours, cooled and again weighed. The obtained values were then put into the formula given below to determine percent water absorption by both the blocks ^[14].

 

% water absorption =   W_Saturation – W_{Oven Dried} / W_{Oven Dried} X 100

 

Where,

W_Saturation = Weight of block after saturation

W_{Oven Dried}= Weight of block after drying

 

RESULT- As the durability of concrete is affected by the cracks leading to corrosion of reinforcing bars, the general method of repairing is time consuming and expensive, so bio based calcite precipitation has been proposed as an alternative and sustainable, environment friendly cracks repair technique. The bio agent selected for this purpose was based on its tolerance to high pH, and continuous formation of dense CaCO₃ in the liquid medium. According to analysis and study of blocks by the visual examination of concrete core, the estimated size of cracks varied from 0.3 to 0.5 mm ^[9]. Total 12 urease positive Bacillus subtilis strains were isolated from soil sample. Out of these 12 isolates Bacillus subtilis BH3 strain was found to be most potent.

 

Optimization- The optimum temperature required for growth of Bacillus subtilis was 35⁰C. Table 1 and Fig. 1 shows the optimization of temperature by using 3 different temperature ranges.

 

Table 1: Effect of temperature on growth and urease activity of Bacillus subtilis strain

S. No.	Bacterial strains	Temperature range (0C)	Growth measurement	Urease activity	Interpretation
			OD600 nm
1.	BH3	25	1.51	Color changes to mild pink	The maximum urease activity with effective growth was observed at temperature range of 350C
2.		35	1.52	Color changes to intense pink
3.		45	1.519	Color changes to light pink

BH3= Bacillus subtilis strain

 

 

 

Fig. 1: Effect of temperature on growth of Bacillus subtilis strain

 

The specific urea concentration supporting the growth of selected ureolytic bacterial strain (BH3) was 5%. The Table 2 and Fig. 2 show the effect of urea concentration on the bacterial growth and urea degrading ability of it.

 

Table 2: Effect of urea conc. on growth and urease activity of Bacillus subtilis BH3 strain

 

 

Fig. 2: Effect of urea conc. on growth of Bacillus subtilis strains

 

Spectroscopic growth curve- The growth curve of Bacillus subtilis was elucidated using the partially optimized condition. Table 3 and Fig. 3 indicate the growth curve of B. subtilis isolates, which is considered as the selected isolate of this research paper. The maximum OD was seen between 6 to 10 hours and referred as log phase. Nephlometric reading showed that the cultures reached the stationary phase after 10 hours. After 24 hours the bacterial growth was inhibited due to media component depletion and the release of secondary metabolite that may be toxic to viable cells.

 

Table 3: Nephlometric absorbance values to construct growth curve of screened isolate of B. subtilis

 

Fig. 3: Growth curve of screened potent isolate of B. subtilis

 

Studies on concrete crack healing potential of BH3 strains- In this investigation, the 3 concrete blocks, Block 1, Block 2, and Block 3 were used for autogenous healing by the selected strain of Bacillus subtilis BH3. Block 1 with crack size of 0.45 mm width was injected with 10⁵ CFU/ ml along with silica gel and calcium lactate showed the healing of cracks at 21^st day of inoculation and the width of crack was reduced to 0.18 mm.

Similarly the Block 2 with crack size of 0.52 mm when injected with 10⁶ CFU/ml showed the efficient healing on 19^th day of inoculation, whereas the Block 3 with crack size 0.48 mm width, when injected with 10⁴ CFU/ml, showed the healing on 23^rd day of inoculation with the reduction in crack size up to 0.25 mm.

Table 4: Concrete crack healing potential of B. subtilis strain in block 1 (10⁵ cells/ml)

S. No.	Date	Initial Characteristics	Final characteristic                 (Width)	Interpretation
1.	22nd January	Grade: M20 Size:100 X 60 mm Curing: 28 days Crack developed by: plastic  shrinkage type Width: 0.45 mm	0.45 mm	Block 1, inoculated with the 105 cells/ ml of BH 3 strain, showed the effective healing within 21 days from the day of inoculation
2.	23rd January		0.45mm
3.	24th January		0.44 mm
4.	25th January		0.43 mm
5.	26th January		0.41 mm
6.	27th January		0.4 mm
7.	28th January		0.4 mm
8.	29th January		0.38 mm
9.	30th January		0.37 mm
10.	31st January		0.37 mm
11.	1st February		0.35mm
12.	2nd February		0.34 mm
13.	3rd February		0.31 mm
14.	4th February		0.28 mm
15.	5th February		0.27 mm
16.	6th February		0.25 mm
17.	7th February		0.23 mm
18	8th February		0.2 mm
19.	9th February		0.19 mm
20.	10th February		0.19 mm
21.	11th February		0.18 mm

 

Fig. 4: Crack healing study in bacteria treated cracked concrete block (Block 1)

 

Table 5: Concrete crack healing potential of B. subtilis strains in block 2 (10⁶cells/ml)

S. No.	Date	Block characteristics	Final characteristic                 (Width)	Interpretation
1.	22nd January	Grade: M20 Size:100 X 60 mm Curing: 28 days Crack developed by: plastic  shrinkage type Width: 0.52 mm	0.52 mm	Block 2, inoculated with the 106  cells/ ml of BH 3 strain, showed the effective healing within 19 days from the day of inoculation
2.	23rd January		0.52 mm
3.	24th January		0.52 mm
4.	25th January		0.518mm
5.	26th January		0.516  mm
6.	27th January		0.514 mm
7.	28th January		0.51 mm
8.	29th January		0.48 mm
9.	30th January		0.47 mm
10.	31st January		0.47 mm
11.	1st February		0.46 mm
12.	2nd February		0.44 mm
13.	3rd February		0.44 mm
14.	4th February		0.4 mm
15.	5th February		0.4 mm
16.	6th February		0.4 mm
17.	7th February		0.39 mm
18.	8th February		0.39 mm
19.	9th February		0.38 mm

 

Fig. 5: Crack healing study in bacteria treated cracked concrete block (Block 2)

 

Table 6: Concrete crack healing potential of B. subtilis strain in block 3 (10⁴cells/ml)

 

S. No.	Date	Initial Characteristics	Final characteristic                 (Width)	Interpretation
1.	22nd January	Grade: M20 Size:100 X 60 mm Curing: 28 days Crack developed by: plastic  shrinkage type Width: 0.48 mm	0.48 mm	Block 3, inoculated with the 104 cells/ ml of BH 3 strain, showed the effective healing within 23 days from the day of inoculation
2.	23rd January		0.48 mm
3.	24th January		0.45 mm
4.	25th January		0.44 mm
5.	26th January		0.43  mm
6.	27th January		0.42 mm
7.	28th January		0.38 mm
8.	29th January		0.38 mm
9.	30th January		0.35 mm
10.	31st January		0.34 mm
11.	1st February		0.3 mm
12.	2nd February		0.3 mm
13.	3rd February		0.3 mm
14.	4th February		0.29 mm
15.	5th February		0.29 mm
16.	6th February		0.29 mm
17.	7th February		0.28 mm
18	8th February		0.28 mm
19.	9th February		0.28 mm
20.	10th February		0.26 mm
21.	11th February		0.26 mm
22.	12th February		0.25 mm
23.	13th February		0.25 mm

 

Fig. 6: Crack healing study in bacteria treated cracked concrete block (Block 3)

 

 

Water absorption test by using saline buffer- The saline buffer is used in this research work so as to determine the increased resistance of concrete block towards the water penetration and this test was conducted at a laboratory level. According to this test, the normal concrete block had shown the higher water absorption as compared to the concrete block treated with potent ureolytic bacterial strain. Table 7 numerically represents the bacterial influence on the water permeability of blocks.

 

Table 7: Water saturation test results of bacteria healed cracked concrete

Blocks No.	Weight (Saturation)	Weight (Dried)	% water absorption
Block 1	192.73	183.70	4.91%
Block 2	188.05	174.80	7.58%
Block 3	152.30	140.10	8.70%
Control	131.1	119.4	9.79%

 

DISCUSSION- This study has revealed the BH3 strain of bacterial species Bacillus subtilis isolated from commercial construction sites, have the high ureolytic activity and can tolerate the pH range up to 12, which becomes the primary factor for bio-cementation. Salmabanu L and Suthar G ^[2] stated this genus of endolithic bacteria can resist the pH range up to 13 and persist for the long duration with efficient ureolytic activity. The isolated and screened potent isolate (BH3) was identified phenotypically and biochemically same as done by Achal et al. ^[11] and  Cheng et al. ^[15] while the species level characterization was achieved by 16s gene sequencing technique. From the further optimization studies of BH3 strain, it was clear that it shows the effective urea degrading activity at temperature 35⁰C with 5% urea concentration. Similar conclusion was given by Steubing ^[12], who stated that change in temperature and nutritionally sound components can also affect the CaCO₃ precipitation efficiency by ureolytic bacteria. Growth curve of this potent strain was determined nephlometrically that showed the log phase of this strain during initial 6-10 hours and become stationary after 10 hours further reduction in the growth rate after 24 hours. The cell count in the range 10⁵ CFU/ml when injected in block 1 with the crack width size of 0.45 mm shows the significant reduction in crack size upto 0.18 in 21 days after inoculation which was more effective as compared to other 2 concrete block injected with 10⁶ and 10⁴ CFU/ml cells. A similar finding was reported by Bai and Varghese ^[16] during an experimental investigation on the strength   properties of fly ash based bacterial concrete.

The bacteria treated concrete block shows the significant reduction in its water penetration ability compared to normal concrete block due to the deposition of CaCO₃ matrix like structure around the bacterial cells thereby blocking the pores and inhibiting the water percolation through it. As with constant development in the field of civil engineering and increasing CO₂ emission, considerable efforts has been devoted to this alternative concept of bio healing of concrete.

 

CONCLUSIONS- Our research paper describes that due to its self-healing abilities, eco-friendly nature, resistance to water absorption thereby enhancing the strength of building materials along with prevention of steel corrosion, the microbial concrete technology had proved to be better than the conventional technologies. It may also be termed as “Smart Bio Material”. The overall development of strength in bio-concrete is attributed to significant reduction in water absorption capability by using potent bacterial strain of Bacillus subtilis immobilized by means of silica gel and supplemented with calcium lactate as food source. The efficient healing was shown with 10⁵ cells, in block 1 with the reduction in crack size from 0.43 mm to 0.29 mm width at 21^st day of inoculation.

More exploratory works at large scale should be undertaken to determine the efficacy of bio cementation for consolidations of building. To make the process economical, microbial additives can repaired by industrial growth of cells by employing the products as lactose mother liquor and corn step liquor as nutrient source. Also, the durability of bacterial concrete should be studied under the various weathering conditions.

 

ACKNOWLEDGMENT- The author is grateful to P.G. Department of Microbiology, Dhote Bandhu Science College, India for their help and support in carrying out present study. Technical assistance and advice was provided by Dr. D. A. Chouhan so I would like to thank him for his encouragement.

 

CONTRIBUTION OF AUTHORS

Research concept- Prof. D. A. Chouhan

Research design- Prof. D. A. Chouhan

Supervision- Prof. D. A. Chouhan

Data collection- Diksha H. Jokyani

Data analysis and interpretation-  Diksha H. Jokyani

Literature search- Diksha H. Jokyani

Writing article- Diksha H. Jokyani, Prof. D. A. Chouhan

Critical review- Prof. D. A. Chouhan

Article editing- Prof. D. A. Chouhan

Final approval- Prof. D. A. Chouhan

 

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