ASIAN JOURNAL OF CIVIL ENGINEERING (BUILDING AND HOUSING) VOL. 11, NO.

1 (2010) PAGES 43-55

PERFORMANCE OF STANDARD GRADE BACTERIAL (BACILLUS SUBTILIS) CONCRETE

S. Sunil Pratap Reddy , M.V. Seshagiri Rao , P. Aparna and Ch. a Sasikala Department of Civil Engineering,CJITS, Jangaon, Warangal, India b Department of Civil Engineering, JNTU, Hyderabad, India c Center for Environment, JNTU, Hyderabad, India
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b c c

ABSTRACT
The objective of the present investigation is to obtain the performance of the concrete by microbiologically induced special growth/filler. One such thought has lead to the development of a very special concrete known as Bacterial Concrete where bacteria the induced in the mortars and concrete to heal up the faults. Researchers with is bacterias different proposed different bacterial concretes. Here an attempt was made by using the bacteria Bacillus subtilis. Calcite formation by Bacillus subtilis is a model bacterium, laboratory which can produce calcite precipitates on suitable media supplemented with c aalcium source. Cement mortar cubes with four different cell concentrations were cast control specimen was also cast. This study showed a significant increase in the and 5 compressive observed due to the addition of bacteria for a cell concentration of 10 cells per strength was ml of mixing water. From Scanning Electron Micrography analysis, it is noted that were pores partially filled up by material growth with the addition of the bacteria. Reduction in due to such material growth will obviously increase the material strength. pore cubes with Concrete and without addition of bacteria were cast and it is observed that there is an improvement in the compressive strength for the cubes with the addition of Concrete bacteria. cylinders with and without addition of bacteria were cast and it is observed that is an improvement in the Split tensile strength for the cylinders with the addition there bacteria. From the durability studies, the percentage weight loss and percentage strength of with loss 5% H2SO 4revealed that Bacterial concrete has less weight and strength losses than conventional concrete and it also revealed that bacterial concrete is more durable in terms the Acid Durability Factor and Acid Attack Factor than conventional of concrete. Keywords: Bacterial concrete; bacillus subtilis; compressive strength; durability

1. INTRODUCTION
Concrete is a material, which is by far the most used building material in the world.
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Email-address of the corresponding author: sunilpratap@yahoo.com (S. Sunil Pratap Reddy)

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S. Sunil Pratap Reddy, M.V. Seshagiri Rao, P. Aparna and Ch. Sasikala

Concrete has a large load bearing capacity for compression load, but the material is weak in tension. That is why steel reinforcement bars are embedded in the material to be able to build structures. The steel bars take over the load when the concrete cracks in tension. concrete on other hand protects the steel bars for attacks from the environment and The corrosion prevent to take place. However, the cracks in the concrete form a problem. Here ingress of water and ions take place and deterioration of the structure starts with the the corrosion of the steel. To increase the durability of the structure either the cracks that formed are repaired later or in the design phase extra reinforcement is placed in the are structure that the crack width stay within a certain limit. This extra reinforcement is to ensure only then needed for durability reasons (to keep the crack width small) and not for structural capacity. Especially with current steel prices this extra steel is not desirable. Durability one is reason to prevent cracks or limit crack widths. Other reasons are water tightness of structures, loss of stiffness and aesthetic reasons. If in some way a reliable method could be developed that repairs cracks in concrete automatically, this would increase and durability and functionality developed that repair cracks in concrete enormously. On ensure other the hand it would save a lot of money. Of course repair cracks of cracks that develop in concrete structures would go down. But also the extra steel that is used to limit crack could widthsprobably be saved to a large extent. Cracks widths in concrete structure should be limited, mainly for durability reasons. If cracks widths are too large the cracks need to be repaired or extra reinforcement is needed already in the design. If a method could be developed to automatically repair cracks concrete this would save an enormous amount of money, both on the costs of in fluids fro injection cracks and also on the extra steel that is put in structures only to limit widths. crack For structural reasons this extra steel has no meaning. A reliable self-healing for concrete would lead to a new way of designing durable concrete structures, which is method beneficial for national and global economy. The Bacterial Concrete can be made by embedding bacteria in the concrete that able to constantly precipitate calcite. This phenomenon is called microbiologically are calcite inducedprecipitation. Calcium carbonate precipitation, a widespread phenomenon bacteria, among has been investigated due to its wide range of scientific and technological implications. Calcite formation by Bacillus subtilis is a model laboratory bacterium, can produce calcite precipitates on suitable media supplemented with a calcium source. which common soil bacterium, Bacillus subtilis, was used to induce A 3 precipitation. CaCOprinciples for this application are that the microbial urease hydrolyzes urea to The basic ammonia produce and carbon dioxide, and the ammonia released in surroundings increases pH, subsequently leading to accumulation of insoluble CaCO3. The favorable conditions do directly exist in a concrete but have to be created. A main part of the research will focus not on topic. How can the right conditions be created for the bacteria not only to survive in this concrete but also to feel happy and produce as much calcite as needed to repair the Further cracks. more the bacteria should be suspended in a certain concentration in a medium certain before they are mixed through the concrete ingredients. Optimization is here, which involves experimental needed testing.

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2.1 Materials used The following are the details of the materials used for concrete making. Cement Ordinary Portland cement of 53 grade available in local market is used in the investigation. The cement used has been tested for various properties as per IS:40311988 and found to be confirming to various specifications of IS:12269-1987 having specific gravity of 3.0 Ennore Ennore Sand sand was used to find the compressive strength of cement mortar cubes. Aggregate Coarse Crushed granite angular aggregate of size 20 mm nominal size from local source was used as coarse aggregate having specific gravity of 2.71 Fine Aggregate Natural river sand confirming to IS-383 zone II having specific gravity of 2.60 Water Locally available portable water Confirming to IS 456 is used. Microorganism B s acillus subtilis, a model laboratory bacterium is used. 2.2 Biochemical characteristics of the pure culture bacillus subtilis JC3 Characteristics Bacillus subtilis JC3 Long rods, 0.6-0.8 m in width and 2.0 to 3.0 m in Shape, size, gram stain length, gram positive ( see Figure 2.) Colony morphology (on Irregular, dry, white, opaque colonies (see Figure 1.) nutrient agar plate) Fermentation: Lactose No acid, no gas Dextrose No acid, no gas Sucrose Acid and gas H2S production Nitrate reduction Indole production Methyl Red test Vogesproskauer test Citrate utilization Catalase activity + Gelatin liquefaction + Starch hydrolysis + Lipid hydrolysis + Note: +:- Present -:- Absent

2. EXPERIMENTAL INVESTIGATION

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S. Sunil Pratap Reddy, M.V. Seshagiri Rao, P. Aparna and Ch. Sasikala

Figure 1. Colony morphology of strain JC3 on nutrient agar plate (irregular, dry, white, opaque colonies)

Figure 2. Phase contrast microphotograph of strain JC3 (long rods, 0.6-0.8 m in width and 2.0 to 3.0 m in length, gram positive)

2.3 Culture of Bacteria The pure culture was isolated from the soil sample of JNTU and is maintained constantly nutrient agar slants. It forms irregular dry white colonies on nutrient agar. on Whenever single colony of the culture is inoculated into nutrient broth of 25 ml in 100 required a 0 conical flask and the growth condition are maintained at 37 C temperature and placed ml 125 in rpm orbital shaker. The medium composition required for growth of culture is Peptone : 5 g/lt. NaCl : 5 g/lt. Yeast :3 extract g/lt. 2.4 Maintenance of Stock Stock cultures of Bacillus subtilis JC3 were maintained on nutrient agar slants. The Cultures culture

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was streaked on agar slants with an inoculating loop and the slants were incubated at 37 0 C. After 2-3 days of growth, slant cultures were preserved under refrigeration (4 C) until further use. Subculturing was carried out for every 90 days. Contamination from bacteria other was checked periodically by streaking on nutrient agar plates.

3. TEST ON CONCRETE
Sand and cement was mixed properly. Distilled water and the require amount microorganisms with media were mixed and added to the cement sand mix to make of sand paste. Standard cubes 70.7mm.7mm.7mm were cast and compacted cement vibration machine. All the specimens were cured in water. The compressive strength of in mortar cubes at 3 days, 7 days and 28 days was determined and Scanning Electron the Microscopy (SEM) analysis was made on the broken sample of 28 days cube Micrographs specimen. were obtained with a RUSKA 3,500 Scanning Electron Microscope. process is carried out in electrically operated concrete mixer. The The mixing arematerials laid in uniform layers, one on the other in the ordercoarse aggregate, fine aggregate and cementitious material. Dry mixing is done to obtain a uniform color. Distilled water and 5 require amount of microorganisms( i.e. 10 /ml cell concentration were used) with the were mixed. Standard cubes 100mm x 100mm x 100mm were cast and compacted. All media the specimens were cured in water. The compressive strength of the concrete cubes at 7 14 days, 28 days, 60 days, 90 days, 180 days, 270 days and 365 days was determined. days, Standard cylindrical moulds of size 150mm diameter and 300mm length were cast compacted. Split tensile strength tests were carried out on cylinders using ac and ompression testing machine of 1000 KN capacity as per IS 516:1959. Concrete cubes with and addition without of bacteria were cast. After 28 days of casting, each cube is tested for weight and dimensions . An experimental program was conducted on ordinary Portland cement concrete bacterial concrete. Specimens are subjected to 5% solutions of H SO2. Cubes and 4 continuously immersed in solution. The specimens are arranged in the plastic tubs in are way a such that the clearance around and above the specimen is not less than 30 mm. The solution changed for an interval of every 15 days after taking the measurements. The has been response of the specimens to the solutions was evaluated through change in appearance, weight, compressive strength, thickness and solid diagonals. Two specimens from each were used for testing after every 15 days of immersion. Before testing, each specimen group was removed from the baths, and brushed with a soft nylon brush and rinsed in tap water. This process removes loose surface material from the specimens. For determining resistance of concrete specimens to aggressive environment such as acid attack, the the durability factors are proposed by the author, with the philosophy of ASTM C 666 , the as basis. ASTM C 666 considers the standard test method for resistance of concrete to rapid freezing and thawing and the durability factors are defined in terms of relative dynamic modulus of elasticity. In the present investigation, the author derived the Durability Factors directly in terms of relative strengths. The relative strengths are Acid with respect to the 28 days value (i.e at the start of the test). The Acid Durability always Factors

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S. Sunil Pratap Reddy, M.V. Seshagiri Rao, P. Aparna and Ch. Sasikala

(ADF) can be designed as follows. Acid Durability Factor (ADF) = Sr N / w Mhere, Sr = relative strength at N days, ( % ) N = number of days at which the durability factor is M = number of days at which the exposure is to be needed. Acid attack terminated. test was terminated at 105 days. So, M is 105 in this case. The extent of deterioration at each corner of the struck face and the opposite face is measured in terms of the acid diagonals (in mm) for each of two cubes and the Acid Attack Factor (AAF) per face is calculated as follows. AAF = (Loss in mm on eight corners of each of 2 cubes) / 4 The percentage weight loss and strength loss at 30, 45, 60, 75, 90 and 105 days of immersion has been presented in Table 3. Acid Durability Factors (ADF) and Acid Attack (AAF) are presented in Table 5. In the present investigation M40 concrete mix Factors considered. The mix proportions M40 is designed using I.S.10262is 1982.

4. RESULTS From the given Table 1 the compressive strength of Mortar at 3 days, 7 days and 28 days for different cell concentrations were shown. Figure 3 Shows the Scanning Electron Micrography analysis, referring to Figure 3. it is noted that pores were partially filled up material growth with the addition of the bacteria. Reduction in pore due to such by growth materialwill obviously increase the material strength. The compressive strength of at 7 days, concrete 14 days, 28 days, 60 days, 90 days, 180 days, 270 days and 365 days were given in Table 2. Split tensile strength of bacterial concrete when compared with concrete were conventional given in Table 3. Less percentage of loss in weight and compressive of bacterial concrete when compared with conventional concrete in acid immersion strength were test given in Table 4. The Acid Durability Factor and Acid Attack Factor of concrete and withwithout bacteria were given in Table 5.
Table 1. Effect of the bacillus subtilis bacteria addition on mortar strength Cell concentration/ml of mixing water Nil (control) 104 105 106 107 Average compressive strength of mortar in MPa 3 days 25.55 27.91 29.97 27.40 26.20 %Increase 9.23 11.73 7.24 2.54 7 days 38.34 41.24 44.31 42.14 40.16 %Increase 7.56 13.47 9.91 4.75 28 days 51.81 58.02 61.79 57.21 54.66 %Increase 11.99 16.15 10.42 5.51

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Table 2. Effect of the Bacillus subtilis bacteria addition on standard grade concrete Age (No. of days) 7 14 28 60 90 180 270 365 Compressive strength Conventional Bacterial concrete, N/mm concrete, N/mm 2 35.57 39.48 44.73 51.26 51.19 60.17 55.39 63.35 56.97 66.27 58.37 67.62 59.17 68.84 60.87 70.07

Table 3. Effect of the bacillus subtilis bacteria addition on split tensile strength Age (No. of days) 28 days 60 days 90 days 180 days Conventional concrete 4.51 4.63 4.89 5.07 Bacterial concrete 5.13 5.41 5.50 5.76

Table 4. Weight loss and Strength loss of concrete in acid immersion test weight of cube in kgs and compressive strength is in MPa. Type and percentage of acid: 5% H SO 4 2
Weight and Compressive of strength cube Weight before Weight after % Weight loss Compressive strength before Compressive strength after in % loss compressive strengt h Weight before Weight after % Weight loss Compressive strength before Compressive strength after in % loss compressive strengt h 30 days 2.538 2.509 1.14 51.19 50.71 0.94 2.535 2.512 0.91 60.17 59.76 0.68 45 days 60 days 75 days 2.538 2.356 7.17 51.19 44.72 12.63 2.535 2.380 6.11 60.17 55.85 7.18 90 days 2.538 2.313 8.87 51.19 42.77 16.45 2.535 2.339 7.73 60.17 54.0 3 10.2 105 days 2.538 2.274 10.4 0 51.19 40.0 9 21.68 2.535 2.308 8.95 60.17 51.86 13.81

Conventional concrete 2.538 2.538 2.451 2.396 3.4 5.59 3 51.19 51.19 49.07 47.13 4.14 7.93

Bacterial concrete 2.535 2.535 2.464 2.417 2.8 4.66 60.17 60.17 58.73 57.21 2.39 4.92

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S. Sunil Pratap Reddy, M.V. Seshagiri Rao, P. Aparna and Ch. Sasikala

Table 5. Acid durability factor and acid attack factor Days of immersion 30 45 60 75 90 105 30 45 60 75 90 105 Relative strength S 99.06 95.86 92.07 87.77 83.55 78.32 99.32 97.61 95.08 92.82 89.80 86.19 N M ADF Total loss in mm on 8 corners 6 17 25 34 41 49 5 10 15 22 29 35 AAF

Conventional concrete 30 45 60 75 90 105 30 45 60 75 90 105 105 105 105 105 105 105 105 105 105 105 105 105 28.30 41.08 52.61 62.69 71.61 78.32 28.38 41.83 54.33 66.30 76.97 86.19 0.19 0.53 0.78 1.06 1.28 1.53 0.16 0.31 0.47 0.69 0.91

Bacterial concrete

1.09

SEM. 1 Magnified view of SEM micrograph mortar without any of bacteria

SEM. 2 Magnified view of SEM micrograph of mortar with bacteria of 10 /ml cell concentration

SEM. 3 Magnified view of SEM micrograph mortar with bacteria of 105 /ml cell of concentration

SEM. 4 Magnified view of SEM micrograph of mortar with bacteria of 106 /ml cell concentration

bacteria (SEM analysis)

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PERFORMANCE OF STANDARD GRADE BACTERIAL...

7 SEM. 5 Magnified view of SEM micrograph of mortar with bacteria of 10 /ml cell concentration

Figure 3. Improvement in hydrated structure of cement-sand mortar using

a P M , h t g n e r t S e v i s s e r p m o C

80 70 60 50 40 30 20 10 0 7 14 28 60 90 180 270 365 Age ( No. of Days )

Conventional Concrete Bacterial concrete

Figure 4. Variation of compressive strength with age

a P M , h t g n e r t s e l i s n e t t i l p S

7 6 5 4 3 2 1 0 28 60 90 180 Age (No. of days)

Bacterial Concrete Conventional Concrete

Figure 5. Variation of split tensile strength with age

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S. Sunil Pratap Reddy, M.V. Seshagiri Rao, P. Aparna and Ch. Sasikala

s s o l t h g i e w e g a t n e c r e P

1 2 1 0 8 6 4 2 0 3 0 4 5 6 7 0 5 Age (No. of days) 9 0 1 5 0

Figure 6. Variation of percentage weight loss with age

Conventional Concrete Bacterial Concrete

e v i s s e r p m o C n i s s o l e g a t n e c r e P

25 2 0 h t g e r t s 15 10 5 0 3 0 45 6 75 Age0( No. of days ) 9 0 105

Figure 7. Variation of percentage loss in compressive strength with age

Bacterial Concrete Conventional Concrete

1.8 1.6 r o t c a F k c a t t A d i c A 1.4 1.2 1 0. 8 0. 6 0. 4 0. 20 3 0 45 6 75 0 Age ( No. of days ) 9 0 105

Bacterial Concrete Conventional Concrete

Figure 8. Variation of acid attack factor with age

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5. DISCUSSION ON TEST RESUTLS 5.1 Compressive Strength of Cement Mortar Referring to Table-1 the compressive strength at 3 days, 7 days and 28 days for different cell concentrations were shown. It is observed that the compressive strength of cement 5 showed mortar significant increase by 16.15% for cell concentration of 10 cells per ml of 5 mixingSo, for the further investigation bacteria with a cell concentration of 10 cells per water. of ml mixing water was used. Figure 3. shows the Scanning Electron Micrography analysis, it noted that pores were partially filled up by material growth with the addition of the bacteria. is Reduction in pore due to such material growth will obviously increase the material strength. 5.2 Compressive strength of concrete The compressive strength of concrete at 7 days, 14 days, 28 days, 60 days, 90 days, 180 days, 270 days and 365 days were given in Table 2. It is observed that with the addition of bacteria the compressive strength of concrete showed significant increase by 14.92% at 28 days. 5.3 Split tensile The Split strength Tensile Strength on standard cylindrical specimens at 28 days, 60 days, 90 and days180 days were given in Table-3. It is observed that with the addition of bacteria there a issignificant increase in Split Tensile Strength. 5.4 Loss in weight and loss in compressive The Loss strength in Weight and Loss in Compressive Strength at different ages were given in Table 4. With the addition of bacteria it is observed that there is less percentage of loss in weight and compressive strength. 5.5 Acid durability factor and acid attack The values of Acid Durability Factor and Acid attack Factor were given in Table-5. factor the addition of bacteria it is observed that bacterial concrete is more durable in terms of With Acid Durability Factor than conventional concrete and bacterial concrete is less in terms attacked of Acid Attack Factor than conventional concrete. 6. CONCLUSIONS
Based on the present experimental investigation, the following conclusions are drawn 6.1. Bacillus subtilis can be produced from lab which is proved to be a safe and cost effective. 6.2. The addition of bacillus subtilis bacteria improves the hydrated structure of mortar cement 6.3 . The compressive strength of cement mortar is maximum with the addition of 5 . bacillus bacteria for a cell concentration of 10 cells per ml of mixing water. So, subtilis 5 bacteria with a cell concentration of 10 cells per ml of mixing water was used in the

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S. Sunil Pratap Reddy, M.V. Seshagiri Rao, P. Aparna and Ch. PERFORMANCE OF STANDARD GRADE BACTERIAL... Sasikala

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Remediation, investigation 70th Annual Technical Conference and Exhibition of the Society of . 6.4. The addition of bacillus subtilis bacteria increases the compressive strength Petroleum Engineers, Dallas, Texas, Oct 22-25, 1995. 10. concrete. InK, Williams A E, Warmintoncompressive strength is increased Bachmeier standard grade concrete the J and Bang, S.S. Urease activity of 14.92% at 28 days by addition of bacillus subtilis bacteria when compared to in upto Microbiologically-induced calcite precipitation Journal of Biotechnology, conventional concrete. The addition of bacillus subtilis bacteria showed 171-181. 93(2002) improvement 11. significant in the split tensile strength than the conventional induced sealant Day JL, Panchalan RK, Ramakrishnan V. Microbiologically 6.5. concrete. crack From the durability studies, the Proceedingsweight loss and percentage strength percentage of the 16 th Engineering for concrete with revealed that Bacterial concrete has less weight and strength loss 5% H 2 Seattle, WA, 2003. conference, remediationSO 4 Mechanics than the conventional 12. losses P, Mandal S, Chattopadhyay BD, and Pal S. Use of Microorganisms to Ghosh 6.6. concrete. the Strength of out in the investigation Proceedings of Durability studies carried Cement-Sand Mortar. through acid attack test with improve H2SO 5% 4revealed International Concrete and Construction, ICACC-2004, India. pp. 983that bacterial Conference on Advances in concrete is more durable in terms of Acid Factor than Durability conventional concrete and bacterial concrete is less attacked in terms 988 Acid Attack Factor than conventional EF, and Bang SS, SEM Investigation 13. of Santhosh KR, Ramakrishnan V, Duke . 6.7. concrete. above it can be concluded Cement Proceedingscan be easily cultured From the Calcite Precipitation in that bacillus subtilis of the nd Internationa of Microbial safely and used in improving the performance characteristics of Canada, 2000. Conference on Cement Microscopy, pp. 293-305, Montreal, 22 l concrete.

1. 2. 3 . 4. 5.

6. 7. 8 . 9.

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