Effect of Bacteria Inoculums on Compressive Strength

The use of bio-concrete is increasing in the present day context and researchers are working on strength and durability characteristics of concrete using bacteria species which have shown calcite precipitation. Three different species of bacteria namely P. Fluorescence, B. Pumilis and B. Subliis that have calcite precipitation properties have been investigated in this study. The investigations were carried first on cement mortar (CM) cubes using these three bacteria species suspension of 20%; 40% and 60% having colony forming units P. Fluorescence (108 CFU/ml), B. Pumilis (106 CFU/ml) and B. Subtilis (108 CFU/ml) respectively. The 40% suspension in all the three cases has shown increased compressive strength as compared to 20% and 60%. The compressive strength measured showed increase (CS) of 18%; 12% for P. Fluorescence; B. Subtilis and decrease of 35% with B. Pumilis respectively. B. Subtilis with optimized 40% suspension having CFU10x108/ml showed 4.32% ; 5.56%; and 3.81% increase in CS of CC cubes with 3 days; 7 days and 28 days respectively and 5.92% overall increase in CS of CC cubes as compared to the 3 days CS of control cube.


Introduction
The concrete is the modern material used globally to fulfill the needs of infrastructure development. The research on this material is as old as the invention of cement itself. The maintenance of concrete structures is of significance to have a long structural life [1]. Therefore, CS and durability studies are important in the shelf-life of concrete.
Micro-cracks are the main cause to the ingress of moisture and deterioration leading to structural failure [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. It has been reported in literature that mineralproducing bacteria could be used for sand consolidation as well as for the repair of limestone monuments [20,21,22]. The pores and crack filling were extensively studied for the maintenance; leakages and durability point of view [23,24]. The strain B. Subtilis was used by many researches in the formation of calcium silicate hydrated gel by means of adsorbing silicate using chemically modified B. Subtilis (CMBS) [25,8]. The Reports say 28% improvement in the CS of CMBS incorporated concrete compared to control concrete with optimum concentration of 10 6 cells/ml. The 7 day, 14 day and 28 days CS, was carried out using portland cement with mortar cubes and concrete cubes with cell concentration of E.Coli from 10¹ to 10 7 when compared with control (without E.Coli) did not show any change [8].
Similarly B. Subtilis showed no improvement in CS when used with concentration of 10 5 [3]. However, inoculation with B. Sphaericus result in increase of CS as well as improvement of split tensile test strength. It has been reported that CS has increased by 30.76%, 46.15%, and 32.21% respectively on Day 3, 7 and 28 respectively with B. Sphaericus inoculation, and during the same period split tensile test strength has been increased by 13.75%, 14.28% and 18.35% respectively [26]. The increase in 28 [27]. The increase in CS at 7 and 28 days were found to be 28.53% and 17.21%, using Spirulina and 33.99% and 24.67%, using B. Subtilis respectively, when compared to control concrete without bacteria [28,29]. The literature review shows that there is a further scope to work in the direction of obtaining durable concrete using bacteria inoculation by conducting pin pointing experiments using different bacteria species which are known to precipitate calcite.

Isolation and Classification of Strains
The isolation and maintenance of pure culture for the selected bacterial species B. Subtilis, P. Fluorescence, B. Pumilis by dilution pour plate method as per the standard procedures outlined [29,17,30] and were regularly subcultures at regular interval for the purity and maintained at the department of Biotechnology KUD Dharwad.

Bacterial Species
B. Subtilis, P. Fluorescence, B. Pumilis are used which were procured from Department of biotechnology and microbiology KUD, Dharwad. The bacterial species in suspension was brought to the laboratory with concentration as given in Table 1 & 2.

Cement Mortar (1:3)
Mortar cubes (70.6mm×70.6mm×70.6mm) were prepared as per standards specified by IS: 4031 part 6. The compressive strength of the mortar cubes at 3 days, 7 days and 28 days was determined. Mortar cubes with and without additions of bacteria were cast.

Concrete Cubes
The concrete mix cubes of M25 grade were cast in accordance with IS 10262-2012 of dimensions (150*150*150) mm respectively. The bacteria inoculums were 40% of the total water content i.e. 300 ml. Slump test is carried out as per the procedure outlined in IS 456-2000. Similarly a slump of 75mm for M30 grade concrete is recorded using B. Subtilis [7]. Curing was carried out in accordance with IS 10262:2012. CS is measured at 3, 7 & 28 days.   [18] have also shown the similar results of increasing CS of 25% in 28 days with B. Subtilis. The review also supports the use of B. Subtilis species [3,7,10,16,18,23,14,15]. The strength improvement is due to growth of filler material within the pores of the cement-sand matrix. There is gain in strength after cooling in all grades of controlled and bacterial concretes may be due to the absorption of moisture from the surrounding medium which leads to extra hydration. The moisture content has a significant bearing on the strength of concrete. The CS increase is 18.3% and 12.2% respectively for B. Sphaericus and Sporosarcina Pastuerii. The increased CS with B. Subtilis strains is also reported when used in combination in fiber concrete [10].

CS of Concrete Cubes with Bacillus Subtilis
Depending upon the results of CS of mortar cubes with selected bacterial speciesand analyzing, high % increase in CS is showed by B. Subtilis. Therefore, concrete cubes using B. Subtilis only are casted and subjected to CS test and the results are placed in Fig 4. The addition of B. Subtilis showed noticeable increase in the compressive strength of concrete B. Subtilis with optimized 40% suspension having CFU 10x10 8 /ml showed 4.32%; 5.56%; and 3.81% increase in CS of CC cubes with 3 days; 7 days and 28 days respectively and 5.92% overall increase in CS of CC cubes as compared to the 3 days CS of control cube. The increase in CS is in conformity with the results of [16,24]. However the studies in bio concrete are at the infant stage requiring more pin pointing experiments to solve the field problems of variability's like temperature, water cement ratio monitoring; compaction etc. The most challenging in bio concrete is to address issues of supplying the designated bacteria right in time & proportion during mixing of concrete and practicability of handling the bacteria.

Conclusion
The 40% suspension in all the three cases has shown increased compressive strength as compared to 20% and 60%. The compressive strength of CM 1:3 proportion showed an increase (CS) of 18%; 12% for P. Fluorescence; B. Subtilis and decrease of 35% with B. Pumilis respectively. B. Subtilis with optimized 40% suspension having CFU 10x10 8 /ml showed 4.32%; 5.56%; and 3.81% increase in CS of CC cubes with 3 days; 7 days and 28 days respectively, and 5.92% overall increase in CS of CC cubes as compared to the 3 days CS of control cube. Therefore it is concluded that B. Subtilis has shown its superiority in CS indicating it has produced more calcite to fill up the pores and give better bond between the ingredients of CC.