Isolation and Identification of Bacillus Strains for Bioconcrete

Crack formation is a very common phenomenon in concrete structures which allows the water and chemicals through the cracks and decreases the durability and strength. For repairing the cracks developed in the concrete, it requires maintenance and special type of treatment. So, to overcome this problem an autonomous self healing mechanism is introduced into the concrete which helps to repair the cracks with the help of Bacillus bacteria by producing calcium carbonate crystals which seals the micro cracks and pores in the concrete. For the isolation of bacteria nearly eighty soil samples were collected from soils of extreme environments and cultured in three different media. A total of two thousand seventy colonies were observed and fifty five Bacillus colonies were isolated and identified by Gram staining and different biochemical tests.


Introduction
Concrete is the most commonly used construction material, but most of the structures are prone to cracking with time and different reasons such as material limitations, design gaps, construction practices as well as exposure conditions to the environment. Cracking is a common phenomenon in concrete due to the relatively low tensile strength [1]. Cracking in the surface layer of concrete mainly reduces its durability and can lead to damage of the mineral matrix and corrosion of steel. Because of these disadvantages an alternative technique for the improvement of the durability of concrete which biologically produces calcium carbonate crystals to seal the cracks on the surface of the concrete by using different types bacterial strains was introduced. Though concrete is quite strong mechanically, it suffers from several drawbacks, such as low tensile strength, permeability to liquid and consequent corrosion of reinforcement, susceptibility to chemical attack and low durability [2]. Self healing concrete biologically produces calcium carbonate crystals to seal the cracks which appear on the surface of the concrete [3]. In this process selected types of genus Bacillus along with calcium based nutrient in the presence of oxygen and the soluble calcium is converted to insoluble calcium carbonate by ureolytic activity [4]. When cracks appears on the concrete structure water starts to seep through spores of the bacteria which enable to start microbial activities after contact with the water and oxygen. The soluble nutrients are converted to insoluble calcium carbonate which solidifies on the cracked surface thereby sealing it up [5]. It impersonate the process of bone healing of fractures in the human body are naturally restored by osteoblast cells that mineralize to reform the bones [6][7][8][9] and bacterial based self healing agent is believed to remain hibernated within the concrete up to 200 years [10], [11].This paper mainly reports the investigation of microorganisms isolated from extreme environments and are known to survive in alkaline environments, some are able to produce urease enzyme which involve in microbial induced calcite precipitation (MICP) which is also called as bio-mineralization. MICP is a natural phenomenon associated with a wide range of bacterial species in an alkaline environment rich in Ca+ [12]. The addition of urea to the microorganism allows the conversion of urea to dissolved inorganic carbon and ammonium, subsequently releasing the ammonium to the environment [13]. This biomineralogy concept leads to the potential invention of a new material i.e, Bacterial concrete, an inherent and self repairing biomaterial that can remediate the cracks and fissures in concrete [3]. The mechanism of microbial calcium carbonate precipitation occurs worldwide in natural systems such as, oceans, microbial mats, biofilms and stromatolites [14][15][16][17],especially in oceans [18], [3].

Material and methods
In order to identify right bacteria for the development of bioconcrete it is necessary to isolate urease producing bacteria for the production of calcium carbonate which was used for the development of bioconcrete.

Isolation of bacterial colonies from soil samples
Soil samples were collected from brick kilns, furnaces, rocks of different areas of Warangal district i.e, in Hanamkonda, Bheemaram village Bairanpally, Gopalpur, Yerragollahpahad, Marigadi, Chowdaram and in Khammam Dist (Maddhulapally village) and marine samples from Vishakapatnam are cultured in nutrient agar media and Zobell medium by pour plate method and incubated for 24 hrs at 37 0 C. The isolated bacterial cultures were separated and sub cultured several times to obtain pure cultures.

Morphological, biochemical studies of bacterial isolates
Bacterial colonies obtained by pure culture technique are subjected for identification tests such as Gram staining, Endospore staining and biochemical tests such as catalase, oxidase, starch hydrolysis, eosin methylene blue, macconkey media, indole production, urease test etc for the identification of Bacillus species. To characterize all the bacterial colonies according to conventional, physiological and biochemical characterization tests were carried out as described in Bergey's Manual of Systemic Bacteriology [19].

Results & discussion 3.1 Isolation of Bacterial colonies from soil samples
Eighty soil samples were collected and cultured in different media for the growth of bacteria, approximately two thousand seventy bacterial colonies were observed and fifty five bacillus colonies were isolated and were used for screening of potent strains.

Screening and biochemical studies of bacterial isolates
Fifty five bacterial strains obtained by pure culture technique are subjected for identification tests such as Gram staining, Endospore staining and biochemical tests such as catalase, oxidase, motility test, starch hydrolysis, simmon's citrate agar, macconkey media, gas production, indole production, Urease test etc for the identification of Bacillus species. The biochemical test results for the fifty five bacterial strains obtained were reported in the following table:  Samples which showed better results in calcium carbonate precipitation media were further used for quantitative analysis and after that selected bacterial samples were studied for morphological, molecular identification upto the species level. Among the 13 isolates, four samples were selected based on their better precipitation in calcium carbonate precipitating media and were identified as Bacillus species by biochemical tests. Molecular identification of these four isolates were performed by amplification and sequencing the 16S rRNA gene and confirmed as Bacillus species which was done by National Chemical Laboratory (Pune), the sequences thus obtained was submitted in National Centre for Biotechnology Information (NCBI GenBank Accession Numbers -MN809595, MN849173, MN849426 and MN849881). The selected four isolates CJ-9 , CJ-21 , CJ-26, CJ-28 were found to be more efficient and belongs to Bacillus, based on nucleotide homology and phylogenetic analysis the strain CJ-9 is identified as (Bacillus thuringenisis), CJ-21 is (Bacillus albus), CJ-26 is (Bacillus mycoides) and CJ-28 is (Bacillus anthracis). The isolated bacteria which were obtained are screened quantitatively by gram staining, endospore staining and for the urease test. Urease produced by bacteria is widely known to precipitate calcium carbonate, one of the main components of concrete, which is referred as microbial concrete enzyme. To remediate building materials urease needs to be active and stable in alkaline environment (pH 9-11) that also include high temperature [6]. The screening for urease producing bacteria was conducted by using urease agar medium which changes the colour from pale yellow to pink indicates positive urease activity. Several studies have reported that urease agar base can be used as a quick method for primary screening of urease producing bacteria which is suitable for biocementation purposes [20][21][22]. Urease agar base contains urea and phenol red which acts as a pH indicator. When urea is hydrolysed by the bacteria, ammonia is released and becomes accumulated in the medium which increases the pH of the environment making it alkaline [23].All the isolated bacteria of the present study were identified as Bacillus and most of the calcifying bacteria belong to the Bacillus genera. The genus Bacillus has been mostly used for the biological development of calcium carbonate based minerals as, which is considered as a ureolytic bacteria. The formation of calcium carbonate by using this type of bacteria is because of the hydrolysis of urea to carbondioxide and ammonia which increases the pH of the medium at the cell surface and promotes formation of calcium carbonate crystals [24], [25], [6]. Similar results were reported by Hammed et al, [23] for the formation of calcium carbonate by sporosarcina pasterii. Numerous studies have mainly adopted the use of Sporosarcina pasteurii as their preferred ureolytic bacteria for MICP process because it is non-pathogenic and has quick capability to produce urease [26][27][28][29][30][31][32]. All the bacterial isolates selected for the present study were capable of forming endospores. Endospores are special resistant dormant, tough, and non-reproductive structures produced by some bacteria within the cell in the phylum Firmicutes [33], [34]. Endospores are extremely resistant to heat, chemicals, radiation, desiccation, enzymatic destruction and are capable of surviving in hostile environmental conditions and they can germinate into a vegetative cell within 90 min [35]. By the formation of endospores, bacteria can withstand large mechanical and chemically induced stresses during concrete mixing [36]. Ercole et al, [37] studied the bacteria for calcification and used Bacillus species for the development of bioconcrete.

Conclusion
From the results it was evident that from the isolation and identification studies, the selected four isolates CJ-9, CJ-21, CJ-26, CJ-28 in the present study were found to be more efficient and belongs to Bacillus, based on nucleotide homology and phylogenetic analysis the strain CJ-9 is identified as (Bacillus thuringenisis), CJ-21 is (Bacillus albus), CJ-26 is (Bacillus mycoides) and CJ-28 is (Bacillus anthracis) and they were investigated and used for further studies in concrete application.