T uo ' c s x Y

= = = = = = = = =

Total tensile force at a section Deflection at service load Strain Strain at ultimate strength of plain concrete Strain in concrete Strain in steel Curvature Normalised strain Noramalised Stress

CHAPTER - I INTRODUCTION

1.0 Introduction Concrete is the most widely used construction material. Despite its versatility in construction, it is known to have several limitations. It is weak in tension, has limited ductility and little resistance to cracking. Based on the continuous research carried out around the globe, various modifications have been made from time to time to overcome

the deficiencies of cement concrete. The ongoing research in the field of concrete technology has lead to the development of special concrete considering the speed of construction, the strength of concrete, the durability of concrete and the environmental friendliness with industrial material like fly ash, blast furnace slag, silica fume, metakeolin etc. Recently, it is found that microbial mineral precipitation resulting from metabolic activities of favorable microorganisms in concrete improved the overall behavior of concrete. The process can occur inside or outside the microbial cell or even some distance away within the concrete. Often bacterial activities simply trigger a change in solution chemistry that leads to over saturation and mineral precipitation. Use of these Bio mineralogy concepts in concrete leads to potential invention of new material called Bacterial Concrete.

1.1 History of Microbiology Bacteria are microscopic organisms, single-celled prokaryotic creatures. Bacteria come in different shapes and the sizes. Bacteria are ubiquitous in every habitat on Earth, growing in soil, acidic hot springs, radioactive waste, water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and animals. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water; in all, there are approximately five nonillion (51030) bacteria on Earth (Whitman et al. 1998, Vol.95) forming much of the world's biomass.

Bacteria were first observed by Antoine van Leeuwenhoek in 1676, using a single-lens microscope of his own design. He called them "animalcules" and published his observations in a series of letters to the Royal Society. The name bacterium was introduced much later, by Christian Gottfried Ehrenberg in 1838. Louis Pasteur demonstrated in 1859 that the fermentation process is caused by the growth of microorganisms. Along with his contemporary, Robert Koch, Pasteur was an early advocate of the germ theory of disease. There are broadly speaking two different types of cell wall in bacteria, called Gram-positive and Gram-negative. The names

originate from the reaction of cells to the Gram stain, a test longemployed for the classification of bacterial species. In the laboratory, bacteria are usually grown using solid or liquid media. Solid growth media such as agar plates are used to isolate pure cultures of a bacterial strain. However, liquid growth media are used when measurement of growth or large volumes of cells are required. Growth in stirred liquid media occurs as an even cell suspension, making the cultures easy to divide and transfer, although isolating single bacteria from liquid media is difficult. The use of selective media (media with specific nutrients added or deficient or with antibiotics added) can help identify specific organisms. Bacterial growth follows three phases. When a population of bacteria first enters a high-nutrient environment that allows growth, the cells need to adapt to their new environment. The first phase of

growth is the lag phase, a period of slow growth when the cells are adapting to the high-nutrient environment and preparing for fast growth. The lag phase has high biosynthesis rates, as proteins necessary for rapid growth are produced. The second phase of growth is the logarithmic phase (log phase), also known as the exponential phase. The log phase is marked by rapid exponential growth. The rate at which cells grow during this phase is known as the growth rate, and the time taken for the cells to double is known as the generation time. During log phase, nutrients are metabolised at maximum speed until one of the nutrients is depleted and starts limiting growth. The final phase of growth is the stationary phase and is caused by depleted nutrients. The cells reduce their metabolic activity and consume non-essential cellular proteins.

1.2 Classification of Bacteria 1.2.1 Classification on the Basis of Shapes Bacteria are usually classified on the basis of their shapes. Broadly, they can be divided into Rod-shaped bacteria (Bacilli), Sphere-shaped bacteria (Cocci) and Spiral-shaped bacteria (Spirilla). 1.2.2 Classification on the Basis of Gram Strain This classification is based on the results of Gram Staining Method, in which an agent is used to bind to the cell wall of the bacteria, they are Gram-positive and Gram-negative. 1.2.3 Classification on the Basis of Oxygen Requirement

This classification is based on the requirement of oxygen for the survival of the bacterium. They are Aerobic (Use molecular oxygen as terminal electron acceptor) and Anaerobic (Do not use molecular oxygen as terminal electron acceptor).

1.3 Various bacteria used in the concrete are i) Bacillus pasteurii ii) Bacillue sphaericus iii) Escherichia coli iv) Bacillus subtilis ( used in the present study ) 1.4 Bacillus subtilis JC3 Researchers with different bacteria proposed different bacterial concretes. The various bacteria used in the concrete are Bacillus pasteurii, Bacillue sphaericus, E.coli etc. In the present study an attempt was made by using the bacteria Bacillus subtilis strain no. JC3. The main advantage of embedding bacteria in the concrete is that it can constantly precipitate calcite. This phenomenon is called microbiologically induced calcite precipitation (MICP). Calcium

carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological implications. Bacillus subtilis JC3 is a laboratory cultured soil bacterium and its effect on the strength and durability is studied here.

1.5

Polyphasic

characterization

of

strain

JC3

(Ms. P.Aparna, (Ph.D.) thesis submitted to JNTUH)

1.5.1 Colonial characteristics On nutrient agar plate, colonies of strain JC3 were round, convex, smooth and translucent. Size of the colony may reach up to 2-3 mm diameter after 24hrs of incubation in dark aerobic conditions at 37oC. The organism produced a bluish green pigment that diffused into the medium. 1.5.2 Morphology Individual cells of strain JC 3 were Gram positive, oval to rods, 0.6-0.8 m in width and 2.0 to 3.0 m in length, motile and multiplied by binary fission.

Fig 1.5.1 Colony morphology of strain JC3 on nutrient agar plate

Fig 1.5.2 Phase contrast microphotograph of strain JC3

1.5.3 Biochemical characteristics Various biochemical tests done on the organism are given in Table.1.5.1 Table 1.5.1 Biochemical characteristics of the pure culture Bacillus subtilis JC3 Bacillus subtilis JC3 Characteristics Shape, size, gram Long rods, 0.6-0.8 m in width and 2.0 stain to 3.0 m in length, gram positive Colony morphology Irregular, dry , white, opaque colonies (on 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

1.6 MAINTENANCE OF STOCK CULTURES Stock cultures of Bacillus subtilis JC3 are maintained on nutrient agar slants. The culture is streaked on agar slants with an inoculating loop and the slants are incubated at 370C. After 2-3 days of growth, slant cultures are preserved under refrigeration (40C) until further use. Sub culturing is carried out for every 90 days. Contamination

from other bacteria is checked periodically by streaking on nutrient agar plates.

1.7 BACTERIAL CONCRETE The concept of bacterial concrete was first introduced by V.Ramakrishnan et al. A novel technique is adopted in remediating cracks and fissures in concrete by utilizing microbiologically induced calcite (CaCO3) precipitation. Microbiologically induced calcite

precipitation (MICP) is a technique that comes under a broader category of science called biomineralization. Bacillus subtilis JC3, a common soil bacterium can induce the precipitation of calcite. As a microbial sealant, CaCO3 exhibited its positive potential in selectively consolidating simulated fractures and surface fissures in granites and in the consolidation of sand. Microbiologically induced calcite precipitation is highly desirable because the calcite precipitation induced as a result of microbial activities, is pollution free and natural. The technique can be used to improve the compressive strength and stiffness of cracked concrete specimens. The bacterial concrete makes use of calcite precipitation by bacteria. The phenomenon is called microbiologically induced calcite precipitation (MICP). The pioneering work on repairing concrete with MICP is reported by the research group of Ramakrishnan V and others at the South Dakota School of Mines & Technology, USA. The MICP is a technique that comes under a broader category of science called biomineralization. It is a process by which living

organisms or bacteria form inorganic solids. Bacillus subtilis JC3, a common soil bacterium, can induce the precipitation of calcite. Under favorable conditions Bacillus subtilis JC3, when used in concrete, can continuously precipitate a new highly impermeable calcite layer over the surface of the already existing concrete layer. The precipitated calcite has a coarse crystalline structure that readily adheres to the concrete surface in the form of scales. In addition to the ability to continuously grow upon itself, it is highly insoluble in water. It resists the penetration of harmful agents (chlorides, sulphates, carbon dioxide) into the concrete thereby decreasing the deleterious effects they cause. Due to its inherent ability to precipitate calcite continuously, bacterial concrete can be called a Smart Bio Material for repairing concrete. The MICP comprises a series of complex biochemical reactions. It is selective and its efficiency is affected by the porosity of the medium, the number of cells present and the total volume of nutrient added. The phosphate buffer or urea-CaCl2 has been found effective as nutrients. The bacteria precipitate calcite in the presence of nutrients. The optimum pH for growth of B. pasteurii is around 9. The alkaline environment of concrete with pH around 12 is the major hindering factor for the growth of bacteria. However, B. pasteurii has the ability to produce endospores to endure an extreme environment, as observed by V.Ramakrishnan and the research team. The microbial modified mortar or concrete has become an important area of research for high-performance construction

materials. Ghosh et al. investigated the effects of incorporating a

facultative anaerobic hot spring bacterium on the microstructure of a cementsand mortar. Environmental scanning electron microscopic (ESEM) views and image analysis (IA) of the bacteria modified mortar (thin-section) showed significant textural differences with respect to the control (without bacteria) samples.

Fig 1.7.1 Magnified image of Rod shaped impressions consistent with the dimensions of B. pasteurii, spread around the calcite crystals. (courtesy : ASM MicrobeLibrary.org)

CHAPTER II REVIEW OF LITERATURE

The Literature available reveals that bacteria can be used to enhance the performance of the concrete. The concept of bacterial concrete was first introduced by V.Ramakrishnan, a novel technique