J. Microbiol. Biotechnol. (2010), 20(11), 1571–1576 doi: 10.4014/jmb.1006.06032 First published online 14 September 2010 Characterization of Two Urease-Producing and Calcifying Bacillus spp. Isolated from Cement Achal, Varenyam 1,2 * , Abhijit Mukherjee 1 , and M. Sudhakara Reddy 1 Department of Biotechnology, Thapar University, Patiala 147 004, India Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology and Geography, Urumqi 830 011, China Received: June 23, 2010 / Revised: August 10, 2010 / Accepted: August 11, 2010 Two bacterial strains designated as CT2 and CT5 were isolated from highly alkaline cement samples using the enrichment culture technique. On the basis of various physiological tests and 16S rRNA sequence analysis, the bacteria were identified as Bacillus species. The urease production was 575.87 U/ml and 670.71 U/ml for CT2 and CT5, respectively. Calcite constituted 27.6% and 31% of the total weight of sand samples plugged by CT2 and CT5, respectively. Scanning electron micrography analysis revealed the direct involvement of these isolates in calcite precipitation. This is the first report of the isolation and identification of Bacillus species from cement. Based on the ability of these bacteria to tolerate the extreme environment of cement, they have potential to be used in remediating the cracks and fissures in various building or concrete structures. Keywords: Bacillus, cement, 16S rRNA, urease, calcite precipitation, crack remediation Microorganisms are active in a wide range of mineralization processes and have been involved in the deposition of minerals throughout the history of the Earth [14]. Bacteria from soils, freshwater, and saline habitats have frequently been reported to be able to precipitate calcium carbonate both in natural and in laboratory conditions. This capability has been related to the formation of marine calcareous skeletons, carbonate sediments, and soil carbonate deposits [26, 29]. According to Boquet et al. [11], calcium carbonate precipitation is a general phenomenon in the bacterial world, and under suitable conditions, most bacteria are able to precipitate calcite crystals. Microbiologically induced calcium carbonate precipitation (MICCP) is composed of a series of complex biochemical reactions, including concomitant participations of microbial urease (urea amidohydrolase; E.C. 3.5.1.5), and high pH. The microbial urease hydrolyzes urea to produce ammonia and carbon dioxide, and the ammonia released into the surroundings subsequently increases the pH, leading to accumulation of insoluble calcium carbonate [33]. The biomediated production of calcite crystals by calcifying bacteria has great applicable value for the restoration of deteriorated calcareous monuments and remediation of cracks and fissures in structures. Species of the Bacillus group are able to precipitate calcite on their cell constituents and in their microenvironment by conversion of urea into ammonia and carbon dioxide. Microbial mineral precipitation technologies have already been used for consolidation of sand columns [3, 33], for repair of limestones [35], and to a small extent for remediation of cracks in concrete [8, 10, 28]. Despite the importance of these bacteria in remediation of cracks and fissures, very few bacteria have been exploited. Microorganisms inhabit all possible locations including extremes, and exhibit growth and reproduction in such environments [31]. There is a need to explore extreme alkaline environments to isolate indigenous bacteria that can survive in concrete structures for effective biocalcification. In this investigation, two bacterial strains have been isolated from cement samples and their abilities to produce urease and calcite tested. Furthermore, these strains have been identified based on physiological and molecular characteristics. MATERIALS AND METHODS Sample Collection Cement samples were collected from the commercial bags and placed in radiation-sterilized polypropylene bottles (HiMedia, Mumbai). The physicochemical parameters of the cement, such as pH, specific gravity, silica content, and alumina content, were analyzed using standard methods (Table 1). *Corresponding author Phone: +86-15199137383; Fax: +86-991-7885300; E-mail: varenyam@gmail.com