60 International Journal of Biotechnology for Wellness Industries, 2016, 5, 60-69 ISSN: 1927-3037/16 © 2016 Lifescience Global Production of Bioagent for Calcium-Based Biocement Viktor Stabnikov 1,2,* 1 School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore 2 Department of Microbiology and Biotechnology, National University of Food Technologies, 68 Volodymyrska Street, Kiev 01601, Ukraine Abstract Biocements and biogrouts are developing extensively as new materials alternative to cement and toxic chemical grouts. The most popular type of biocement is a mixture of urease-producing bacteria, urea and calcium salt. Thus, development of biotechnology to produce biomass of urease-active bacteria for large-scale biocementation is an important biotechnological task. Two strains of urease-producing bacteria, Yaniella sp. VS8 and Bacillus sp. VS1 that synthesized inducible and constitutive urease, respectively, were used in the present study. It was shown that low cost biomass of urease-active bacteria can be produced from the hydrolyzed excessive activated sludge of municipal wastewater treatment plant. The biomass of Yaniella sp. VS8 grown in this medium diminished the hydraulic conductivity of sand from 4.8×10 -4 m/s to 5·10 -8 m/s after several biotreatments with solution of 1.5 M urea and 0.75M СаCl2. Keywords Bacillus sp., Yaniella sp., Activated sludge, Biocement, Biogrout. INTRODUCTION New area of Biotechnology, Construction Biotechnology, is developing in two directions: 1) biotechnological production of construction materials, and 2) biotechnology of construction processes [1]. Biocements and biogrouts are developing extensively as new construction materials alternative to cement and chemical grouts. Most popular type of biocement and biogrout produce calcium carbonate minerals due to activity of urease-producing bacteria (UPB) in the presence of urea and calcium ions [1-9]. The most popular type of biocementation and biogrouting is based on microbially-induced calcium carbonate precipitation (MICCP). It is a sequence of the following steps: 1) adhesion of cells of urease-producing bacteria on the soil particle/rock surface; 2) creation a microgradient of carbonate/bicarbonate concentration and the pH in the site of cell attachment due to hydrolysis of urea by urease; and 3) formation of calcium carbonate crystals that are attached to the soil particle or rock surface [1, 8]. (NH 2 ) 2 CO + 2 H 2 O + CaCl 2 UPB ! " !! CaCO 3 # + 2 NH 4 Cl Biomass of urease-producing bacteria is one of the major components of the biocement. Therefore, the important task for the development of biocementation biotechnology is the method to obtain UPB biomass for large-scale applications using cheap raw materials. *Address correspondence to this author at the School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore and Department of Microbiology and Biotechnology, National University of Food Technologies, 68 Volodymyrska Street, Kiev 01601, Ukraine; Tel/Fax: +38-097-9460237; E-mail: vstabnikov@ntu.edu.sg, vstab@nuft.edu.ua The medium ATCC 1376 NH4-YE, which contains yeast extract, 20 g; (NH 4 ) 2 SO 4 , 10 g; 0.13 M Tris buffer (pH 9.0), 1L, is recommended for cultivation of urease- producing bacteria Sporosarcina pasteurii (former Bacillus pasteurii) and is most often used for laboratory studies of biocementation. This medium was used for cultivation of Sporosarcina pasteurii ATCC 11859 and Bacillus pasteurii ATCC 6453 [10-12]. Sometimes, Tris buffer is replaced with distilled water, but the pH of the medium is adjusted to 9.0 [9]. Very often ammonium sulfate is replaced with urea, 20 g/L, and addition of urea to medium for cultivation of UPB is usually recommended. Liquid medium is sterilized by autoclaving for 15 – 20 min at 121 o C, meanwhile solution of urea has to be sterilized by filtration through 0.2 µm Millipore filter to avoid thermal decay. The strain of Sporosarcina pasteurii DSMZ 33 was grown in Nutrient Broth supplemented with 2 % (w/v) urea [13]. Tryptic Soy Broth with urea, 20 g/L, was recommended for cultivation of Sporosarcina pasteurii DZM 33 by Collection of Microorganisms and Cell Cultures, Braunschweig, Germany [14, 15]. Yeast extract with urea was used for cultivation of Bacillus sphaericus LMG 225 57 [5, 16]. Nutrient Broth supplemented with 2% urea was used for cultivation of Sporosarcina pasteurii NCIMB 8841 [17]. Sometimes, medium contains both ammonium sulfate and urea [18, 19]. In some studies, extra source of carbon was added to medium to enhance the conditions for cultivation of UPB. For example, 100 mM sodium acetate was added to the medium containing yeast extract, urea, and ammonium sulfate for cultivation of enrichment culture of UPB [20], and calcium acetate, 26 g/L, was added to the medium containing Nutrient Broth, 3 g/L,