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Applied and Environmental Microbiology, August 2003, p. 4901-4909, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4901-4909.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Strain-Specific Ureolytic Microbial Calcium Carbonate Precipitation

Frederik Hammes,^1, Nico Boon,¹ Johan de Villiers,² Willy Verstraete,^1* and Steven Douglas Siciliano^1,

Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, B-9000 Ghent, Belgium,¹ Department of Earth Sciences, University of Pretoria, 0001 Pretoria, South Africa²

Received 5 December 2002/ Accepted 2 May 2003

During a study of ureolytic microbial calcium carbonate (CaCO₃) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO₃ crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K_m) and maximum hydrolysis rates (V_max) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.

Present address: Department of Microbiology and Molecular Ecotoxicology, Swiss Federal Institute for Environmental Science and Technology, CH-8600 Duebendorf, Switzerland.

Present address: Department of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada.

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