Bacterially-Induced Calcite Precipitation via Ureolysis

  • Authors: Chiung-Wen Chou 1, Ahmet Aydilek 2, Eric Seagren 3, Timothy Maugel 4
    Affiliations: 1: Dept. of Civil and Environmental Engineering, University of Maryland, College Park, MD, 20740; 2: Department of Civil and Environmental Engineering, University of Maryland , College Park, MD, 20740; 3: Department of Civil and Environmental Engineering, University of Maryland, College Park, MD, 20740; 4: Laboratory for Biological Ultrastructure, University of Maryland , College Park, MD, 20740
  • Citation: Chiung-Wen Chou, Ahmet Aydilek, Eric Seagren, Timothy Maugel. 2008. Bacterially-induced calcite precipitation via ureolysis.
  • Publication Date : November 2008
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During bacterially-induced calcium carbonate precipitation via ureolysis, Sporosarcina pasteurii (formerly Bacillus pasteurii) uses urea as its nitrogen and energy sources (11) , releasing ammonium and dissolved inorganic carbon (carbonate and bicarbonate) as by-products and creating an alkaline environment.  This change in the ambient pH creates conditions under which calcium carbonate precipitation becomes favorable via homogeneous nucleation (14) .  In addition, if free calcium is available, it may be attracted to cells due to the negatively charged cell surface.  In the presence of carbonate, calcium carbonate precipitation on the cell surfaces results via heterogeneous nucleation.    

Presented in Fig. 1, as the baseline, are scanning electron micrographs of untreated sand: (A) with pretreatment before scanning electron microscope (SEM) analysis,  and (B) focused on the surface of the sand particle   .  Note the rough and angular surface of the untreated sand.    

Figure 2 shows scanning electron micrographs of biocemented sand:   (A) without pretreatment before SEM analysis, (B) focused on the area within the solid square boundary in Fig. 2A, and (C) focused on the area within the dotted square boundary shown in Fig. 2B.  Note the heavy cementation formed between the sand particles and on the surface of the sand (Fig. 2A and 2C), and the crystals with flaky morphologies between the sand particles (Fig. 2B).    

Figure 3 provides scanning electron micrographs of the surface of biotreated sand: (A) without pretreatment before SEM analysis,  (B) with pretreatment before SEM analysis, and (C) focused on the area within the solid square boundary in Fig. 3B. Note that with the SEM pretreatment procedure (Fig. 3B and 3C), the surface of the sand particle is revealed to be angular, similar to the untreated sand (Fig. 1).  In addition, rod-shaped microbes can clearly be seen in close association with the sand particles.  


Stock streak-plate cultures of S. pasteurii 11859 were grown on ATCC 1832 medium agar (12) .  To prepare the inoculum for the biocalcification experiments, S. pasteurii 11859 cells were grown to the late exponential phase on Tris-YE medium (15) , washed twice using Tris-HCl buffer (pH 9), and then resuspended in the buffer.    

To create a controlled environment that could induce uniform calcium carbonate formation within the pore spaces of a model porous medium, a hydraulically completely-mixed biofilm reactor (CMBR) packed with a poorly-graded silica sand was used.  The autoclaved CMBR was aseptically packed with sterilized sand.  The specimen was then saturated with Tris-HCl buffer, after which the column was inoculated using a 2 x 107 CFU/ml bacterial suspension and left to sit stagnant for 12 hours to allow the microorganisms to attach to the sand. After the stagnant period, a urea-CaCl2 medium (15) was fed into the column at an influent flow rate of 1.5 ± 0.1 ml/min, with a recycle flow rate of 20 ml/min.  Subsequently, key parameters (urea, ammonium, calcium, pH, flow rate, head loss, dissolved oxygen, and cell numbers) were monitored until the column was completely plugged, as indicated by high head loss across the column.  

An SEM was used to examine the formation of bio-induced precipitation and biofilms on the surface of the sand matrix.  Specimens preserved from different experimental conditions were first oven-dried at 40 ° C for 3 days.  Subsequently, some specimens went through a pretreatment procedure (e.g., Fig. 1A, 1B, 3B, 3C), followed by mounting and coating, while other specimens were directly mounted and coated for SEM analysis without pretreatment to avoid dissolution of the authigenic minerals (e.g., Fig. 2A–C).  The specimens subjected to pretreatment were initially washed with 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer.  Subsequently, the samples were chemically fixed in a 2% solution of glutaraldehyde prepared in 10 mM PIPES buffer, followed by rinsing in the same buffer to remove excess fixative.  Next, a second fixation reagent, a 1% solution of osmium tetroxide in 10 mM PIPES buffer, was added to the samples, followed by a rinsing with double distilled water.  The specimens were then dehydrated in sequential ethanol solutions and, to minimize surface tension, they were soaked sequentially in mixtures of 100% ethanol and hexamethyldisilazane (HMDS), followed by 100% HMDS.  Finally, the samples were covered with a minimal amount of 100% HMDS, placed in a desiccator, and allowed to dry for several days.  During the mounting procedure, numerous grains of the dry samples were mounted onto labeled specimen stubs using double sticky adhesive.  Finally the samples were coated with a 60:40 gold-palladium alloy using a high vacuum evaporator (Denton DV 503).  The coat thickness was 25 nm.  


Natural cementation of geological formations occurs constantly over time due to physiochemical and biological reactions.  Indeed, numerous examples of microbiologically-mediated mineral precipitation (biomineralization) in the natural soil environment have been documented (1, 3, 5–7) .  These microbially-mediated reactions result in relatively insoluble compounds that can contribute to soil cementation.  One of these natural processes is microbially-mediated calcium carbonate precipitation, such as occurs via ureolysis, as described above.  In addition to playing a role in the natural soil environment, the bacterially-induced calcite precipitation process illustrated here has also been investigated for biomediation of soil geotechnical properties (4, 8, 15) , repair of concrete or calcareous stone (2, 9, 12, 13) , or immobilization of environmental contaminants (10) .  


This material is based upon work supported by the National Science Foundation under grant no. CMS-05-28171.  Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.  


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