Microbiological precipitation of CaCO3

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Abstract

The process of microbial mineral plugging in porous media is common in nature. We examined physical and biochemical properties of CaCO3 precipitation induced by Bacillus pasteurii, an alkalophilic soil microorganism. X-ray diffraction analysis quantified the composition of the mineral deposited in sand and identified the CaCO3 crystal as calcite. Examination by scanning electron microscopy identified bacteria in the middle of calcite crystals, which acted as nucleation sites. The rate of microbiological CaCO3 precipitation correlated with cell growth and was significantly faster than that of chemical precipitation. Biochemical properties of urease (urea amidohydrolase, E.C. 3.5.1.5) from B. pasteurii that was indirectly involved in CaCO3 precipitation were examined to understand the kinetics of the microbiological process. Urease from B. pasteurii exhibited a relatively low affinity for urea at pH 7.0 with a Km of 41.6 mM and Vmax of 3.55 mM min−1 mg−1 protein and increased affinity at pH 7.7 with a Km of 26.2 mM and Vmax of 1.72 mM min−1 mg−1 protein. Results of kinetic studies indicate that urease activity and its affinity to urea are significantly high at the pH where calcite precipitation is favorable. Our findings further suggest a potential use of the microbial calcite precipitation process in remediation of the surface and subsurface of porous media.

Introduction

Microbial metabolic activities often contribute to selective cementation by producing relatively insoluble organic and inorganic compounds intra- or extracellularly. Some microorganisms produce glycocalyx on the cell wall which remains in the natural environment even after cell death ([18], [21]), while others accumulate inorganic compounds such as phosphorites, carbonates, silicates and iron and manganese oxides (Beveridge et al., 1983, [12], [16], [27], [26]). In a natural setting, precipitation processes continue at a slow rate over geological time, plugging cracks in highly permeable rock formations ([14], [15]). The importance of selective cementation has been widely recognized in Petroleum, Geological and Civil Engineering. It has been documented that cracks in rock formations, especially in oil reservoirs, could be remediated by microorganisms ([33], [11]).

In our previous studies ([13], [34]), Bacillus pasteurii induced a mineral plugging in surface fractures and fissures of granite. B. pasteurii uses urea as an energy source and produces ammonia which increases pH in the proximal environment, causing Ca2+ and CO32− to precipitate as CaCO3 (Kroll, 1990). The microbial process was found to be most effective in remediating fissures with an average width of 2.7 mm. Among the filling materials that were mixed with B. pasteurii for fissure remediation of granite, the silica (10%) and sand (90%) mixture produced the highest compressive strength and lowest permeability. Thus, the potential of this technique as a long-term remediation tool is highly feasible for cementation of various structural formations. Results of our studies further suggested that microbial plugging could be greatly enhanced using microorganisms with high urease enzyme activities indirectly involved in CaCO3 consolidation.

We present physical and biochemical evidence of microbial mineral cementation, in which microbial metabolic activities play an important role. Microbial sedimentation of CaCO3 on the surface and subsurface of sand columns was examined by scanning electron microscopy and X-ray diffraction quantitative analysis. The biochemical processes were investigated by comparing kinetics of microbiological and chemical CaCO3 precipitation. Furthermore, urease activity and its Michaelis–Menten kinetics were evaluated at different pH values, which is a key factor in calcite precipitation.

Section snippets

Microorganism and growth conditions

B. pasteurii ATCC 6453 was used throughout the study. Medium (Tris–YE) for stock and pilot cultures contained the following ingredients l−1 of glass distilled water: Tris–HCl, 130 mM (pH 9.0); (NH4)2SO4, 10 g; and yeast extract, 20 g; to which 1.5% agar was added to obtain a solid medium for the stock culture. Individual ingredients were autoclaved separately and mixed afterward to avoid precipitation. CaCO3 precipitation experiments were carried out in liquid medium (urea–CaCl2) containing the

XRD analysis of microbiologically-induced CaCO3 precipitation

Table 1 summarizes the results of XRD quantitative analyses of four different sand samples. The most abundant compound was clearly quartz, the main component of sand. CaCO3 crystals were identified as calcite, not aragonite which is more common in seawater or magnesium-rich aqueous solutions (Berner, 1975, [26]). Calcite constituted 30.2% of the total weight of the column plugged by bacteria, but none was detected in column samples without live cells. In columns 1, 2 and 3 which were supplied

Factors involved in microbiological CaCO3 precipitation

The solubility of CaCO3 is affected by ionic strength in the aqueous medium ([32], [31]). In calcite precipitation, the overall equilibrium reaction isCa2++CO32−⇄CaCO3At 25°C, the solubility of CaCO3 is estimated to be 3.8×10−9 mol l−1 of water at zero ionic strength, which increases to 6.3×10−7 mol l−1 at ionic strength equivalent to seawater. Carbonate ion is produced in water by the following equilibrium reactions:HCO3⇆CO32−+H+HCO3+OH⇄CO32−+H2OIn urea–CaCl2 medium, NH4+ and Cl react

Acknowledgements

This research was funded by a grant from the National Science Foundation. We express our sincere appreciation to Dr. M.R. Islam and Dr. V. Ramakrishnan who have provided helpful comments and suggestions for the research. We also acknowledge Dr. E.F. Duke and Dr. B.L. Davis of the Engineering and Mining Experiment Station at the South Dakota School of Mines and Technology for their technical assistance in SEM and XRD. Special thanks go to D. Johnston of the South Dakota Department of

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