Imaging Biologically Induced Mineralization in Fully Hydrated Flow Systems
Logan Schultz,1 Betsey Pitts,1 Andrew C. Mitchell,1 2 Alfred B. Cunningham,1
and Robin Gerlach1 * 1 Center for Biofilm Engineering, P.O. Box 173980, Montana State University, Bozeman, MT 59717 2 Institute of Geography and Earth Sciences, Aberystwyth University, SY23 3DB, UK
*
robin_g@biofilm.montana.edu
Introduction A number of proposed technologies involve the controlled
implementation of biologically induced carbonate mineral precipitation in the geologic subsurface. Examples include the enhancement of
soil stability [1], immobilization of
groundwater contaminants such as strontium and uranium [2], and the enhancement of oil recovery and geologic carbon sequestration via controlled permeability reduction [3]. Te most significant challenge in these technologies remains to identify and better understand an industrially, environmentally, and economically viable carbonate precipitation route. One
of the most promising routes is ureolytic
biomineralization, because of the ample availability of urea and the controllable reaction rate. In this process, ureolytic bacteria hydrolyze urea, leading to an increase in pH. In the presence of calcium, this process favors the formation of solid calcium carbonate, as illustrated in the following equations:
CO(NH2)2 + H2O à NH2COOH + NH3 à 2 NH3 + CO2 (Urea hydrolysis)
CO2 + 2 OH– ßà CO32– + H2O (Carbonate ion formation)
CO32– + Ca2+ ßà CaCO3 (solid) (Precipitation is favored at high pH)
(1) 2 NH3 + 2 H2O ßà 2NH4+ + 2OH– (pH increase) (2) (3) (4)
Tis process relies on molecular-level chemical and biological processes that must be better understood for large-scale implementation. Researchers at the Center for Biofilm Engineering at
Montana State University (USA) and Aberystwyth University (UK) have conducted several biomineralization experiments in simulated porous media reactors. Microscopy has proven to be one of the most useful analytical tools in these studies, providing the ability to non-invasively visualize, differentiate, and quantify the various components, including the cells, cell matrix, and mineral precipitates. Because of the possibility of real-time observation and the lack of dehydration artifacts, microscopy has been tremendously useful for elucidating the temporal and spatial relationships of these components.
Methods and Materials Te soil bacterium Sporosarcina pasteurii was used to
induce ureolysis. For each study, the flow reactor was inoculated with a culture of S. pasteurii, and the cells were allowed to
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attach for 1.5 hours before flow was started. Te flow medium, consisting of nutrient broth, urea, and dissolved calcium, was pH-adjusted to 6.0 to avoid immediate mineral precipitation. A Nikon SMZ 1500 stereo microscope was used to provide
a view of the biomineralized reactor and to quantify the volume fraction of precipitates within the pore networks. A Leica TCS-SP2 AOBS confocal laser scanning microscope (CLSM) was used to evaluate the micro-scale interaction between the crystalline surfaces and the biofilm. Flow medium was collected at the outlet of the reactor and analyzed for Ca, NH4+, and pH. Tis article discusses some techniques and examples of imaging biomineralization in flow reactors with these tools.
Results Stereo Microscopy. To characterize the spatial and tem-
poral development of precipitates in a network of pores, the stereo microscope was placed over the reactor system (Figure 1). Troughout the 15-hour experiment, images of one region
near the inlet were taken at regular time intervals, allowing for a time-lapse video of the mineral growth to be constructed. Tis video provided insight into nucleation and precipitation kinetics. At the end of the experiment, when the reactor had been terminally plugged, images were taken along the length of the reactor. Tese images, displayed in Figure 2 below, highlight a gradient in mineral size from inlet to outlet. From images, such as those in Figure 2, quantitative data
about the process can be obtained. Using ImageJ soſtware [4], the precipitates in each field of view were counted and measured. Subsequent X-ray diffraction analysis of the precipitates showed that calcite was the predominant mineral. Assuming the precipitates to be the density of calcite (2.71 g/cm3) and spherical in shape, the total amount of calcium immobilized within the reactor was estimated to be 49.1 mg. Knowing the chemical composition of the inlet and outlet, in addition to the fluid flow rate, the total amount of calcium immobilized was estimated to be 82 mg based on an effluent mass balance. Because the image-analysis method did not account for the smallest precipitates (that is, less than approximately 0.02 mm diameter) or the entirety of the densely precipitated inlet region (see Figure 2a), this low estimate makes sense. Our analysis suggests,
though, that quantitative image-analysis
approaches can be effective for reactors in which image clarity is not affected by large numbers of precipitates. A solubility constant of each measured crystal was
estimated for the given molar surface area, and the crystal solubility was calculated for each region of the reactor, based on relationships outlined by Stumm et al. [5] and Mitchell and Ferris [2]. Te results shown in Figure 3 suggest that the crystal
doi:10.1017/S1551929511000848
www.microscopy-today.com • 2011 September
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