Imaging Biologically Induced Mineralization
induced mineralization processes and aided our ability to understand and ultimately control
Glass capillary flow systems, previously
the overall process. used
in our
Figure 1: (a) Schematic of the 2-dimensional reactor with a network of 1 mm pores. Flow moved from left to right across the system. The bacteria were introduced through the injection port, and the effluent samples were extracted through the sample port for chemical and biological analyses. (b) Photograph of the reactor under observation by the Nikon SMZ 1500 stereo microscope during biomineralization experiment.
laboratory, were used for this purpose (Figure 4) [6]. Te 0.17 mm thick glass walls allow for non-invasive CLSM. Te Leica TCS-SP2 AOBS confocal microscope employing a Leica 63× water immersion lens (2 mm working distance) was used. Imaris image-analysis soſtware (Bitplane Scientific Soſtware, St. Paul, MN) was used to compile the z-stacks into 3-dimensional images. Aſter the system was run
for 2 days and visible minerals had formed, fluorescent stains were applied to the reactors
to differentiate the various components. In the first study, SYTO-11 and C2-dichlorotriazine (C2d) (Invitrogen, Carlsbad, CA) were used. C2d (red) is expected to bind to alcohols, polysaccharides, and amines (
invitrogen.com) and thus reveal the compounds most likely associated with the extracellular matrix; SYTO-11 (green) is a nucleic acid stain that is expected to bind to bacterial cells. Figure 5 shows several images of cells and extracellular
matrix surrounding what appear to be mineral precipitates (calcite). Te cells appear to be attached preferentially to the calcite surface over the glass surface. Figure 5a displays a stack of CLSM images at low magnification. Te image spans the whole width (1 mm) of the capillary flow cell and shows clusters of cells and matrix surrounding dark areas (filled with calcite mineral). Figure 5b shows a three-dimensional reconstruction of clusters of bacteria and extracellular polymeric substances, which, as seen in Figures 5c and 5d, surround areas without fluorescence suggesting that calcium carbonate precipitates
Figure 2: Selected stereomicroscopy images taken from above the reactor and focused on mineral formation on the interior surface of the glass cover slip. (a) Inlet region showing the greatest degree of biomineralization, (b) 2 cm into the reactor, (c) 5 cm into the reactor, and (d) outlet region (approx. 8 cm into the reactor). Note the size gradient from inlet to outlet. Images such as these provided the data for size, density, and solubility calculations as a function of reactor location and retention time.
solubility increased with distance into the porous media reactor because of the larger free surface energy of the smaller crystals. Confocal Laser Scanning Microscopy (CLSM). Te
large-scale processes observed by stereo microscopy and chemical analysis of the flow medium at the outlet of the reactor are ultimately dictated by processes that occur on a much smaller scale, demanding the ability to visualize microbe-mineral
interactions on a smaller scale. Confocal
laser scanning microscopy provided higher-resolution differentiation of the components involved in the biologically
2011 September •
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Figure 3: Estimated crystal solubility constant (KsO) as a function of the estimated crystal surface area based on image analysis. Calculations were performed using the methods outlined by Stumm et al. [5] and Mitchell and Ferris [2].
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