Imaging Biologically Induced Mineralization
the fluorescent stains were unable to penetrate these regions. Tere appears to be a higher proportion of red (propidium iodide) stained “dead” cellular material associated with the calcite crystal surface and the bottom of the biofilm proximal to the glass surface in Figure 6b. Tis suggests that solute transport in these environments might be limited, possibly because of the development of regions with low solute permeability or biological uptake of nutrients by the biofilm above.
Discussion Tese studies have highlighted the usefulness of techniques
microscopic at two different scales to better
understand the process of biomineralization quantitatively and qualitatively. Stereomicroscopy was used to analyze the size and
distribution of calcite precipitates, the overall immobilization of dissolved calcium, and the solubility of precipitates as a function of position. Te limitations of resolution (that is, not accounting for the smallest crystals) in addition to the limited ability to quantify the most densely precipitated inlet region led to an underestimate of overall calcium immobilization compared to a solution chemistry-based estimate. However, the benefits of this in situ method to quantify mineral deposition and substrate conversion with respect to the location along the flow path are significant. Controlling the spatial distribution of calcium carbonate precipitates can provide an invaluable tool for the development of strategies to manipulate porous media permeability and reactive transport on large scales. For example, during soil stabilization [1], contaminant co-precipitation [2], and geologic carbon sequestration [7,8] it will be important to either promote or prevent biomineral clogging at a certain distance from the injection point. Te original confocal scanning laser microscopy work by
Pitts and Stewart [6] demonstrating the power of observing biofilms in-situ using CLSM was expanded in this study to include biomineral formation. Te findings of this paper show that mineral surfaces can provide a suitable environment for microbial attachment and that the effect of bacteria may extend beyond the alteration of bulk fluid chemistry (for example, ureolysis). Further, attached microbial communities may have significant local effects on the chemical, biological, and nucleation environment at the mineral surface.
Future Work Ongoing investigations are seeking to gain a better
understanding of the association of bacteria throughout the process of precipitate formation. For instance, it is unclear at this point whether cells attach prior to mineral nucleation or if calcium carbonate precipitates form prior to bacterial attachment on the mineral surface. Additional experimental work will also focus on understanding the distribution of cells in the interior of the mineral precipitates; the characterization of geochemical gradients within the systems, for example, using pH-indicator dyes; and the influence of temperature and pressure on biomineral formation.
Acknowledgments Tis work was supported by the U.S. Department of Energy
(DOE) Zero Emissions Research Technology (ZERT) program, award no. DE-FC26-04NT42262; the DOE Office of Science, Environmental Remediation Science Program (ERSP), contract no. DE-FG02-09ER64758; the U.S. Department of Energy EPSCoR program under grant no. DE-FG02-08ER46527; and
2011 September •
www.microscopy-today.com 15
the National Science Foundation Award No. DMS-0934696. Te authors also acknowledge funding for the establishment and operation of the Environmental and Biofilm Mass Spectrometry Facility at Montana State University through the Defense University Research Instrumentation Program (DURIP, Contract Number: W911NF0510255) and the MSU Termal Biology Institute from the NASA Exobiology Program (Project NAG5-8807). Imaging facilities were provided by the Center for Biofilm Engineering, Bozeman, MT, and the confocal microscope was funded through a grant from the M.J. Murdock Charitable Trust.
References [1] V Whiffin, L van Paassen, and M Harkes, Geomicrobiol J 24 (2007) 417–23.
[2] A Mitchell and G Ferris, Environ Sci Technol 40 (2006) 213–26.
[3] G Ferris, L Stehmeier, A Kantzas, and F Mourits, J Can Petrol Technol 13 (1996) 57–67.
[4] W Rasband, ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA.
http://rsb.info.nih.gov/ij/. 1997–2009.
[5] W Stumm and J Morgan, Aquatic Chemistry, Wiley, New York, 1996, p. 1022.
[6] B Pitts and P Stewart, Microscopy Today 16(4) (2008) 18–22.
[7] A Mitchell, K Dideriksen, L Spangler, A Cunningham, and R Gerlach, Environ Sci Technol 44 (2010) 5270–76.
[8] A Cunningham, R Gerlach, L Spangler, and A Mitchell, Energy Procedia 1 (2009) 3245–52.
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