Suitability of lectin binding studies for the characterization of redox-active microbial environmental biofilms.

Aquatic environmental microbial biofilms grow in a broad range of redox environments from oxic to methanogenic, and they often also establish internal redox gradients. In technical applications, biofilms are also subjected to controlled redox conditions. Studies on biofilms often make use of fluorescence microscopic imaging techniques together with lectin binding analysis to gain insights into structure, composition, and functions of the biofilms. Here we studied the direct influence of redox potentials on fluorescence lectin binding analyses (FLBA) for two commonly used lectin-fluorophore conjugates. An effect of the electrical potential on signal intensity was observed and found to be statistically significant. The signal intensity changes however, remained within the range of a few percent total. A significant drop in intensity was only observed for extremely oxidizing potentials, typically not found under environmental conditions. Our results showed that the fluorophore itself and not the lectin binding to the respective glycoconjugate causes fluorescence changes. The two tested lectin-fluorophores are shown to be suitable for studying the distribution and composition of EPS in environmental biofilms or technical applications and under varying redox conditions.

Submicron-Scale Heterogeneities in Nickel Sorption of Various Cell-Mineral Aggregates Formed by Fe(II)-Oxidizing Bacteria.

Fe(II)-oxidizing bacteria form biogenic cell-mineral aggregates (CMAs) composed of microbial cells, extracellular organic compounds, and ferric iron minerals. CMAs are capable of immobilizing large quantities of heavy metals, such as nickel, via sorption processes. CMAs play an important role for the fate of heavy metals in the environment, particularly in systems characterized by elevated concentrations of dissolved metals, such as mine drainage or contaminated sediments. We applied scanning transmission (soft) X-ray microscopy (STXM) spectrotomography for detailed 3D chemical mapping of nickel sorbed to CMAs on the submicron scale. We analyzed different CMAs produced by phototrophic or nitrate-reducing microbial Fe(II) oxidation and, in addition, a twisted stalk structure obtained from an environmental biofilm. Nickel showed a heterogeneous distribution and was found to be preferentially sorbed to biogenically precipitated iron minerals such as Fe(III)-(oxyhydr)oxides and, to a minor extent, associated with organic compounds. Some distinct nickel accumulations were identified on the surfaces of CMAs. Additional information obtained from scatter plots and angular distance maps, showing variations in the nickel-iron and nickel-organic carbon ratios, also revealed a general correlation between nickel and iron. Although a high correlation between nickel and iron was observed in 2D maps, 3D maps revealed this to be partly due to projection artifacts. In summary, by combining different approaches for data analysis, we unambiguously showed the heterogeneous sorption behavior of nickel to CMAs.

Synchrotron-based chemical nano-tomography of microbial cell-mineral aggregates in their natural, hydrated state.

Chemical nano-tomography of microbial cells in their natural, hydrated state provides direct evidence of metabolic and chemical processes. Cells of the nitrate-reducing Acidovorax sp. strain BoFeN1 were cultured in the presence of ferrous iron. Bacterial reduction of nitrate causes precipitation of Fe(III)-(oxyhydr)oxides in the periplasm and in direct vicinity of the cells. Nanoliter aliquots of cell-suspension were injected into custom-designed sample holders wherein polyimide membranes collapse around the cells by capillary forces. The immobilized, hydrated cells were analyzed by synchrotron-based scanning transmission X-ray microscopy in combination with angle-scan tomography. This approach provides three-dimensional (3D) maps of the chemical species in the sample by employing their intrinsic near-edge X-ray absorption properties. The cells were scanned through the focus of a monochromatic soft X-ray beam at different, chemically specific X-ray energies to acquire projection images of their corresponding X-ray absorbance. Based on these images, chemical composition maps were then calculated. Acquiring projections at different tilt angles allowed for 3D reconstruction of the chemical composition. Our approach allows for 3D chemical mapping of hydrated samples and thus provides direct evidence for the localization of metabolic and chemical processes in situ.