Mapping of Heavy Metal Ion Sorption to Cell-Extracellular Polymeric Substance-Mineral Aggregates by Using Metal-Selective Fluorescent Probes and Confocal Laser Scanning Microscopy

Abstract

b Biofilms, organic matter, iron/aluminum oxides, and clay minerals bind toxic heavy metal ions and control their fate and bioavailability in the environment. The spatial relationship of metal ions to biomacromolecules such as extracellular polymeric substances (EPS) in biofilms with microbial cells and biogenic minerals is complex and occurs at the micro-and submicrometer scale. Here, we review the application of highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy (CLSM) that were originally developed for use in life sciences and propose their suitability as a powerful tool for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates (CEMAs). The benefit of using metal fluorescent dyes in combination with CLSM imaging over other techniques such as electron microscopy is that environmental samples can be analyzed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. In this minireview, we present data for a group of sensitive fluorescent probes highly specific for Fe 3؉ , Cu 2؉ , Zn 2؉ , and Hg 2؉ , illustrating the potential of their application in environmental science. We evaluate their application in combination with other fluorescent probes that label constituents of CEMAs such as DNA or polysaccharides and provide selection guidelines for potential combinations of fluorescent probes. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural, hydrated state will further our understanding of the behavior of metals in environmental systems since it allows for identifying bonding sites in complex, heterogeneous systems. Biofilms are the dominant form of microbial life on Earth (1), and the organic material that is present in biofilms significantly impacts the cycling and sequestration of toxic heavy metals in the environment (2, 3). The underlying sorption and complexation mechanisms are difficult to evaluate (4), since biofilms are highly dynamic and complex structures that consist of diverse biomacromolecules (5) and the in situ heavy metal distributions are readily influenced by common invasive analysis approaches such as sequential extractions for the determination of different metal fractions. Here, we introduce a promising approach for studying the distribution and sorption of heavy metals in biofilms and cell-extracellular polymeric substance (EPS)-mineral aggregates (CEMAs) under close-to-natural conditions using confocal laser scanning microscopy (CLSM) in combination with highly selective metal ion-sensitive fluorescence probes. This approach is frequently used for metal detection in cell biology but was rarely applied in environmental and geomicrobiology research due to a former lack of highly selective fluorescence probes and due to the complexity of environmental biofilms and CEMAs.Biofilms are mainly composed of water, microorganisms, and an EPS matrix (6) that consists of mostly polysacchari...

Potential Role of Nitrite for Abiotic Fe(II) Oxidation and Cell Encrustation during Nitrate Reduction by Denitrifying Bacteria

“…4E to H), seem to originate from a thin, electron-dense, and nanometer-size grained mineral layer at the outer rim of the EPS capsule. The organic EPS polymers likely block kinks and steps of the initially formed mineral nuclei and thus prevent the formation of larger crystals within the EPS layer (55). We indeed observed that the minerals within the capsule, visible as filamentous structures, were generally much smaller grained than those on the surface.…”

“…In the case of BoFeN1, the Fe(III) mineral precipitation starts in the periplasm, continues on the cell surface, and then terminates in the cytoplasm (13). In all cultures, mineral precipitation at the cell surface was probably initially avoided by a protective EPS layer surrounding the cells, which potentially complexed Fe(III) and/or inhibited crystal nucleation and crystal growth because of binding of Fe ions by the organics (55). When nitrite diffuses through this EPS layer, green rust and goethite minerals form on the outside of the EPS layer, catalyzing Fe(II) oxidation and Fe(III) mineral formation (31,46,69).…”

A Survival Strategy for Pseudomonas aeruginosa That Uses Exopolysaccharides To Sequester and Store Iron To Stimulate Psl-Dependent Biofilm Formation

“…In order to know whether iron can bind to Psl under physiological conditions, particularly in biofilms, we applied a highly selective and sensitive iron-specific fluorescent probe to label iron and to detect the localization of iron in the Psl matrix of P. aeruginosa biofilms (58). Biofilms after 1 day of growth under iron-replete (100 M) or iron-limiting (2 M) conditions were stained by Psl-staining lectin HHA-FITC (green fluorescence) and an ironspecific fluorescent probe (red fluorescence).…”

Fractionation of Fe isotopes during Fe(II) oxidation by a marine photoferrotroph is controlled by the formation of organic Fe-complexes and colloidal Fe fractions

“…Fe(III) minerals similar to ferrihydrite precipitate in isotopic equilibrium with Fe(II/III) interm . soluble based on binding with a fluorescent sensor that does not bind to Fe(III) in minerals (Hao et al, 2013;Wu et al, 2014). This phase was co-localized to EPS, organics excreted by bacteria into their surroundings, based on the spatial overlap of fluorescence from a fluorescent Fe(III)-sensor and a fluorescent dye that binds EPS in confocal laser scanning microscopy (CLSM) images.…”