Evaluation of Electron Microscopic Sample Preparation Methods and Imaging Techniques for Characterization of Cell-Mineral Aggregates

146

140

Microbial nitrate-dependent, Fe(II) oxidation (NDFO) is a ubiquitous biogeochemical process in anoxic sediments. Since most microorganisms that can oxidize Fe(II) with nitrate require an additional organic substrate for growth or sustained Fe(II) oxidation, the energetic benefits of NDFO are unclear. The process may also be self-limiting in batch cultures due to formation of Fe-oxide cell encrustations. We hypothesized that NDFO provides energetic benefits via a mixotrophic physiology in environments where cells encounter very low substrate concentrations, thereby minimizing cell encrustations. Acidovorax sp. strain 2AN was incubated in anoxic batch reactors in a defined medium containing 5 to 6 mM NO 3 ؊ , 8 to 9 mM Fe 2؉ , and 1.5 mM acetate. Almost Microbially driven, Fe(III)/Fe(II) redox transitions in the environment have a dramatic impact on iron's solubility, mineralogy, sorption characteristics, and overall geochemical properties (21,29,45). Microbially mediated redox transformations of Fe can also affect the biogeochemical cycling of other key nutrients (e.g., C, S, P, and N) (15, 29), trace metals (9, 49), metalloids (12), and the fate of organic pollutants (25) and contaminant metals (8), including those released from industrial and mining areas (30). Anaerobic, microbial Fe(III) reduction has been studied extensively over the last 30 years (31, 45), and much is known about the microbiology and geochemistry of that process.Chemotrophic, anoxic Fe(II) oxidation under circumneutral conditions, on the other hand, was first described only 15 years ago (42), and little is known about the microbiology of the process or its actual significance in the environment. Microbial nitrate-dependent, Fe(II) oxidation (NDFO) has been demonstrated in a variety of sediments, microbial consortia, and pure cultures, including both autotrophic and heterotrophic cultures. With respect to autotrophic growth via NDFO, for example, the mesophilic enrichment culture originally described by Straub et al. (42) has been maintained for over a decade under autotrophic conditions and is capable of near complete oxidation of Fe(II) and reduction of NO 3

Section: Methodsmentioning

confidence: 99%

Enhanced Growth of Acidovorax sp. Strain 2AN during Nitrate-Dependent Fe(II) Oxidation in Batch and Continuous-Flow Systems

Chakraborty

Roden

Schieber

et al. 2011

146

140

“…Twenty-eight-day-old samples were chemically fixed using a half-strength Karnovsky solution (20); placed on holey, carboncoated, electron microscopy copper grids; dehydrated in subsequent steps with an increasing concentration of isopropanol; and finally dried in a Bal-Tec CPD030 critical point dryer. A detailed description of the sample preparation steps is given by Schaedler et al (24). Dried samples were mounted on aluminum stubs using double-sided carbon tape.…”

Section: Methodsmentioning

confidence: 99%

Extracellular Iron Biomineralization by Photoautotrophic Iron-Oxidizing Bacteria

Miot

Benzerara

Obst

et al. 2009

156

113

Iron oxidation at neutral pH by the phototrophic anaerobic iron-oxidizing bacterium Rhodobacter sp. strain SW2 leads to the formation of iron-rich minerals. These minerals consist mainly of nano-goethite (␣-FeOOH), which precipitates exclusively outside cells, mostly on polymer fibers emerging from the cells. Scanning transmission X-ray microscopy analyses performed at the C K-edge suggest that these fibers are composed of a mixture of lipids and polysaccharides or of lipopolysaccharides. The iron and the organic carbon contents of these fibers are linearly correlated at the 25-nm scale, which in addition to their texture suggests that these fibers act as a template for mineral precipitation, followed by limited crystal growth. Moreover, we evidence a gradient of the iron oxidation state along the mineralized fibers at the submicrometer scale. Fe minerals on these fibers contain a higher proportion of Fe(III) at cell contact, and the proportion of Fe(II) increases at a distance from the cells. All together, these results demonstrate the primordial role of organic polymers in iron biomineralization and provide first evidence for the existence of a redox gradient around these nonencrusting, Fe-oxidizing bacteria.

“…In contrast to scanning electron microscopy (SEM) or transmission electron microscopy (TEM), where even the most careful specimen preparation cannot prevent artifacts (74,75), CLSM and scanning transmission X-ray microscopy (STXM) are powerful tools for in situ quantitative or semiquantitative analysis of environmental samples such as biofilms in their hydrated state (24,76,77). These techniques yield reliable information on biofilm structure (78,79), chemical composition (24,80), and heavy metal adsorption (24,28).…”

Section: Applicationsmentioning

confidence: 99%

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

Hao

Kappler

et al. 2013

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...