Julie Cosmidis

Magnetotactic bacteria form magnetite from a phosphate-rich ferric hydroxide via nanometric ferric (oxyhydr)oxide intermediates

The iron oxide mineral magnetite (Fe3O4) is produced by various organisms to exploit magnetic and mechanical properties. Magnetotactic bacteria have become one of the best model organisms for studying magnetite biomineralization, as their genomes are sequenced and tools are available for their genetic manipulation. However, the chemical route by which magnetite is formed intracellularly within the so-called magnetosomes has remained a matter of debate. Here we used X-ray absorption spectroscopy at cryogenic temperatures and transmission electron microscopic imaging techniques to chemically characterize and spatially resolve the mechanism of biomineralization in those microorganisms. We show that magnetite forms through phase transformation from a highly disordered phosphate-rich ferric hydroxide phase, consistent with prokaryotic ferritins, via transient nanometric ferric (oxyhydr)oxide intermediates within the magnetosome organelle. This pathway remarkably resembles recent results on synthetic magnetite formation and bears a high similarity to suggested mineralization mechanisms in higher organisms.

Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria.

Significance Cyanobacteria are known to promote the precipitation of Ca-carbonate minerals by the photosynthetic uptake of inorganic carbon. This process has resulted in the formation of carbonate deposits and a fossil record of importance for deciphering the evolution of cyanobacteria and their impact on the global carbon cycle. Though the mechanisms of cyanobacterial calcification remain poorly understood, this process is invariably thought of as extracellular and the indirect by-product of metabolic activity. Here, we show that contrary to common belief, several cyanobacterial species perform Ca-carbonate biomineralization intracellularly. We observed at least two phenotypes for intracellular biomineralization, one of which shows an original connection with cell division. These findings open new perspectives on the evolution of cyanobacterial calcification. Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria.

Characterization of Ca-phosphate biological materials by scanning transmission X-ray microscopy (STXM) at the Ca L 2,3 -, P L 2,3 - and C K-edges

Several naturally occurring biological materials, including bones and teeth, pathological calcifications, microbial mineral deposits formed in marine phosphogenesis areas, as well as bio-inspired cements used for bone and tooth repair are composed of Ca-phosphates. These materials are usually identified and characterized using bulk-scale analytical tools such as X-ray diffraction, Fourier transform infrared spectroscopy or nuclear magnetic resonance. However, there is a need for imaging techniques that provide information on the spatial distribution and chemical composition of the Ca-phosphate phases at the micrometer- and nanometer scales. Such analyses provide insightful indications on how the materials may have formed, e.g. through transient precursor phases that eventually remain spatially separated from the mature phase. Here, we present scanning transmission X-ray microscopy (STXM) analyses of Ca-phosphate reference compounds, showing the feasibility of fingerprinting Ca-phosphate-based materials. We calibrate methods to determine important parameters of Ca-phosphate phases, such as their Ca/P ratio and carbonate content at the ∼25nm scale, using X-ray absorption near-edge spectra at the C K-, Ca L2,3- and P L2,3-edges. As an illustrative case study, we also perform STXM analyses on hydroxyapatite precipitates formed in a dense fibrillar collagen matrix. This study paves the way for future research on Ca-phosphate biomineralization processes down to the scale of a few tens of nanometers.

Entrapped elemental selenium nanoparticles affect physicochemical properties of selenium fed activated sludge.

Selenite containing wastewaters can be treated in activated sludge systems, where the total selenium is removed from the wastewater by the formation of elemental selenium nanoparticles, which are trapped in the biomass. No studies have been carried out so far on the characterization of selenium fed activated sludge flocs, which is important for the development of this novel selenium removal process. This study showed that more than 94% of the trapped selenium in activated sludge flocs is in the form of elemental selenium, both as amorphous/monoclinic selenium nanospheres and trigonal selenium nanorods. The entrapment of the elemental selenium nanoparticles in the selenium fed activated sludge flocs leads to faster settling rates, higher hydrophilicity and poorer dewaterability compared to the control activated sludge (i.e., not fed with selenite). The selenium fed activated sludge showed a less negative surface charge density as compared to the control activated sludge. The presence of trapped elemental selenium nanoparticles further affected the spatial distribution of Al and Mg in the activated sludge flocs. This study demonstrated that the formation and subsequent trapping of elemental selenium nanoparticles in the activated sludge flocs affects their physicochemical properties.

Self-assembly of biomorphic carbon/sulfur microstructures in sulfidic environments

In natural and laboratory-based environments experiencing sustained counter fluxes of sulfide and oxidants, elemental sulfur (S0)—a key intermediate in the sulfur cycle—can commonly accumulate. S0 is frequently invoked as a biomineralization product generated by enzymatic oxidation of hydrogen sulfide and polysulfides. Here we show the formation of S0 encapsulated in nanometre to micrometre-scale tubular and spherical organic structures that self-assemble in sulfide gradient environments in the absence of any direct biological activity. The morphology and composition of these carbon/sulfur microstructures so closely resemble microbial cellular and extracellular structures that new caution must be applied to the interpretation of putative microbial biosignatures in the fossil record. These reactions between sulfide and organic matter have important implications for our understanding of S0 mineralization processes and sulfur interactions with organic carbon in the environment. They furthermore provide a new pathway for the synthesis of carbon-sulfur nanocomposites for energy storage technologies.

Calcium-Phosphate Biomineralization Induced by Alkaline Phosphatase Activity in Escherichia coli: Localization, Kinetics, and Potential Signatures in the Fossil Record

Bacteria are thought to play an important role in the formation of calcium-phosphate minerals composing marine phosphorites, as supported by the common occurrence of fossil microbes in these rocks. Phosphatase enzymes may play a key role in this process. Indeed, they may increase the supersaturation with respect to Ca-phosphates by releasing orthophosphate ions following hydrolysis of organic phosphorus. However, several questions remain unanswered about the cellular-level mechanisms involved in this model, and its potential signatures in the mineral products. We studied Ca-phosphate precipitation by different strains of Escherichia coli which were genetically modified to differ in the abundance and cellular localization of the alkaline phosphatase (PHO A) produced. The mineral precipitated by either E. coli or purified PHO A was invariably identified as a carbonate-free non-stoichiometric hydroxyapatite. However, the bacterial precipitates could be discriminated from the ones formed by purified PHO A at the nano-scale. PHO A localization was shown to influence the pattern of Ca-phosphate nucleation and growth. Finally, the rate of calcification was proved to be consistent with the PHO A enzyme kinetics. Overall, this study provides mechanistic keys to better understand phosphogenesis in the environment, and experimental references to better interpret the microbial fossil record in phosphorites.

The Iron Wheel in Lac Pavin: Interaction with Phosphorus Cycle

Lac Pavin is a crater lake, characterized by water column stratification, with oxygenated shallow waters lying above anoxic and ferruginous deep waters. In the deep waters, ferrous iron, Fe(II)aq, is the main dissolved cation, with concentrations up to 1 mM. Iron is efficiently confined below the oxic-anoxic boundary due to the formation of insoluble ferric iron species, Fe(III)s, by oxidation with O2 and other oxidants (e.g., NO3−, Mn(IV)). The Fe(III)s particles settle down and are reduced in the anoxic waters and at the lake bottom by reaction with organic matter to soluble Fe(II)aq. It then diffuses upward in the water column and finally is re-oxidized to Fe(III) at the redox boundary. This process, known as the “iron wheel”, is described in the present paper that reviews available data for dissolved and particulate matter in the water column, settling particles collected by sediment traps and sediment cores. Detailed analyses for some major and trace element concentrations, along with iron speciation and isotope composition, high-resolution microscopy, and geochemical modeling provide a picture of biogeochemical cycling in this Fe-rich aqueous system. At Lac Pavin the P and Fe cycles are tightly coupled. Orthophosphate is sorbed onto Fe oxyhydroxides and/or precipitated as Fe(II)-Fe(III)-phosphates at the redox interface, confining P ions in the deep anoxic waters. Deeper in the water column, particulate Fe concentrations progressively increase due to Fe(II) phosphate (vivianite) formation. In the sediment, Fe is buried as various ferrous minerals, such as vivianite, pyrite and siderite.

In Vitro and in Silico Evidence of Phosphatase Diversity in the Biomineralizing Bacterium Ramlibacter tataouinensis

Microbial phosphatase activity can trigger the precipitation of metal-phosphate minerals, a process called phosphatogenesis with global geochemical and environmental implications. An increasing diversity of phosphatases expressed by diverse microorganisms has been evidenced in various environments. However, it is challenging to link the functional properties of genomic repertoires of phosphatases with the phosphatogenesis capabilities of microorganisms. Here, we studied the betaproteobacterium Ramlibacter tataouinensis (Rta), known to biomineralize Ca-phosphates in the environment and the laboratory. We investigated the functional repertoire of this biomineralization process at the cell, genome and molecular level. Based on a mineralization assay, Rta is shown to hydrolyse the phosphoester bonds of a wide range of organic P molecules. Accordingly, its genome has an unusually high diversity of phosphatases: five genes belonging to two non-homologous families, phoD and phoX, were detected. These genes showed diverse predicted cis-regulatory elements. Moreover, they encoded proteins with diverse structural properties according to molecular models. Heterologously expressed PhoD and PhoX in Escherichia coli had different profiles of substrate hydrolysis. As evidenced for Rta cells, recombinant E. coli cells induced the precipitation of Ca-phosphate mineral phases, identified as poorly crystalline hydroxyapatite. The phosphatase genomic repertoire of Rta (containing phosphatases of both the PhoD and PhoX families) was previously evidenced as prevalent in marine oligotrophic environments. Interestingly, the Tataouine sand from which Rta was isolated showed similar P-depleted, but Ca-rich conditions. Overall, the diversity of phosphatases in Rta allows the hydrolysis of a broad range of organic P substrates and therefore the release of orthophosphates (inorganic phosphate) under diverse trophic conditions. Since the release of orthophosphates is key to the achievement of high saturation levels with respect to hydroxyapatite and the induction of phosphatogenesis, Rta appears as a particularly efficient driver of this process as shown experimentally.