3‐D analysis of bacterial cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches

The formation of cell‐(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2‐D microscopy techniques, the 3‐D and internal structure remain obscure. In this study, we examined the 3‐D structure of cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3‐D microscopy techniques. We obtained 3‐D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4–200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike‐ or platelet‐shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell‐(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell‐(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3‐D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X‐ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a comprehensive understanding of structure and composition of the various Fe‐precipitates and their association with bacterial cells and EPS.     

Surface chemistry and reactivity of bacteriogenic iron oxides from Axial Volcano, Juan de Fuca Ridge, north-east Pacific Ocean

Iron oxides were collected from the caldera of Axial Volcano, a site of hydrothermal vent activity along the Juan de Fuca Ridge. Mineralogical inspection using X‐ray diffraction (XRD) revealed the majority of samples to be 2‐line ferrihydrite, with one of the samples corresponding to poorly ordered goethite. Examination using environmental scanning electron microscopy (ESEM) found the constituents of the iron oxides to consist predominantly of bacterial‐like structures that resembled the iron oxidizing bacteria Leptothrix ochracea, Gallionella ferruginea and a novel PV‐1 strain. X‐ray photoelectron spectroscopy (XPS) detected the presence of Fe, O, C, N, Ca, Si and P on all the samples with the exception of poorly ordered goethite, where Ca and P were absent, in addition to a weak N peak. Binding energy shifts of the Fe 2p and O 1s peaks were indicative of ferrihydrite and hydroxyl functional groups, while the presence and speciation of the C 1s peak was attributed to the presence of bacteria. Use of acid‐base titration data modelling in conjunction with a linear programming regression method (LPM) indicated that the iron oxides are composed of heterogeneous surface functional groups. Differences in iron oxide reactivity values correlated with differences in the bacterial and mineral fabric of the samples. The diverse surface chemistry and high reactivity of these iron oxides may be important in the global cycling of various elements throughout the oceans due to their presence along widespread mid‐ocean ridges.     

Enumeration of Thiobacilli within pH-Neutral and Acidic Mine Tailings and Their Role in the Development of Secondary Mineral Soil

The Lemoine tailings of Chibougamau, Quebec, Canada, were deposited as a pH-neutral mineral conglomerate consisting of aluminum-silicates, iron-aluminum-silicates, pyrite, chalcopyrite, and sphalerite. These tailings are colonized by an active population of Thiobacillus ferrooxidans which is localized to an acid zone occupying 40% of the tailings' surface. This population peaked at 7 × 10⁸ most probable number per gram of tailings during July and August 1990 and extended to a depth of 40 cm from the surface. Examination of samples over this depth profile by transmission electron microscopy and electron dispersive spectroscopy revealed a microbially mediated mineral transition from sulfides (below 40 cm) to chlorides and phosphates (at the surface). Silicate minerals were unaltered by microbial action. Transmission electron microscopy showed a tight association between Thiobacillus species and the sulfide minerals, which helps account for their prominence in tailings environments. Accurate enumeration of T. ferrooxidans from tailings required the disruption of their bonding to the mineral interface. Vortexing of a 10% aqueous suspension of the tailings material prior to most-probable-number analysis best facilitated this release. Even though heavy metals were highly mobile under acidic conditions at the Lemoine tailings, it was evident by transmission electron microscopy and electron dispersive spectroscopy that they were being immobilized as bona fide fine-grain minerals containing iron, copper, chlorine, phosphorus, and oxygen on bacterial surfaces and exopolymers. This biomineralization increased with increasing bacterial numbers and was most evident in the upper 3 cm of the acidic zone.     

Adsorption of Colloidal Iron by Bacteria

The adsorption of iron from a positive-iron sol by species of seven bacterial genera was examined by electron microscopy. All species precipitated the iron from the sol, and the bacterial cells became encrusted with iron. This was related to iron deposition in surface water supplies.     

Microbial adaptation to iron: a possible role of phosphatidylethanolamine in iron mineral deposition

Pseudomonas fluorescens multiplied in a minimal mineral medium supplemented with iron(III) (5 mm) complexed to citrate, the sole source of carbon, with no apparent diminution in cellular mass. Atomic absorption studies of different cellular fractions and supernatant at various growth intervals revealed that the trivalent metal was initially internalized. At approximately 41 h of incubation, the soluble cellular extract contained 9.5% of the iron originally found in the growth medium. However, as bacterial multiplication progressed, most of the metal was deposited as an extracellular insoluble gelatinous residue. Phosphatidylethanolamine appeared to be an important organic constituent of this precipitate. X-ray fluorescence and diffraction studies revealed that iron(III) was deposited as amorphous hydrated oxide. Scanning electron microscopy and energy dispersive X-ray microanalysis of the pellet aided in the identification of irregular shaped bodies rich in iron and oxygen that were associated with carbon-containing elongated structures. Examination of the bacterial cells by a transmission electron microscope equipped with an electron energy loss spectrometer indicated the deposition of iron within the cells.     

Arsenic in Biogenic Iron Minerals from a Contaminated Environment

High arsenic levels have been found in some water samples from the Iron Quadrangle, a main gold, manganese and iron mining region in Brazil. In this work, we used transmission electron microscopy coupled to energy-dispersive X-ray analysis to show arsenic in bacteriogenic iron minerals (BIOS) collected in this region. Two types of iron bacteria stalks and several morphologically different filamentous sheaths of bacteria were found, most containing arsenic. Bacterial stalks were partially coated by spherical precipitates probably deposited after stalk secretion. Arsenic/iron ratios were the same independently of the amount of spherical precipitates, suggesting that arsenic incorporation is independent of bacterial metabolism. Additionally, arsenic seems to be saturated in these minerals, since the arsenic/iron ratio was the same under different arsenic concentrations.     

Effects of digestion with chondroitinases upon mucosaccharide stainings of rabbit cartilage as revealed by electron microscopy

Summary The colloidal iron staining reactions of acid mucosaccharides were studied by electron microscopy in tracheal cartilage tissues of the rabbit and those subjected to digestion with chondroitinase ABC or AC. The colloidal iron reactive acid mucosaccharides are localized in close association with filamentous structures of the cartilage matrix and in cytoplasmic granules of chondrocytes. The major moieties of these mucosaccharides are susceptible to digestion with either of the chondroitinases and appear, therefore, to be chondroitin sulfates and related mucosaccharides.     

Bacterial destruction of mica during bioleaching of kaolin and quartz sandsby Bacillus cereus

Growth and metabolic activities of Bacillus cereus were found to cause the extraction of iron atoms from the octahedral position in mica in the kaolin sample (49%) and in the quartz sands sample (17%) after 3 months of bioleaching, while aluminium removal was only 5%. Mica destruction was detected in kaolin and quartz sands samples by X-ray diffraction analysis and also by i.r. adsorption spectroscopy in quartz sands samples. The structural changes obtained were confirmed by scanning electron microscopy (SEM) analysis. The SEM pictures show a different morphology in the boundary region of mica grains before and after bioleaching. Bacterial destruction effects were feeble in the interlayer sites and were specially directed to split planes, which are occupied by a number of bacterial cells. The biological destruction of mica with phengite composition after iron removal led to development of illite, which was detected by energy-dispersion microanalysis (EDS). Illite development caused also the enrichment of the kaolin sample by fine-grained fraction.     

Effects of acetic acid treatment on plant chromosome structures analyzed by atomic force microscopy

Acetic acid treatment has been frequently used to remove cellular contaminants from plant chromosome samples for structural analyses by scanning electron microscopy and atomic force microscopy (AFM). We evaluated the effects of various concentrations of acetic acid treatments on barley chromosome structures by using AFM. The long-term 45% acetic acid treatment significantly damaged the chromosome structures, although the treatment effectively removed the cellular contaminants. On the other hand, the treatment with 15% acetic acid could not obtain sufficiently clean chromosome samples and the chromosome surface structures could not be observed. In contrast, we obtained clean chromosome preparation without severe damage by using an intermediate concentration (30%) of acetic acid treatment. In the centromeric region, we could observe fiber structures with a width of 100 nm, which were composed of ca. 50-nm granules and aligned to the axes of chromosomes. Thus, AFM analysis of chromosomes appropriately treated with acetic acid will provide important insights into the organization of higher-order structures of plant chromosomes.     

Scanning electron microscopy and epifluorescence investigation of bacterial colonization of marine sand sediments

Scanning electron microscopy (SEM) was employed for the investigation of microorganisms living in marine sand sediments. Epifluorescence, as well as sediment analyses, gave further data on the parameters of the sediment samples.SEM revealed a correlation between the site and density of bacterial colonization and the microtopography of the individual sand grains.Sand grains with a medium roundness showed the greatest density of bacterial colonization. Protected surface sites were favored in the colonization process. The mode of bacterial attachment varied; mostly the barren sand grain surface was colonized. However, bacteria were also observed close to or within detritus or attached to diatoms. Many of the attaching bacteria observed were found to produce polymer strands.In some cases special structures were discovered which could serve bacterial attachment. Entire colonies attached by means of polymer nets, and disc-shaped bacteria were observed.     

Electron Microscope Studies of Deoxyribonucleic Acid Synthesis in Escherichia coli: Localization of [3H]Thymidine in Deoxyribonucleic Acid-Membrane Complexes

The attachment of deoxyribonucleic acid to the membrane in Escherichia coli 15 T(-) cells incubated with [(3)H]thymidine was studied by electron microscopy. Isolated deoxyribonucleic acid-membrane complexes were prepared from synchronized and unsynchronized cells during the exponential or stationary phase of growth and were examined by autoradiography. After short pulses with [(3)H]thymidine, a specific enrichment in radioactivity was observed in areas of membranous structures in exponentially growing cells. In contrast, the grain tracks produced in autoradiographs of chromosomes from cells in stationary phase were randomly distributed. The autoradiographic patterns are, therefore, evidence that deoxyribonucleic acid replication is closely related to the bacterial membrane.     

Electron acceptors for anaerobic oxidation of methane drive microbial community structure and diversity in mud volcanoes

Mud volcanoes (MVs) emit globally significant quantities of methane into the atmosphere, however, methane cycling in such environments is not yet fully understood, as the roles of microbes and their associated biogeochemical processes have been largely overlooked. Here, we used data from high‐throughput sequencing of microbial 16S rRNA gene amplicons from six MVs in the Junggar Basin in northwest China to quantify patterns of diversity and characterize the community structure of archaea and bacteria. We found anaerobic methanotrophs and diverse sulfate‐ and iron‐reducing microbes in all of the samples, and the diversity of both archaeal and bacterial communities was strongly linked to the concentrations of sulfate, iron and nitrate, which could act as electron acceptors in anaerobic oxidation of methane (AOM). The impacts of sulfate/iron/nitrate on AOM in the MVs were verified by microcosm experiments. Further, two representative MVs were selected to explore the microbial interactions based on phylogenetic molecular ecological networks. The sites showed distinct network structures, key species and microbial interactions, with more complex and numerous linkages between methane‐cycling microbes and their partners being observed in the iron/sulfate‐rich MV. These findings suggest that electron acceptors are important factors driving the structure of microbial communities in these methane‐rich environments.     

Fine visualization of filamentous structures in the bacterial cytoplasm

A new method was established for fine visualization of bacterial subcelluar filamentous structures by freezing the bacterial cells to displace cytoplasmic matrix granules to the periphery. This method was successfully applied in immunoelectron microscopy and electron microscopic tomography, and should be applicable for further studies of bacterial architecture and nanotransportation.     

Ex vivo gingival-biofilm consortia

AIMS: To develop a protocol for harvesting ex vivo samples of gingival-biofilm consortia and to investigate their basic characteristics. METHODS AND RESULTS: Gingival epithelial cells with attached biofilm were collected from healthy subjects by taking a smear. The bacterial viability was estimated via the alteration of the membrane permeability and metabolic activity via the double/single-stranded nucleic acid ratio using a confocal laser-scanning microscope. Morphological analysis was performed by scanning and transmission electron microscopy. Additionally, microbiological estimations were made. The electron microscopy revealed fimbriae-mediated adhesion and the formation of a biofilm matrix. Most bacteria were viable and had a high metabolic activity. CONCLUSIONS: The presented study offers an easy to follow approach for harvesting samples of gingival-biofilm consortia. The latter differs considerably from the supragingival plaque in viability and zonal distribution. Related to free-living and in vitro-grown biofilms, the gingiva-associated biofilm revealed an atypically high metabolic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm fragments should possess the basic features of the entire gingiva-associated biofilm; which as yet cannot be simulated in vitro. Thus, samples of ex vivo gingival-biofilm consortia can be used to investigate the resistance of oral biofilms against antibiotics and biocides.     

Structure and composition of ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells

Ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated by high resolution transmission electron microscopy, electron diffraction and chemical analysis. Hemosiderin particles isolated from thalassemic spleens also have been studied. The results show that there is a marked difference in structure and composition of the biomineral phases. Human ferritin and hemosiderin particles are single domain crystals of hydrated iron (III) oxide (ferrihydrite). Lattice fringes were low in contrast and often discontinuous within the central regions of the core. Heat treatment of human ferritins results in a 5 A shrinkage in particle size and an increase in the single crystalline nature of the core. In contrast, lattice images and electron diffraction of limpet and bacterial cores show no evidence of long-range crystallographic order. Chemical analysis indicates a high inorganic phosphate (Pi) (Fe/Pi = 1.71) content in bacterial ferritin compared with human ferritin (thalassemic) (Fe/Pi = 21.0). The high Pi content of bacterial ferritin suggests a hydrated amorphous iron (III) phosphate mineral core. Structural disorder within the limpet and bacterial cores may be associated with increased Pi content and increased oxidation in Fe(II), resulting in rapid mineral deposition. Growth of the iron (III) oxide cores in human ferritin is discussed on the basis of high resolution electron microscopy results.     

Structural bases for the invasion of the causative agents of experimental Pseudomonas aeruginosa sepsis into the bloodstream from the primary septic focus

The soft tissues around the implant with a suspension of P. aeruginosa were studied by light and electron microscopy. Severe damages to structures of the histohematic barrier, phagocytes were revealed as was the bacterial invasion into thrombosed vessels of the microcirculatory bed. The mechanism of the micrometabolism as playing the key role in the bacterial penetration from the primary septic focus into the common bloodstream is discussed.     

Poly-β-hydroxybutyrate accumulation in bacterial consortia from different environments

The aim of the present study was to examine soil samples from various vegetation zones in terms of physicochemical properties, microbial communities, and isolation and identification (by polymerase chain reaction and transmission electron microscopy) of bacteria producing poly-β-hydroxybutyrates (PHBs). Soil samples were analysed originating from zones with heterogeneous environmental conditions from the Romanian Carpathian Mountains (mountain zone with alpine meadow, karstic zone with limestone meadow, hill zone with xerophilous meadow, and flood plain zone with hygrophilic meadow). Different bacterial groups involved in the nitrogen cycle (aerobic mesophilic heterotrophs, ammonifiers, denitrifiers, nitrifiers, and free nitrogen-fixing bacteria from Azotobacter genus) were analysed. Soil biological quality was assessed by the bacterial indicator of soil quality, which varied between 4.3 and 4.7. A colony polymerase chain reaction technique was used for screening PHB producers. With different primers, specific bands were obtained in all the soil samples. Some wild types of Azotobacter species were isolated from the 4 studied sites. Biodegradable polymers of PHB were assessed by negative staining in transmission electron microscopy. The maximum PHB granules density was obtained in the strains isolated from the xerophilous meadow (10-18 granules/cell), which was the most stressful environment from all the studied sites, as the physicochemical and microbiological tests proved.     

Direct image-based correlative microscopy technique for coupling identification and structural investigation of bacterial symbionts associated with metazoans

Coupling prokaryote identification with ultrastructural investigation of bacterial communities has proven difficult in environmental samples. Prokaryotes can be identified by using specific probes and fluorescence in situ hybridization (FISH), but resolution achieved by light microscopes does not allow ultrastructural investigation. In the case of symbioses involving bacteria associated with metazoan tissues, FISH-based studies often indicate the co-occurrence of several bacterial types within a single host species. The ultrastructure is then relevant to address host and bacterial morphology and the intra- or extracellular localization of symbionts. A simple protocol for correlative light and electron microscopy (CLEM) is presented here which allows FISH-based identification of specific 16S rRNA phylotypes and transmission electron microscopy to be performed on a same sample. Image analysis tools are provided to superimpose images obtained and generate overlays. This procedure has been applied to two symbiont-bearing metazoans, namely, aphids and deep-sea mussels. The FISH protocol was modified to take into account constraints associated with the use of electron microscopy grids, and intense and specific signals were obtained. FISH signals were successfully overlaid with bacterial morphotypes in aphids. We thus used the method to address the question of symbiont morphology and localization in a deep-sea mussel. Signals from a type I methanotroph-related phylotype were associated with morphotypes displaying the stacked internal membranes typical for this group and three-dimensional electron tomography was performed, confirming for the first time the correspondence between morphology and phylotype. CLEM is thus feasible and reliable and could emerge as a potent tool for the study of prokaryotic communities.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

Culturable microorganisms associated with Sishen iron ore and their potential roles in biobeneficiation

With one of the largest iron ore deposits in the world, South Africa is recognised to be among the top ten biggest exporters of iron ore. Increasing demand and consumption of this mineral triggered search for processing technologies, which can be utilised to “purify” the low-grade iron ore minerals that contain high levels of unwanted potassium (K) and phosphorus (P). This study investigated a potential biological method that can be further developed for the full biobeneficiation of low-grade iron ore minerals. Twenty-three bacterial strains that belong to Proteobacteria, Firmicutes, Bacteroidetes and Actinobateria were isolated from the iron ore minerals and identified with sequence homology and phylogenetic methods. The abilities of these isolates to lower the pH of the growth medium and solubilisation of tricalcium phosphate were used to screen them as potential mineral solubilisers. Eight isolates were successfully screened with this method and utilised in shake flask experiments using iron ore minerals as sources of K and P. The shake flask experiments revealed that all eight isolates have potentials to produce organic acids that aided the solubilisation of the iron ore minerals. In addition, all eight isolates produced high concentrations of gluconic acid followed by relatively lower concentrations of acetic, citric and propanoic acid. Scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) analyses also indicated extracellular polymeric substances could play a role in mineral solubilisation.