G. Villemin

Chemical and structural (2D) linkage between bacteria within activated sludge flocs

This study describes the complexity of the activated sludge floc structure using four methods: (i) microscopic observation of flocs in situ and after specific staining, (ii) optimization of floc dispersion by sonication of pure bacterial strains, (iii) analysis of polymers released from sonicated sludges, and (iv) floc size distributions after different sonication times. The sonication of activated sludge at 37 W for 60 s was found to be best for dispersing flocs and minimizing bacterial cell lysis. The polymers released from flocs were mainly proteins, with polysaccharides and DNA. Electron microscopy showed that a polysaccharide gel connected the cells together. Raw activated sludges give a continuous distribution of particle sizes (1.2–600 μm). The floc size distribution in sonicated sludge samples was used to build a model of floc structure showing that the predominating macroflocs (125 μm) are formed from 13 μm microfloc aggregates, which are made up of smaller particles (2.5 μm).

Composition, structure and size distribution of suspended particulates from the Rhine River

Fluvial suspended particulates collected from the Rhine River were investigated in terms of composition, structure and size distribution. Elemental analysis and Diffuse Reflectance Infrared Spectroscopy reveal that most particulate organic matter is formed from material derived from microorganisms. Transmission Electron Microscopy observations on resin-embedded samples and structural characterization from break-up experiments, show that fluvial particulate matter should be viewed as fractal aggregates organized by bacterial exopolymeric substances. The shape of particulate size distribution suggests that the formation and dynamics of suspended particulate matter are controlled mainly by physical processes. Finally, particulate growth and structure are consistent with a cluster-cluster aggregation scheme.

Ferric chloride and lime conditioning of activated sludges: an electron microscopic study on resin-embedded samples.

Ferric chloride and lime conditioning of sewage sludge is usually interpreted in terms of enhanced aggregation of sludge components. In this study, transmission electron microscopy was used to investigate the conditioning mechanism at a submicronic scale. Samples were collected from two municipal wastewater treatment plants at different stages of the process, embedded in an epoxy resin, and sectioned with an ultramicrotome. Transmission electron microscopy examination of thin sections revealed that clumps of crystalloids develop on external surfaces of flocs after the application of both conditioners. This precipitate creates a rigid structure around the flocs which, upon mechanical dewatering, transmits the stresses applied to the inner parts of flocs. The porous structure of the precipitate may also participate to the withdrawal of water as a draining media. Energy dispersive X-ray spectroscopy revealed that Fe, P, and Ca are the dominant elements in the precipitate with Fe/P and Fe/Ca elemental ratios close to 2.8-3 and 1.1-3, respectively.

Biodegradation of naphthalene in montmorillonite/polyacryamide suspensions

The biodegradation of naphthalene in the presence of Na montmorillonite particles, of montmorillonite/polyacrylamide complexes and of polyacrylamide alone was studied in batch assays under aerobic conditions. The concentrations of naphthalene used were below aqueous phase saturation. Adsorption-desorption experiments with montmorillonite and montmorillonite/polyacrylamides complex indicated that little or no naphthalene was adsorbed. Adsorption appeared totally reversible and desorption was rapid, reaching completion in less than 24 hours. Naphthalene was rapidly degraded by an adapted strain of Pseudomonas cepacia (10-25 hours) as a function of the initial viable bacteria count of the inoculum. The kinetics of naphthalene degradation in the presence of montmorillonite/polyacrylamide complexes or even with montmorillonite alone (with no naphthalene adsorbed), were slower (0.64 mg l −1 h −1 ) than in clayless aqueous solution (0.84 mg l −1 h −1 ). Dissolved non-biodegradable polyacrylamide had no effect on naphthalene degradation. These results indicate that particles decrease the rate of naphthalene breakdown by Pseudomonas cepacia . This may be caused by the fact that montmorillonite particles are smaller on average than bacteria cells. Montmorillonite particles may cover bacteria and so limits nutrient mass transfer.

In situ ageing of fine beech roots (Fagus sylvatica) assessed by transmission electron microscopy and electron energy loss spectroscopy: description of microsites and evolution of polyphenolic substances.

Root biomass is quantitatively and qualitatively important in most ecosystems, but its contribution to the pool of organic matter in the soil is not clear. This work was designed to specify root ageing on an ultrastructural scale by transmission electron microscopy combined with microanalysis by electron energy loss spectroscopy. This approach is very suitable for studying the soil/plant interface, and for semi‐quantitative analysis of the evolution of polyphenolic substances during root evolution. Three root segments were studied according to a gradient of root senescence: the apical and basal segments of the mycorrhiza and the mycorrhiza‐carrier root. Each segment contained a certain proportion of senescent cells, some of which were of fungal origin, and this proportion increased as the root aged. In the three segments, the soil/plant interfaces were differentiated, and the micro‐organisms observed in situ were described. Senescent root cells contained many polyphenolic substances and our results showed that these substances were, according to the root segment, differently associated with Ca, N and Si. When all these ultrastructural data are correlated with more global data, they can be usefully applied to root cell physiology, microbiology and pedology. This approach makes it possible to specify the evolution of organic matter in situ in soils whatever its origin.

Localization and characterization by electron energy loss spectroscopy (EELS) of the brown cellular substances of beech roots

Abstract Degradation of roots from beech trees was investigated by a novel approach: localization and elemental characterization of predominant brown substances were performed at ultrastructural scale using electron energy loss spectroscopy (EELS). This technique allowed us to detect light elements such as C and N, main constituents concerned in the degradation of plant tissues, within cellular constituents observed with a transmission electronic microscope, after having localized them by light microscopy. The results showed that there was no relationship between the brown pigmentation of the cellular substances and the presence of N, a relationship previously found in studies of degrading leaves. Other elements such as Si and Ca were also found to be involved in these root degradation processes. This approach seem powerful for detailed descriptions of the process of root degradation in soil.

Evolution morphologique des litières de hêtre (Fagus sylvatica L.) et transformation des biopolymères, lignine et polysaccharides, dans un mull et un moder, sous climat tempéré (forêt de Fougères, Bretagne, France)

Morphological evolution of beech litter (Fagus sylvatica L.) and biopolymer transformation (lignin, polysaccharides) in a mull and a moder, under temperate climate (Fougeres forest, Britany, France). Can. J. Soil Sci. 85: 405-416. This study was conducted on a mull and a moder from two beech stands, 27 and 87 yr old, respectively, in the Fougeres forest. In each stand, five profiles were subdivided in OL, OF and OH (moder only) layers and A 1 subhorizons. In the mull OL-OF layer, the organic matter was quickly degraded by white-rot fungi together with bacteria and earthworms. A fungal attack occurred in the moder OL and OF layers, whereas bacteria seemed efficient in the OF layer and especially so in the OH layer, where they appeared responsible for lignin and structural polysaccharide degradation. Lignin degradation was indicated by: (i) the decrease of all phenolic monomers, (ii) methoxyl group loss and (iii) an increase in the vanillic acid/aldhehyde ratio. The production of microbial exo-polysaccharides at the expense of the structural polysaccharides, revealed by an increase of the mannose/xylose ratio, was also supported by transmission electron microscopy observations. The decrease in the lignin phenols and structural polysaccharides continues in the underlying A 1 1 and A 1 2 layers. In the mull, earthworm activity results in a complex organo-mineral association, whereas in the moder, enchytraeid worm activity is responsible for mixing of inherited organic aggregates associated with minerals.

Ultrastructural patterns of beech leaf degradation by Sporotrichum pulverulentum

Degradation of beech leaves by the white rot fungus Sporotrichum pulverulentum was investigated for 4 weeks under laboratory conditions. Observations by transmission electron microscopy revealed that the degradation patterns depended on the nature of the foliar tissues and on the stage of decay. Fibres, parenchyma cells and specific zones of the sclerenchyma tissue, corresponding to the cells located between vessels, were strongly degraded, while vessels and epidermal cells were more resistant to degradation. During the early stages of degradation, a selective removal of components from the lignin and hemicellulose-rich layers was observed in the cell walls of sclerenchyma fibres and parenchyma cells. At a more advanced stage of decay, a simultaneous disappearance of all cell wall layers (primary wall, secondary wall, middle lamella and cell corners) occurred, irrespective of cell type. In parenchyma cells, removal of the intracellular brown pigments occurred prior to degradation of the cell walls, while in epidermis, the cell walls were altered first. In sclerenchyma cells surrounding the vessels, hyphae were found to be closely associated with decayed areas, while in all the other tissues, there was no contact of the fungus with lignocellulosic compounds and polyphenols, suggesting a diffusion of the fungal degradative enzymes. A fibrillar mucilagenous matrix, often detected between hyphae and cell walls, might make this diffusion easier. Cellulase activity was detected in the decayed tissues by the release of reducing sugars localized in the vicinity of the leaf cell walls and often somewhat distant from the hyphae. The role of the fungal enzymes involved in degradation of cell wall components and brown pigments in beech leaves is discussed.

Soil Microstructures Examined Through Transmission Electron Microscopy Reveal Soil-Microorganisms Interactions

Research over the last few decades has shown that the characterization of microaggregates at the micrometer scale using Transmission Electron Microscopy (TEM) provides useful information on the influence of microorganisms on soil functioning. By taking soil heterogeneity into account, TEM provides qualitative information about the state of bacteria and fungi (e.g., intact state of living organisms, spores, residues) at the sampling date within organo-mineral associations, from the soil-root interface to the bulk soil, and in biogenic structures such as casts. The degree of degradation of organic matter can be related to the visualized enzymatic potential of microorganisms that degrade them, thus indicating organic matter dynamics within soil aggregates. In addition, analytical TEM characterization of microaggregates by EELS (Electron Energy Loss Spectroscopy) or EDX (Energy Dispersive X-rays spectroscopy) provides in situ identification of microbial involvement in the biogeochemical cycles of elements. Furthermore, micrometer characterization associated with other methodologies such as Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) or soil fractionation, enables monitoring both incorporation of biodegraded litter within soil aggregates and impacts of microbial dynamics on soil aggregation, particularly due to production of extracellular polymeric substances. The present focused review suggests that such an approach using micrometer characterization of soil microhabitats provides relevant qualitative and quantitative information when monitoring and modelling microbial processes in dynamics of organo-mineral associations.