Effects of temperature and dissolved oxygen on sludge properties and their role in bioflocculation and settling.

Effects of temperature (mesophilic (35 °C) vs. thermophilic (55 °C)) and dissolved oxygen (DO) concentration (under thermophilic conditions) on sludge properties and their role in bioflocculation and settling were studied using well-controlled sequencing batch reactors fed with a synthetic wastewater comprised of glucose and inorganic nutrients. Under a similar DO level, thermophilic sludge had a poorer bioflocculating ability and settleability than that of mesophilic sludge. Under a thermophilic condition, an increase in DO level led to a poorer settleability and a slightly improved bioflocculating ability. A poorer settleability was related to a higher level of filaments. Analysis of bound extracellular polymeric substances (EPS) indicates that thermophilic sludge had a higher level of total bound EPS content than that of mesophilic sludge under a similar DO level, and an increase in DO resulted in an increase in total bound EPS content in thermophilic sludge. Surface analysis of sludge by X-ray photoelectron spectroscopy (XPS) suggests that significant differences in the surface concentrations of elements N, C, O were observed between thermophilic and mesophilic sludge, implying significant differences in bound EPS composition. The results of gel permeation chromatography indicate that the weight-averaged molecular weight (M(w)) of bound EPS covered a range of 1159 Da to 13220 Da. The distribution of EPS "species" at floc surfaces was shown by transmission electron microscopy (TEM) to be uneven; different kinds of nanoscale materials were distributed in a patchy manner at the floc-water interface. The results suggest that it is the role of specific EPS molecules rather than the quantity of bound EPS that determine the difference in bioflocculation behavior between thermophilic and mesophilic sludge. The strategy of increasing the DO level could not solve the biomass separation problems associated with thermophilic sludge.

Soft X-ray spectromicroscopy of nickel sorption in a natural river biofilm.

Scanning transmission X-ray microscopy (STXM) at the C 1s, O 1s, Ni 2p, Ca 2p, Mn 2p, Fe 2p, Mg 1s, Al 1s and Si 1s edges was used to study Ni sorption in a complex natural river biofilm. The 10-week grown river biofilm was exposed to 10 mg L(-1) Ni(2+) (as NiCl(2)) for 24 h. The region of the biofilm examined was dominated by filamentous structures, which were interpreted as the discarded sheaths of filamentous bacteria, as well as a sparse distribution of rod-shaped bacteria. The region also contained discrete particles with spectra similar to those of muscovite, SiO(2) and CaCO(3). The Ni(II) ions were selectively adsorbed by the sheaths of the filamentous bacteria. The sheaths were observed to be metal rich with significant amounts of Ca, Fe and Mn, along with the Ni. In addition, the sheaths had a large silicate content but little organic material. The metal content of the rod-shaped bacterial cells was much lower. The Fe on the sheath was mainly in the Fe(III) oxidation state. Mn was found in II, III and IV oxidation states. The Ni was likely sorbed to Mn-Fe minerals on the sheath. These STXM results have probed nano-scale biogeochemistry associated with bacterial species in a complex, natural biofilm community. They have implications for selective Ni contamination of the food chain and for developing bioremediation strategies.

Effect of solids retention time on structure and characteristics of sludge flocs in sequencing batch reactors.

The effect of solids retention time (SRT) (4-20 d) on sludge floc structure, size distribution and morphology in laboratory-scale sequencing batch reactors receiving a glucose-based synthetic wastewater was studied using image analysis in a long-term experiment over one year. Floc size distribution (>10 microm) could be characterized by a log-normal model for no bulking situations, but a bi-modal distribution of floc size was observed for modest bulking situations. In each operating cycle of the SBRs, the variation in food /microorganisms ratio (0.03-1.0) had no significant influence on floc size distribution and morphology. The results from a long-term study over one year showed that no clear relationship existed between SRT and median floc size based on frequency. However, sludge flocs at the lower SRTs (4-9 d) were much more irregular and more variable in size with time than those at higher SRTs (16 and 20 d). The level of effluent-suspended solids at lower SRTs was higher than that at higher SRTs.

A review of biofouling and its control in membrane separation bioreactors.

Membrane separation technology is increasingly becoming an important innovation in biological wastewater treatment. Biofouling of the membrane is a major factor affecting the efficient and economic operation of membrane separation bioreactors (MBRs). This review summarizes the state-of-the-art progress in understanding the mechanisms and factors affecting membrane biofouling and the strategies for biofouling control. Biofouling mechanisms include the adsorption of soluble and suspended extracellular polymers on membrane surfaces and in membrane pores, the clogging of membrane pore structure by fine colloidal particles and cell debris, and the adhesion and deposition of sludge cake on membrane surfaces. Design and operating conditions of membrane modules and materials, hydrodynamic conditions in MBRs, process and environmental conditions of activated sludge systems, and the physicochemical properties of the wastewater are the dominant factors determining membrane biofouling. Current strategies to control biofouling include periodic relaxation, backwashing, chemical cleaning, and possible manipulation of hydrodynamic conditions and sludge properties. Achieving full integration of MBRs in wastewater treatment technology requires further research and development. Fundamental information on the bacteria, colloid, and membrane interaction, developed through multimethod and multiscale approaches, is particularly needed.

Electron-optical characterization of nano- and micro-particles in raw and treated waters: an overview.

State-of-the-art information is presented on the analysis, by transmission electron microscopy (TEM), of aquatic colloidal particles in the size range of 3 to 500 nm least dimension, with a focus on nanoparticles (1-100 nm). Case studies include selections from both natural waters and waters undergoing treatment. The "species" of nano-particles receiving the greatest attention are: humic substances, polysaccharide fibrils, hydrous iron oxides, viruses, clay minerals, refractory cell debris, and heavy metal agglomerates on biological surfaces. Artifacts and how to both detect and minimize them are outlined. Correlative use of TEM with other imaging techniques is emphasized, along with associated spectroscopy. Noted is the potential of computerized image analysis for quantifying colloids on a "per colloid species" basis, using water samples centrifuged onto electron microscope grids.

Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms.

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.

Effect of solids retention time on floc structure.

Correlative microscopy was applied to study the influence of solids retention time on activated sludge floc structure. Conventional optical microscopy revealed flocs at lower SRTs (4 and 9 days) to be irregular in shape while flocs at higher SRTs (16 and 20 days) had a more spherical and compact structure. Flocs were examined by environmental scanning electron microscopy (ESEM) and by transmission electron microscopy (TEM) coupled with energy-dispersive spectroscopy (EDS). Distinctive differences in floc structure and the arrangement of EPS were revealed. Flocs from higher SRTs were less hydrated and were found to possess a dense EPS layer that covers much of the surface. Extracellular osmiophilic granules present in these flocs indicate that the cells at the higher SRT may produce more lipid-like material. This EPS layer appears to decrease the floc surface roughness and protects the interior cells from disruption by changes in the external environment. Sludge flocs at higher SRTs were found to be physically more stable than those at lower SRTs.

Interparticle interactions affecting the stability of sludge flocs.

Interparticle interactions affecting the stability of sludge flocs taken from laboratory-scale sequencing batch reactors at different solids retention times (SRTs) were investigated in batch experiments by varying the pH, ionic strength, cation valence, and urea and ethylenediaminetetraacetate concentrations of suspending solutions. The ultrastructure of sludge floc surfaces was observed by transmission electron microscopy. Changes in dissociation constants of sludge flocs under different conditions indicated that ionic interactions and hydrogen bonds held flocs together and compensated for the negative influence of electrostatic interactions on the stability of sludge flocs. Ionic interactions and hydrogen bonds were two dominant forces that maintained the stability of sludge flocs at lower SRTs; other mechanisms, such as physical enmeshment and van der Waals and/or hydrophobic interactions, were more important in controling the stability of sludge flocs at higher SRTs. Sludge flocs at higher SRTs (16 and 20 days) were physically more stable than those at lower SRTs (4 and 9 days). A conceptual model of floc structure, based on interparticle interactions, for describing the stability of sludge flocs is proposed. The floc matrix is proposed to consist of two physically distinct regions that are defined by the arrangement of extracellular polymeric substances (EPS). These are likely to be differentially affected by the agents applied to manipulate interparticle forces. Thus, the heterogeneity in the packing of and the type of EPS reflects the stability of the floc.

Surface properties of sludge and their role in bioflocculation and settleability.

The influence of sludge retention time (SRT) on the extracellular polymeric substances (EPS) and physicochemical properties (hydrophobicity and surface charge) of sludge was studied using laboratory-scale sequencing batch reactors (SBRs) fed a synthetic wastewater containing glucose and inorganic salts. Sludge surfaces were more hydrophobic (larger contact angle) and less negatively charged at higher SRTs (16 and 20 d) than at lower SRTs (4 and 9 d). The ratio of proteins to carbohydrates within the EPS of the sludges increased as the SRT increased from 4 to 12 d corresponding to the changes in the physicochemical properties of the sludge. The protein:carbohydrate ratio remained constant at SRTs of 16 and 20 d. A transition in sludge properties appeared to occur between the upper range of low- (9 d) and lower range of high-SRTs. The total EPS content, however, was independent of the SRT. A higher sludge volume index (SVI), an indication of poorer settleability or compression, was associated with a larger amount of total EPS but no significant correlation between SVI and the surface properties of sludge was observed. A more hydrophobic and less negatively charged surface corresponded to lower levels of ESS. These results indicate that it is the surface properties, hydrophobicity, surface charge and composition of EPS, of sludge, rather than the quantity of EPS, that govern bioflocculation. In contrast, the EPS content is more important in controlling the settleability of sludge.

Structure/function/activity relationships in marine snow. Current understanding and suggested research thrusts.

Marine snow and marine snow components contribute to the mucilage phenomenon in the northern Adriatic Sea. Of special relevance is the matrix material, composed of extracellular polymeric substances, which are packaged into fibrils of colloidal dimensions. These 0.005 micron diameter fibrils are physical units of mucilage which can be visualized by transmission electron microscopy (TEM). They form polymer bridges between the various biotic and mineral components of marine snow, creating three-dimensional networks which affect floc porosity, density and settling behaviour. Recent observations of the matrix by TEM reveal complex fibril-delimited channels and capillary systems which partially traverse marine snow flocs and which are postulated to play roles in anomalous settling. Considering the marine snow floc as a microecosystem, the relationships between ultrastructure, chemistry and environmental properties are being explored. On the assumption that colloidal matrix materials, including those released into the bulk water, might provide advance information on anomalous floc behaviour, two new methods are recommended for monitoring the northern Adriatic Sea. One is a technique for chemical quantification of colloidal organic carbon. The other uses TEM, applied to water fractions derived from cascade ultracentrifugation, to estimate fibril quantities as a proportion of colloidal organic carbon.

Relationship between the Intracellular Integrity and the Morphology of the Capsular Envelope in Attached and Free-Living Marine Bacteria.

The integrity of the intracellular structures and the presence and dimension of the capsular envelope were investigated in marine snow-associated and marine free-living bacteria by transmission electron microscopy and special fixation techniques. Three categories depending on the presence of internal structures were differentiated. In marine snow, 51% of the marine snow-associated bacterial community was considered intact, 26% had a partly degraded internal structure, and 23% were empty with only the cell wall remaining. For the free-living bacterial community, 34% were intact cells, 42% exhibited damage, and 24% of the cells were lacking any internal structure. We also investigated the morphology and the extent of the bacterial capsular envelope. More than 95% of all intact marine snow-associated bacteria were surrounded by a capsule while (apprx=)55% of empty marine snow-associated bacteria had no capsule. For free-living bacteria, (apprx=)65% of the intact cells had a capsule while (apprx=)80% of the empty free-living bacteria lacked a capsule. Thus there is a clear trend from intact cells which are commonly surrounded by a capsular envelope to empty bacteria for which only the cell wall is remaining. Since bacterioplankton represent the largest living surface in the ocean, it is concluded that the release of intracellular material from bacteria into the environment as well as the release of extracellular capsular material might fuel the dissolved organic matter pool of the ocean.

Floc stabilization for multiple microscopic techniques.

A nondestructive stabilization technique for the characterization of microbial flocs which permits the application of correlative microscopic techniques is described. Flocs embedded in agarose are retained in a porous, resilient medium which allows for the transport, staining, washing, and subsampling of the flocculated material directly within a plankton chamber with minimal or no destructive forces. A single agarose disc can be subdivided into numerous sections for analysis by several microscope types and associated techniques.

The characterization of algal and microbial mucilages and their aggregates in aquatic ecosystems.

The mucilage 'phenomenon' of marine waters, a sporadic but massive accumulation of gelatinous material at and below the water surface, can create serious environmental and economic problems. To address these problems, we must understand better the causes of the phenomenon, its modulation by environmental factors and its adverse effects on ecosystems. In the context of an improved understanding, this brief review describes the means to characterize mucilage types and mucilage aggregates in their native condition, or as close to native as state-of-the-art technology will permit. Biological, chemical and physical factors interact to determine mucilage 'speciation' and thus the specific properties of mucilaginous materials. These factors and their interactions are described briefly in relation to the molecular biology of mucilage synthesis, the formation of submicroscopic 'particles' of mucilage and the morphology of mucilage aggregates. To facilitate current attempts to relate mucilage fine structure to the macroscale morphology of large aggregates (e.g., as found in the Adriatic Sea), attention will be focused on the 'fibril', a ribbon-like colloid rich in polysaccharide molecules. Such colloids (submicrometre particles) present many morphotypes which are identifiable by transmission electron microscopy; several fibril types appear as basic structural units in many kinds of mucilage aggregates in aquatic ecosystems. Attention will also be focused on (1) the problems of coping with analyzing mixtures of highly-hydrated, physically-unstable materials and (2) the detection, assessment and minimization of colloid instability artifacts which have confounded morphological analyses of mucilage aggregates in the past.

Copper resistance in Anabaena variabilis: Effects of phosphate nutrition and polyphosphate bodies.

A copper-resistant Anabaena variabilis strain was obtained after repeated culturing in progressively higher concentrations of Cu(NO3)2. This strain maintained its resistance even after a year of repeated subculturing in copper-free medium. The resistant strain differed from the sensitive parent strain with respect to filament length, cell shape and size, and control of heterocyst formation. The resistant strain was also more resistant to cadmium, zinc, and nickel. Copper distribution studies conducted with atomic absorption spectroscopy revealed that at low copper levels the sensitive strain bound significantly more metal than the resistant strain. At higher copper levels, however, the resistant strain bound large amounts of the metal. Phosphate-loaded resistant cells could grow in higher copper concentrations than phosphate-starved resistant cells. Toluidine blue staining showed that the resistant strain contained more polyphosphate bodies than the sensitive strain; the resistant cells also had higher internal phosphate levels. X-ray microanalysis, however, did not show a direct localization of copper on polyphosphate bodies. More than one mechanism of resistance may exist in this A. variabilis strain.

Size, morphology and composition of particulates in aquatic ecosystems: solving speciation problems by correlative electron microscopy.

Particulates can impact directly on aquatic ecosystems by determining the availability and mode of dispersion of both contaminants and nutrients. An understanding of the mechanisms of such particle-associated phenomena is being augmented by particle analysis technology. In this context, microscopic and spectroscopic techniques, devised for problem solving, are being applied to frequently encountered sub-micrometre particulates which are 'unstable' with respect to methods of sample preparation and storage used routinely for particulates prior to analysis. These unstable aquatic particulates include 'species' sensitive to dehydration and to artificial aggregation induced by surfaces within a fractionation apparatus. These species, as defined broadly, include polysaccharide gels, hydrated humic substances, iron oxyhydroxides, viruses, the smallest micro-organisms and decomposing parts of cells. To develop predictive models of their roles as dispersing agents for contaminants, and to speciate such associations, it is necessary to characterize them in a state as close to the natural as possible. This critical review presents the state-of-the-art in the realistic characterization of hydrated sub-micrometre particulates by correlative electron microscopy (EM) used in conjunction with spectroscopy and minimally perturbing preparatory techniques. Correlative EM is a strategy for using several different kinds of microscopes and accessory techniques in a multi-method context to analyse a given specimen for different kinds of information, including relationships in three dimensions within colloid systems. Sizing, morphology and gross composition are determined on a 'per particle' basis by transmission EM used in conjunction with energy-dispersive spectroscopy, electron diffraction and molecule-specific stains.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular fibril production by freshwater algae and cyanobacteria.

In order to study the ability of freshwater algae and cyanobacteria to form extracellular fibrils, a screening test using ruthenium red (RR) staining was carried out on 28 species. Five of these were examined for growth and production of fibrillar material in culture media of different phosphate (P;) contents. RR-staining and uronic acid determinations at various stages of algal growth were complemented by electron microscopy of the cells and of fibrillar material released into the medium. The lower Pi concentrations enhanced growth of Micrasterias radiata, Eremosphaera sp., and Microcystis aeruginosa, and had little or no effect on growth of a Xanthidium sp. and Scenedesmus quadricauda. Extracellular uronic acid production, which was higher in low Pi medium in M. radiata, M. aeruginosa, and Xanthidium sp., could reach levels of 50 mg/liter or more. Algae with high proportions of RR-positive cells (M. radiata, Eremosphaera sp., Xanthidium sp., and M. aeruginosa) produced high levels of slime-like material and distinct fibrils that were often seen attached to the cell surface and only slowly released into the medium. No such material was found in cultures (or supernatants) of Sc. quadricauda, which also produced relatively low amounts of polyuronic acids. Specific types of filaments, often forming "fascicles" with rectangular arrays of globular particles were observed by negative staining electron microscopy of some algal cultures. RR-positive material was also observed in the cytoplasm and on the cell walls and surfaces of M. radiata and M. aeruginosa.