The Architecture Of Freshwater Flocs

Floc architecture is a major determinant of floc activities and behavior; it can evolve in complexity under the influence of biological activity, leading to the development of specific features which enhance microbial habitat while conferring environmentally relevant activities and behavior to a floc.3 The capacity to analyze floc architecture by microscopical means is evolving quickly,14,16,17,39,58,70 with the microscopical observations being increasingly well integrated into an interdisciplinary context.14,20,21,40 Sample sites for the analysis of floc architecture have included five in Ontario, Canada (Hamilton Harbour, Port Stanley Harbour, Nith River, Sixteen-Mile Creek, and Fourteen-Mile Creek) and the Elbe River in Germany. Current emphasis is placed on relationships between floc architecture and the (a) activities of microbes and (b) surface-active nanoscale subcomponents, the (c) composition and (d) three-dimensional (3D) distribution of the various EPS and the (e) behavior of flocs with regard to the transport of environmentally significant materials. With regard to the 3D distribution of the various families of EPS molecules, there is great interest in EPS contributions to the following: pore structures; barriers which establish chemical gradients; barriers which obstruct viral predators; and transposable layers which can adjust the stickiness at the floc/water interface.

2.6.1 Architecture in Relation to Floc Activities, Properties, and Behavior

In order to understand better the relevance of floc architecture in terms of the floc physical, chemical, and biological behavior, Droppo3 developed a conceptual model of floc form and behavior. The model breaks the floc down into five subcomponents: (a) inorganic particles, (b) biota and bioorganic particles, (c) fibrils, (d) water, and (e) pores. Each of these components is then further subdivided into specific physical characteristics. The associated physical (e.g., transport/settling), chemical (e.g., chemical assimilation/transformation), and biological (e.g., microbial community development) behaviors are then broken out in relation to each of these characteristics (Figure 2.3 to Figure 2.7). While in reality one cannot segregate specific components of a floc and their behaviors (they all work together to influence floc development, structure, and behavior), this modeling approach allows for insight into the significance of the micro- and macro-architecture on outward floc behavior.

In the model3 the inorganic component (Figure 2.3) may represent mineral particles (silt and clay) or structural chemical precipitates which exhibit characteristic electrochemical double layers and are influenced by van der Waals forces.71 Conceptually, inorganic particles will most significantly influence the physical transport of a flocculated particle due to their density effects (they increase floc density and relative settling velocity) and potential for electrochemical flocculation. In addition, however, they also will influence the floc's chemical behavior by affecting the adsorption and transformation of contaminants and nutrients,72,73 and their biological behavior in terms of their ability to act as sites for bacterial colonization and subsequent chemical and biological activity.3,4,16

The biological component (Figure 2.4) is the most dynamic component of a floc as it can influence not only floc development and therefore transport through mediation of electrochemical flocculation, floc density, bacterial attachment, and EPS production (Figure 2.5), but will also have an impact on the chemical, physicochemical, and biological processes operating within the floc through diffusional gradients and biotransformation of nutrients and contaminants.3,16,74-76

There is increasing evidence that the most important component influencing floc behavior is the EPS fibril produced by bacteria (and by some microalgae). Figure 2.5 illustrates the important characteristics of this material and its influence on behavior. Examination of the three-dimensional matrix of EPS within freshwater flocs reveals that it truly represents a framework for the floc. Fibrils are completely integrated within the floc, forming physical and chemical links to adjacent constituent particles. This network strengthens flocs and binds flocs together, giving them a pseudo-plastic rheology.3,15-17,30,77 In addition, the EPS network influences the floc transport behavior through modifying floc density primarily by promoting the retention of water within the floc. This retention is related to the significant surface area of the fibrils, promoting micropores with significant surface tension. The large surface area of the fibrils and retention of pore water also affect the floc's chemical behavior by influencing the uptake of nutrients and contaminants and promoting diffusional and electrochemical gradients for parameters such as contaminants, pH, and redox potential.3,14,16,40,78

Both free flowing and bound water will impact a floc's structure and behavior (Figure 2.6). The free movement of water within a floc can enhance the removal of contaminants and nutrients from water by bacterial actions and general adsorption because of the advective delivery of compounds.79 Movement of water through a floc can also increase its settling velocity due to a reduction in drag around the floc.80 Bound water on the other hand will promote molecular diffusion and electrochemical gradients of contaminants16,78 and will reduce settling velocity due to its impact on reducing floc density. The greater the water content the closer the overall floc's density will be to that of water. Droppo et al.17 showed that most riverine flocs over 300 ^m had densities close to that of water. As such, water retained within a floc matrix will have significant hydrodynamic influences on floc behavior.

As is evident from the above discussion, floc pores as defined by the interparticle and interfibril voids play a significant role in influencing floc structure and behavior (Figure 2.7). Pores, which appear to be devoid of physical structures when imaged by optical microscope techniques, are sometimes observed to be composed of complex matrices of nanoscale fibrillar EPS when imaged by the much higher resolution of transmission electron microscopy.16 Pores are responsible for much of the floc physical, chemical, and biological behavior as they control water content and movement within the floc proper. As a result and as discussed above, pores will influence floc density, transport and chemical and biological activities, and diffusional and electrochemical gradients.79-81 Figure 2.7 provides the resultant behavioral effects of pores.

The pore structure of flocs could very well have a profound effect on predation of the microbiota by viruses, with consequent effects on the population density and speciation of microbiota. Viruses in the nanoscale size range have a high abundance in aquatic environments;82,83 their abundance is sufficient to make them major predators in many microbial niches. Since the rate of lytic infection depends on the rate of virus adsorption to its host,84 the pore structure of a floc may serve to prevent predation by keeping an effective separation distance between virus and microbe. Using transmission electron microscopy, applied to ultrathin sections of flocs, the authors are currently accumulating evidence that viruses may be unable to penetrate into regions of a floc where the packing of fibrils yields a pore structure in the lower part of the nanoscale range.

2.6.2 Relevant Findings for Floc Architecture from the Biofilm Literature

In terms of architecture, biota-rich flocs often resemble biofilms which had been stripped from their substratum and turned back on themselves.16 This is true even when one considers the complex model of biofilm architecture introduced by de Beer et al.45 to relate specific aspects of structure to oxygen distribution and mass transport. Diffusion in a biofilm is hindered relative to diffusion in the nearby bulk solution85 while oxygen distribution can be strongly correlated to structure, being facilitated by voids as it is delivered from the bulk water to microbial cell clusters.45 Interest in relating specific entities of biofilm structure to molecular composition, activities, and physiological phenomena86 has been strong and some resultant case histories are worthy of note for floc specialists.

Because biofilms are attached to a solid substratum, many investigations of biofilm structure/function relationships are technically less complicated than the same work would be for flocs, whose overall morphology becomes distorted when they fall out of suspension onto a rigid surface. This situation has been addressed by the floc stabilization technique of Droppo et al.,39 thus making available an improved capacity to extend to flocs some investigations formerly confined to biofilms.

Consider the following case study of a biofilm and the utility of relating it directly to the transport and transformation activities of flocs. Microbial exopoly-mers (EPS) were demonstrated to provide a mechanism for the bioaccumulation of the herbicide, diclofop methyl.87 This study used confocal laser scanning microscopy (CLSM) to directly visualize accumulation of the herbicide and its breakdown products within a biofilm community. Correlated mass spectroscopic analysis confirmed the accumulation of the herbicide and its breakdown products within the biofilm. The diclofop-degrading biofilm developed distinctive spatial relationships among diverse members of its microbial community, implying that unique con-sortial relationships facilitated diclofop degradation by cooperative interactions.88

Subsequent physiological experiments demonstrated that the EPS could act as a storage site for the herbicide, prior to its degradation.89 These results led to analyses of the three-dimensional distributions of biofilm exopolymers involved in the accumulation of chlorinated organics, using fluorescent probes in conjunction with CLSM.90 Using fluorescent lectins,91 a nearly 1:1 correspondence could be demonstrated between the distribution of regions that accumulated diclofop and regions which bound a lectin which is specific for an EPS polymer containing a-L-fucose.90

This highly evolved case study shows for biofilms what is almost certainly to be evidenced soon for flocs. The matrix material (EPS) binds a chemical of environmental interest, leading to bioaccumulation followed by metabolically directed degradation of the chemical. The microbial consortia develop distinct spatial relationships to promote cooperative interactions among diverse members of the microbial community, in relation to what they sense as either food or toxicant. The biological activity restructures the overall architecture to improve adaptation to stimuli coming from the bulk water. Some of the restructuring consists of the secretion of specific EPS molecules which facilitate the interactions between microbes and an incoming chemical. Given the similarities between biofilm and floc architecture (and the ability of their constituent microbes to adjust that architecture to gain ecological advantage), improved technology should soon permit the kinds of biofilm research done by Wolfaardt et al.87-90 to be done also on flocs.

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