Mikkelsen and Perjrup69 presented a method for determining effective floc density and calculated settling velocity in coastal marine environments using data collected by a LISST. Assuming that floc fraction and the amount of material in suspension that is found in flocs is high then the effective floc density is equal to the total suspended mass divided by the total volume concentration of the flocs.

Although there are many empirical models available for the estimation of floc density and porosity, none of them can be considered as a universal model. This is simply because all these models were developed from their specific conditions such as the type of natural or engineered system, the type of microorganisms, the hydro-dynamic conditions, and the experimental techniques used. Therefore, floc density and porosity must be experimentally determined in all situations. New instrumentation is now available that can determine the size-settling velocity relationship of flocs in suspension, determine the mass of the flocs, and capture them for microscopic analysis.52 With these advances, it should be possible to determine densities in natural environments in situ.

1.3.1 Floc Structure: Correlative Microscopy

Leppard70 defined correlative microscopy (CM) as a strategy of using multiple microscopic techniques which can include conventional optical microscopy (COM), confocal laser scanning microscopy (CLSM), and transmission electron microscopy (TEM), and allow one to detect, assess, and minimize artifacts that might arise from using one technique only. CM has been successfully used by Liss et al.61 with a minimal perturbation approach in studying natural and engineered flocs. A recent minimal perturbation approach49,50 involves the use of sample stabilization in low melting point agarose and a fourfold multi-preparatory technique. The use of a fourfold multiple preparatory technique and CM has revealed how to maintain the structural integrity of the samples through the stabilization, staining, and washing procedures. The use of only one microscopic technique can bias or limit the information acquired because of the artifacts that arise in specific sample preparations and the resolution constraint associated with a particular technique.

The use of COM is the most common microscopic approach in the analysis of external gross-scale floc structure.12,14,31,57,71,72 High resolution TEM is often used to investigate the fine structure of natural and engineered flocs, especially in the study of EPS distribution within floc structure.26,27,72-75 This is generally done by stabilizing samples in a fixing agent such as glutaraldehyde, then embedding in Spurr resin or alternatively a fixation and embedding in Nanoplast; an ultrathin section is then obtained from the embedded sample by slicing with an ultramicrotome and a diamond knife. This ultrathin section (50 to 100 nm) is then placed on a copper grid for further staining (e.g., uranyl acetate) to give better contrast, although at TEM resolution, fibrils, bacterial cells, and other components of floc are visible. TEM can be used in conjunction with energy dispersive spectroscopy (EDS) to detect metal accumulation and to give element abundance in EPS.61,76 The thickness constraint of ultrathin sections (50 to 100 nm or less) in the preparation of TEM images has restricted the floc sample volume, which has a diameter as large as 1 mm, that can be examined at high resolution due to the consideration of cost and time. COM and CLSM images are useful in indicating the number of TEM sections required to be collected for determining the representative images of flocs.61

Nanoplast resin is particularly effective as a stabilization medium since it is a hydrophilic melamine embedding resin that holds the fibrillar EPS in native three-dimensional disposition. Nanoplast omits the solvent dehydration stage, and it forms cross-linkages between matrix colloids prior to structural water loss at the end of the embedding process. Measurements of the dimensions of colloidal matrix material and their 3-D disposition are realistic. Nanoplast has been recently shown to be useful for stabilizing sediment biofilms and the EPS matrix of these structures for observation by CLSM.77 The environmental scanning electron microscope (ESEM) permits observations of fully hydrated samples but there have been no published reports to date that describe microbial flocs using ESEM, except a few reports using ESEM information as an aid for interpreting other observations.76

CLSM is one of the most recent microscopic techniques used to study flocs and has been shown to be a useful technique in bridging the resolution gap between COM and TEM.23,61 Images are scanned with a laser beam and collected in a point-by-point fashion by a photodetector system.78 These collected images are stored in the computer memory for further image processing and analysis. The advantages of CLSM over conventional light microscopy include the reduction of image blurring caused by light scattering, with a concomitant increase in effective resolution. CLSM also allows the examination of a thick specimen such as animal tissue and biological flocs by scanning a series of planar images (X—Y plane) along a vertical axis (Z) one at a time. These series of planar images can be reconstructed in a computer imaging analysis system into a 3D image of the sample.21-23,61 Floc in excess of 500 \xm can be visualized by CLSM. Two photon, or multiphoton laser scanning microscopy (2P-LSM) permits examination of floc and films approaching 1mm in thickness while minimizing photobleaching and phototoxicity. A particularly useful feature of CLSM and 2P-LSM is that these can be used in combination with a variety of fluorescent molecular probes to study the spatial distribution of extracellular polysaccharides, cell viability, pH gradient, proteins, RNA, lipids, and other components of floc nondestructively.77,79 These techniques are increasingly being adapted by researchers to further our understanding of flocs and the relationship between structure, ecology, and the physicochemical properties of flocs and films.23

Other advanced microscopic methods including Raman confocal microspectro-scopy can potentially provide insights into the chemical composition of microbial cells and the heterogeneity associated with spatial distribution associated with structure and the conditions of growth.80,81 Atomic force microscopy (AFM) has emerged as a tool that can provide detailed information on topography of microbial surfaces as well as probing surface properties.82,83 Through the interaction of the AFM tip and the microbial surface, attractive and repulsive forces can be explored (see Chapter 16). Functionalizing the tip with molecular specific probes is a further advance that permits more detailed examination of molecular interactions. Synchrotron-based scanning transmission x-ray microscopy (STXM) has recently been used correlatively with TEM and CLSM on a riverine biofilm to gain high resolution information on the three-dimensional distribution of specific classes of extracellular polymers.84

1.3.2 Extracellular Polymeric Substances

In aqueous environments, bacteria may invest a substantial amount (>70%) of their carbon and energy in the production of extracellular polymeric substances,77,85 indicating an important role of EPS in the functioning of microbial communities and the structures they form. Over the past 20 years, studies have emphasized the composition of the EPS based on analyses of whole sludge or the extracted EPS. More recently, investigators are beginning to focus on detailed investigation of the surface properties and EPS through direct examination, by microscopy, of the whole biopolymeric material rather than the extracted EPS as has been done previously86 (see Chapter 6).

EPS can be classified as capsular EPS (bound) and slime EPS (soluble). The bound EPS is attached tightly to the exterior cell wall, while the soluble EPS is the loosely attached or unattached 'slime' material that can be washed away by centrifugation.87,88 In order to analyze the composition of bound EPS without inducing cell lysis, a variety of extraction methods have been developed. Brown and Lester89 have compared bacterial EPS extraction methods from other sources including chemical methods such as ammonium hydroxide, sodium hydroxide, EDTA, sulfuric acid, and boiling benzene. Mechanical extraction methods such as high-speed centrifugation, ultrasonication, and boiling or autoclaving were also investigated. The influence of various separation and extraction processes, on the constituents and quantities of EPS being extracted, has been reviewed recently, along with a comparison of the analytical measures in common use.90

Cation exchange resin (CER), utilizing both a mechanical and chemical means of extraction, has been found to be effective in terms of minimal cell lysis and nondis-ruptive effects on the EPS.91,92 This extraction method removes divalent cations such as Ca2+ and Mg2+ from the EPS matrix and replaces them with monovalent cations. By removing the divalent cations the EPS becomes less stable thereby allowing the EPS to separate from the cellular material. Subsequent to capsular EPS extraction, proteins,93 carbohydrates,94 acidic polysaccharides,95 and DNA can be measured.

Although quantitative estimations of EPS in microbial structures have been traditionally accomplished through extraction and chemical methods, fluorescent probes are suitable for estimating EPS in situ. In the past, Calcofluor White M2R was used to measure exopolysaccharide production in single bacterial strains such as Azospirillum' Pseudomonas aeruginosa, and Klebsiella pneumoniae.96,97 Similarly, congo red was employed for general light microscopy staining of polysaccharides.98 More recently, researchers have begun to use fluorescently labeled lectins as a method to probe the spatial relationships of EPS within thick heterogeneous microbial structures.99-102 Lectins are a large group of glycoproteins that bind to specific carbohydrates. They are prevalent in nature and are present in plants, bacteria, animals, and humans.103 Plant lectins have been used as both specific and general stains to estimate EPS, as well as to characterize the EPS left on surfaces after the removal of microbes.99-101,104

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