The function-structure relationships of flocs are important to environmental scientists, microbiologists, and engineers. Ultimately, their goals include being able to solve practical problems more effectively, and to provide better information for modeling ecological processes and contaminant transport in aquatic environments and in the operation of engineered systems (e.g., wastewater and drinking water). Methods and analytical tools play a critical role in floc research and in achieving these goals. These are intended to do one of two things: (i) to provide descriptive and quantitative information that may lead to a fuller understanding of flocculation and (ii) to have tools that may be applied to the management of floc processes in engineered and environmental systems.

At present, few standard methods with good reproducibility are available, although several physical, chemical, and microbiological measurement and analytical techniques have been developed. Earlier reviews give a comprehensive review of the methods and techniques for the measurement of physical characteristics for activated sludge1 and an overview of the principles, methods, and applications of particle size analysis in primarily saltwater systems.2 Eisma et al.3 and Dyer et al.4

©2005 by CRC Press 1

conducted a comparative study in the Elbe estuary to evaluate several different in situ methods for determining floc size and settling velocity. More recently, several entries in the Encyclopaedia of Environmental Microbiology5 provide overviews of methods, particularly advanced optical microscopy and molecular tools applied to the study of microbial structures including flocs.6 Common to all these reviews is the wide range of methods employed to determine some of the most basic of parameters that describe flocs in the environment.

In engineered systems, advances have been achieved primarily in studying floc properties (ecology, structure, andphysicochemical characteristics) of individual flocs from full-scale systems and from laboratory-scale reactors that were run under well-controlled conditions. In contrast, studies in the marine and freshwater environments have concentrated on bulk properties such as gross morphology, size, and settling velocity in samples collected with an emphasis on in situ measurements. One reason for the difference between measurements in the natural environment and engineered systems is the availability of flocs and the ease with which they can be sampled intact. Those involved with studying natural systems have tended to focus on the gross properties and behavior of floc. Engineered systems are suited to detailed examination of surface properties and molecular determinants in floc behavior.

In this chapter, we present an overview of the principal methods presently being used in engineered, freshwater, and marine systems. Some aspects of the methods presented can be applied to both natural and engineered systems. Our goal is to provide an insight into the work being carried out in the different aquatic environments so that researchers can consider adapting the techniques presented to their respective fields.

1.1.1 Floc Size

Floc size is a widely measured floc characteristic. Floc size influences properties such as mass transfer (transport and settling),7 biomass separation, and sludge dewatering.8-10 Flocs are generally observed as two-dimensional (2D) projections, and there is no simple means of specifying size or shape.11 However, flocs are highly irregular in shape, porous, and three-dimensional. Equivalent spherical diameter (ESD), frequently calculated from the two-dimensional area, is often used to characterize floc size due to its simplicity and its application in Stokes' law.11-13 Bache et al.11 defined the effective diameter as the geometric mean (dmn*dmax) based on the maximum (dmax) and minimum (dmjn) dimensions across the 2D floc image. Barbusinski and Koscielniak14 and Li and Ganczarczyk15 described floc size based on the average floc diameter defined as one half of the sum of the longest and shortest dimensions of the flocs measured.

Flocs in suspension are found over a range of sizes that describe a continuous distribution. Several standard parameters are available to describe floc size distributions. Median (d50), upper quartile (d25), and mode have all been used to describe the size distribution of flocs in suspension.13,16,17 Due to the open architecture and poorly defined association of particles within a floc, researchers use fractal geometry to describe floc structure.18-23 Depending on the nature and the sizing technique employed, there is no evidence to show which definition is the best representation of floc size. However, researchers should be clear in their definition of floc size when reporting results.

In general flocs range in size from a few microns to a few millimeters when measured by ESD. One exception is large assemblages of diatoms or other biologically derived material. Sometimes referred to as marine snow to differentiate it from more inorganic rich flocs, these patches of aggregated organic material can reach ESDs many orders of magnitude larger than what could be considered normal flocs. When marine snow becomes buoyant during decomposition, as was observed in the Adriatic Sea during the mucilage phenomenon, "floc size" can exceed 1 m.24-27

Many methods and instruments have been developed in the past to measure floc size distributions in natural and engineered systems. One of the earliest methods was the Coulter Counter, which determined the size distribution of particles in suspension. This method was popular in the marine environment as the electrolyte concentration in seawater permitted samples to be analyzed without alteration. However, stresses applied during the counting process can disrupt flocs which raises the issue that this method may be of little value for estimating floc size.28,29

The determination of floc size has relied primarily on imaging of flocs followed by image analysis to ascertain the parameters describing the size distribution.12,30,31 Both microscopic observations and photographic techniques13,32-35 have been used. In situ photography of flocs, although relatively easy to employ, does not allow measurement of very small flocs due to resolution limits. Often these systems can only image down from 50 to 100 ^m, although a 10:1 camera system with a resolution of 10 ^m has been developed.36,37 Recent advances in digital photography should improve the resolution of in situ camera systems. The main advantage of these instruments is their ability to measure floc size with minimal disturbance to the natural stress environment of the flocs. However, they were developed for the natural environment, and may be difficult to apply in an engineered system as they are limited by the concentration of particles in suspension.

Microscopic methods usually incorporate a camera and computerized digitizer to provide images for analysis. The increased resolution of microscopic systems allows for accurate, reproducible, and relatively fast estimates of floc morphological parameters. Specialized techniques such as confocal microscopy and electron microscopy (discussed in detail later in the chapter) allow the internal structure of flocs to be examined. The obvious drawback for microscopic analysis is the requirement to remove flocs from their natural environment and the associated instrument costs.

Common to both photographic and microscopic methods is the requirement to conduct image processing and analysis on the captured image to determine floc size and other descriptive parameters. Image processing and analysis comprises several steps.38 Different algorithms are applied to the digital image to improve the quality of the image and to separate a floc from its background. Each area of coherent pixels with values within a selected range of threshold values is then used to calculate the different parameters used to describe floc size. There are differing views on the number of pixels required to define a particle with values ranging from 3 to 35 pixels.39,40 Several different image analysis systems are available on the market but all are based on the same principles for manipulating a matrix of pixel values. Clear explanations of the methods employed in the analysis are critical for understanding how descriptive parameters such as ESD are generated.

Laser based sizing instruments are now being widely used to determine floc size in situ.41'42 Two different laser techniques have been used, focused beam reflectance measurement (FBRM) and laser diffraction. FBRM instruments (modified ParTec) employ a rotating laser beam to determine the size of particles in the sensing zone.41 When the laser encounters a particle, the beam is reflected for the period of time it takes to traverse the particle. Using the angular velocity of the beam and the duration of the reflected laser pulse, the length of the intersecting particle chord is determined. A chord correction algorithm is then used to determine the size distribution of the particles in suspension. FBRM instruments were designed for process control and are not easily adapted to studies in natural systems. However, they do have the advantage of working at higher concentrations than instruments that rely on light transmission.41

Laser diffraction instruments were first used by Bale and Morris,43 who modified a Malvern particle size analyzer (Malvern Instruments, U.K.) for underwater use. Since then purpose-designed laser diffraction systems have become available, notably the LISST (Sequoia Scientific Inc., WA, United States) and the CILAS (CILAS, France). Laser diffraction instruments are based on the scattering of laser light by particles as the beam transits a known sample zone. The scattering angle is determined by the size of the particle with the scattering angle being small for large particles and large for small particles.42 A series of concentric ring detectors sense the amount of light they are receiving. Using the Mie or Fraunhofer theory of scattering for spheres, these values can then be inverted to yield the particle size distribution.42

Floc size has also been inferred from the settling behavior of flocculated suspensions.4,44 Settling column methods in general measure the equivalent hydraulic diameter of particles in suspension rather than the actual physical size of the suspended particles. Floc size is expressed in terms of the diameter of a sphere with the density of quartz, settling at the same speed as the particle in question.45

1.1.2 Sample Handling and Stabilization

In situ measurement of floc size is clearly preferable due to the fragile nature of flocs. Sample handling may break up existing flocs or promote formation of larger flocs during storage.46 Gibbs and Konwar47 showed that common sampling methods disrupt flocs. Critical to any work with flocs outside their natural environment is sample handling and preparation. The need for microscopic examination of flocs and for laboratory experiments with natural flocs has led to the development of new techniques for removing flocs from their ambient conditions with minimal change in floc size or structure. Considerable efforts have been given to overcome perturbation that may be associated with sampling and specimen preparation.

For floc size measurements not performed in situ, samples are collected in bulk suspension and transported to the laboratory for sizing. Essential to this first step is minimizing the stress applied to the flocs during sampling. Droppo and Ongley12 employed traditional laboratory-used plankton chambers within fluvial systems. By using the plankton chamber as both the sampling and analytical chamber for image analysis, potential perturbations are minimized. Depending on the sizing methods, further floc sampling might be required. Some size measurements using image analysis systems or microscopic observation require subsampling of flocs onto microscope slides. This is normally done using a pipette for which the opening has to be wide enough (2 to 3 mm) to prevent floc breakage and disaggregation.48

Floc stabilization prior to further sample handling has been shown to be effective in preserving floc structural characteristics. Droppo et al.49'50 described a method of utilizing low melting point agarose to physically stabilize microbial flocs before analysis. This technique was found to have no significant effects on floc size distributions. Ganczarczyk et al.51 used a similar approach in physically stabilizing microbial flocs. Optically clear polyacrylamide gels have been used in marine sediment traps to capture flocs intact for later processing.52

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