Enzyme Activity Associated With Microbial Flocs

A number of the studies, including those cited above suggest that the majority of exoenzyme activity detected in the activated sludge process of various wastewater treatment plants is associated with the organic particulate fraction, comprised primarily of flocs derived from the activated sludge process. Protease, PO4ase, lipase, and esterase activities partition strongly with the particulate phase.15,37,41 By separating activated sludge into different fractions using centrifugation, filtration, and sonica-tion, Goel et al.36 determined the activity for alkaline and acid PO4ase, a-glucosidase, and protease that was associated with bacterial cells, free in the bulk solution, and loosely associated with cells or entrapped in the flocs. A major fraction of the total enzyme activity was found to be associated with the flocs. Richards et al.44 and Frolund et al.39 also found that exoenzymes were immobilized in the sludge floc matrix.

Cadoret et al.12 measured extracellular enzyme activities in whole sludge, in dispersed activated sludges, as well as that associated specifically with the floc EPS. The activated sludge from a treatment plant was treated as follows before being assayed as "whole" sludge for enzyme activities: sludge was allowed to settle, then resuspended in deionized water, then homogenized, and the process repeated. Dispersed activated sludge was first prepared as "whole" sludge before subjecting to sonication or cation exchange resin (Dowex-Na+ 50 x 8). EPS was recovered from the supernatant fraction of the centrifuged aqueous phase following removal of the cation exchange resin and associated adsorbed material. They found that 17% of the L-Leu-aminopeptidase, 5% of a-glucosidase, 23% of protease, and 44% of a-amylase activity of sludge dispersed by sonication or cation exchange resin were recovered in the EPS fraction. Most of the enzymes remained associated either to tightly bound EPS or directly bound to the bacterial cell surfaces.

Whiteley et al.37 determined the PO4ases and protease activity in the supernatant fraction of liquor recovered from methanogenic and sulfidogenic sewage sludge reactors following incubation with substrates selective for these enzymes, sonic-ation, and centrifugation. The majority of the exoprotease and PO4ase activity was found associated with the particulate fraction that was released upon sonication. Most enzyme activity was therefore found either associated with or immobilized within the particulate matter.

Pletschke et al.38 determine ATPS activity in the supernatant fraction of liquor recovered from methanogenic and sulfidogenic sewage sludge reactors following sonication and centrifugation. Since >90% of the enzyme activity was released into the supernatant after sonication, it was suggested that the ATPS enzymes were located either intracellularly or immobilized in or on the floc material.

Boczar et al.15 assayed esterase activity in the particulate fraction of activated sludge using substrates of different chain lengths. C4-ester yielded the highest activity of the different substrates tested, with activity decreasing with increasing chain length above C4. The p-nitrophenol esters were hydrolyzed at higher rates than fluorescein esters. Esterase activities determined after freeze-thaw treatment suggested that a significant portion of the fluorescein esters was hydrolyzed by enzymes located on the cell surface or within the cell envelope. In general, the bulk of the esterase activity was associated with the particulate flocs with no significant amount of activity detectable in the bulk solution. Similar results were reported by Teuber and Brodish45 who found that 75% to 99% of the PO4ase, glucosidase, and aminopepti-dase activity was associated with sludge material that pelleted upon centrifugation at 3000 x g.

Van Ommen Kloeke and Geesey41 used a precipitating fluorogenic enzyme substrate ELF-PO4 (Figure 14.1) to localize PO4ase activity in the activated sludge liquor. Since this substrate is converted to a water-insoluble, crystalline, yellow, fluorescent product at the site of organic-P hydrolysis, the position of the resulting fluorescent objects identifies the site of active PO4ase enzymes when viewed by fluorescence microscopy (Figure 14.5). It is evident from the distribution of fluorescence that the PO4ase activity is localized in discrete locations throughout the matrix of the floc particles, possibly reflecting the distribution of PO4ase-producing microorganisms. Some areas are as large as 40 xm in diameter (Figure 14.5, inset), although most are on the order of 5 ¡xm in diameter. The intensity of the fluorescence emanating from these local regions in the floc particles increased linearly over a 40-min period (Figure 14.6). Incubations of 60 min allowed the development of maximum density of ELF crystals before increasing crystal size, and fluorescence intensity caused the fluorescent image of adjacent crystals to merge into a single fluorescent object. Sixty-minute incubations were thus used to determine the density and distribution of PO4ase activity in the flocs.

Frolund et al.39 showed that a large portion of various classes of exoenzymes present in activated sludge were adsorbed to the EPS matrix. When the EPS was treated with a cation exchanger, large quantities of enzyme were released into the bulk aqueous solution. Since the activities of these extracellular enzymes strongly partition with the floc-containing fraction, these enzymes likely have evolved to remain associated with the cell surface or the floc matrix (Figure 14.7). Flocs provide a conditioned environment conducive for extracellular enzyme accumulation and activity outside the cell. Goel et al.36 showed that the floc-bound nature of extracellular alkaline PO4ase, acid PO4ase, a-glucosidase, and protease enabled the enzymes to remain active under anaerobic as well as aerobic phases of operation. Recently, it has been shown that the EPS of activated sludge aggregates inhibited the diffusion of high molecular weight substrates of extracellular enzymes, limiting their availability to the enzymes and the rate of catalysis.12 These results challenge the idea that hydrolysis of the organic polymers is the first and rate-limiting step in the activated sludge process, and support the suggestion by Guellil et al.46 that sorption or uptake of the organic polymers by the exoenzyme-containing floc is a rate-limiting step that precedes hydrolysis (Figure 14.7). Thus, the benefits provided to exoenzymes by EPS



FIGURE 14.5 Epifluorescence microscopic image of ELF crystals precipitated in floc particles recovered from activated sludge and incubated with 100 /M ELF-P for 60 min. (a) Crystals distributed throughout floc matrix. Arrow shows crystal with diameter of approximately 40 /m. (b) Crystals concentrated in one floc particle at densities higher than in adjacent particles.

are, to some extent, offset by the added diffusion resistance the EPS exerts on the enzyme substrates.

Floc formation requires expenditure of energy generated by the bacteria in the system. The main source of this energy is the polymeric organic material in the wastewater aqueous phase that can only be accessed by the microorganisms if first degraded into useable subunits by exoenzymes (Figure 14.8). Since even the smallest

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