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Exoenzymes EPS

FIGURE 14.8 Schematic diagram of pathway of formation of microbial biomass, exoenzymes, and EPS. Polymer subunit intermediate products have different properties than original polymeric compounds. Production of intermediate products may affect P removal efficiency.

floc particles resolved by light microscopy contained regions of ELF fluorescence, exoenzyme production and secretion likely occurs during or possibly even prior to floc formation in the system.

When floc material was homogenized in a tissue homogenizer to disperse bacterial cells, and the preparation incubated with ELF-P, approximately 9% of the cells that were visualized after staining with the nucleic acid stain acridine orange had ELF crystals precipitated in their immediate vicinity.41 PO4ase activity was thus contributed by a small fraction of the floc-associated bacterial community. Of the total area of fluorescence produced by all crystals of ELF evaluated in homogenized floc preparations, approximately 80% was contributed by crystals associated with cells that also reacted positively with SYTO9, a fluorochrome that is taken up by cells with a membrane potential, and thus considered viable.41 The other ELF crystals could not be associated with any other objects in the floc. PO4ase activity was therefore primarily associated with active bacterial cells in the floc.

14.7 IDENTIFICATION OF POPULATIONS OF CELLS RESPONSIBLE FOR EXTRACELLULAR PO4ASE ACTIVITIES WITHIN FLOC-ASSOCIATED MICROBIAL COMMUNITIES

Considerable progress has been made in recent years in identifying the types of bacteria present in activated sludge systems. Besides the poly-P-accumulating bacteria of the genus Acinetobacter, those aligning with the cytophaga-flavobacteria group have also been widely reported in activated sludge systems in different parts of the world.28,47-49 Snaidr et al.50 reported that the cytophaga-flavobacteria group contributed 12% of the DAPI-stained bacterial community of an activated sludge system. Manz et al.47 found a similar fraction of the bacteria present in activated sludge that hybridized with the Bacteria-specific probe EUB338 probed positive with a cytophaga-flavobacter-specific 16S rRNA oligonucleotide probe. The extent to which these groups of bacteria are responsible for extracellular enzymatic activities measured in activated sludge floc has only recently been assessed.41,51

The detection of the cultivable bacterial populations in homogenized activated sludge floc preparations that exhibit extracellular PO4ase activity was facilitated by the development of a new screening technique.51 Adding the precipitating fluorogenic substrate 2-(5'-chloro-2/-phosphoryloxyphenyl)-4-[3H]-quinazolinone (CPQ-PO4) to a bacterial cultivation medium, after medium sterilization but prior to solidification, offered the opportunity to detect PO4ase active colonies following inoculation and incubation of samples of homogenized activated sludge floc preparations plated on this medium (Figure 14.9). Upon enzymatic cleavage, this molecule yields the stable, highly fluorescent precipitate 2-(5'-chloro-2/-hydroxyphenyl)-4-[3H]-quinazolinone (CPQ)52 (Figure 14.9). CPQ excites at 345 nm and fluoresces at 530 nm.51 Phosphatase activity can then be monitored as the colonies grow by viewing the plates under UV light. Another benefit of

FIGURE 14.9 Fluorogenic substrate (a) 2-(5/-chloro-2/-phosphoryloxyphenyl)-4-[3H]-quinazolinone is hydrolyzed to (b) 2-(5/-chloro-2/-hydroxyphenyl)-4-[3H]-quinazolinone to produce a water-insoluble fluorescent precipitate.

FIGURE 14.9 Fluorogenic substrate (a) 2-(5/-chloro-2/-phosphoryloxyphenyl)-4-[3H]-quinazolinone is hydrolyzed to (b) 2-(5/-chloro-2/-hydroxyphenyl)-4-[3H]-quinazolinone to produce a water-insoluble fluorescent precipitate.

this approach is that it distinguishes between activity of secreted (bacteria-free) and cell-associated PO4ase activity, due to the precipitating nature of the probe. Colonies actively secreting PO4ase enzyme into their environment have a fluorescent halo, whereas those PO4ase-positive colonies which have only cell-associated activity do not produce a halo.51 Using this approach, these investigators found that 35% of the cfus that grew on LB/HEPES/CPQP agar medium yielded an intense yellow-green fluorescence, indicating the presence of phosphatase-positive clones (Figure 14.10).

The PO4ase-positive colonies from the homogenized activated sludge preparations displayed two types of fluorescence when plated on the above medium. Some colonies displayed fluorescence that was limited to the area occupied by the colony, whereas, fluorescence displayed by other colonies extended into the surrounding medium producing halos around the colonies. Of all fluorescent colonies obtained on the plates, 36% displayed halos. Isolates displaying these different forms of PO4ase activity on solid medium are shown in Figures 14.11 and Figure 14.12. Isolate A produced a fluorescent halo around its colony (Figure 14.11), whereas Isolate J produced fluorescence that was restricted to the colony (Figure 14.12). Isolates A and J were further evaluated for the distribution of PO4ase activity in suspended batch cultures without added agar. Whereas 36% of the total PO4ase activity produced by Isolate A was recovered with the cell fraction, with the remainder free in the culture menstruum, 99% of the total PO4ase activity produced by Isolate J was recovered with the cell fraction.51 PO4ase activity was induced when cultures of both isolates entered the late exponential growth phase.

Selective identification of PO4ase-positive colonies of bacterial populations in complex microbial communities makes it feasible to sequence the 16S rDNA gene to establish a phylogenetic affiliation for the population. When the DNA of cells from several of the fluorescent colonies recovered from plates inoculated with

(b)
FIGURE 14.10 Colonies formed on LB/HEPES/CPQ-PO4 solid medium after inoculation with activated sludge from a wastewater treatment plant. (a) Colonies under normal white light and (b) CPQ fluorescent, PO4ase-producing colonies.

homogenized activated sludge was extracted and a portion of their 16S rDNA amplified by polymerase chain reaction (PCR) using primer set 1056F/1392R, the resulting amplicons, when sequenced, grouped with the cytophaga-flavobacteria in the phylum cytophaga-flavobacter-bacteroides.41 Three of the isolates reacted positively with the cytophaga-flavobacteria group-specific rRNA probe CF319a. The

FIGURE 14.11 (Color Figure 4.11 appears following page 236.) Colonies of Isolate A growing on LB/HEPES/CPQ-PO4 solid medium. Note halo of fluorescent CPQ that has precipitated some distance away from the colonies reflecting diffusion of PO4ase away from the cells and the colony.

CF319a probe reacted positively with approximately 10% of the DAPI-stained bacteria and 17% to 20% of the ELF-precipitating bacteria associated with homogenized floc material from activated sludge samples.41 These results suggest that although the cytophaga-flavobacteria group resists efforts to cultivate in the laboratory, it is an important contributor of PO4ase activity in activated sludge. Using a combination of molecular approaches, it is possible to establish structure-function relationships among uncultivated members of complex microbial communities such as those established in flocs of the activate sludge process of wastewater treatment.

FIGURE 14.12 (Color Figure 4.11 appears following page 236.) Colonies of Isolate J growing on LB/HEPES/CPQ-PO4 solid medium. Note the fluorescent CPQ that has precipitated remains close to the perimeter of the colonies indicating that the PO4ase enzymes remain closely associated with the cells in the colonies.

FIGURE 14.12 (Color Figure 4.11 appears following page 236.) Colonies of Isolate J growing on LB/HEPES/CPQ-PO4 solid medium. Note the fluorescent CPQ that has precipitated remains close to the perimeter of the colonies indicating that the PO4ase enzymes remain closely associated with the cells in the colonies.

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