Characterization and Distribution of Esterase Activity in Activated Sludge
Introduction
During the activated sludge process, complex communities of microorganisms utilize both intracellular and extracellular enzymes to hydrolyze and ultimately mineralize organic chemicals. Previous workers have used enzymes in activated sludge as indicators of specific populations (Hankin and Sands, 1974), measures of active biomass (Teuber and Brodish, 1977; Richards et al., 1984) and indicators of specific processes such as COD and phosphorus removal (Richards et al., 1984; Lotter and van der Merwe, 1987). However, with a few notable exceptions (Nybroe et al., 1992; Confer and Logan, 1998), information concerning the amount, activities, substrate specificity and cellular localization of hydrolytic enzymes in activated sludge is lacking.
Enzymes such as esterases, lipases, proteases that hydrolyze ester bonds are ubiquitous in the environment, generally have broad substrate specificity and act on a wide variety of natural and xenobiotic substrates. They are easily assayed and have been used as analytical tools to monitor dietary lipid (Van Lith et al., 1989) and to measure microbial abundance and activity in soil, lakewater, wastewater and activated sludge samples (Schnurer and Rosswall 1982; Federle et al., 1986; Chrost, 1989; Boczar et al., 1992; Nybroe et al., 1992). In many of these studies, model substrates such as esters of p-nitrophenol, a-naphthol, fluorescein and the LRA-ZYM system were used to assay esterase activities. The objective of this study was to characterize the variability in activity, location and substrate specificity of esterases in three wastewater treatment plants that receive different ratios of domestic and industrial wastewater. Esterases play an important role in the biodegradation of many natural substances in wastewater including lipids, proteins and as well as many synthetic chemicals. p-nitrophenol and fluorescein esters with alkyl side chains of different lengths were used as model substrates to measure total esterase activities. In addition, polyacrylamide gel electrophoresis (PAGE) using a-naphthol esters was used to characterize the functional diversity, distribution and substrate specificity of these enzymes in the three wastewater treatment plants.
Section snippets
Chemicals
Fluorescein diesters of acetate, butyrate, caproate and caprylate as well as p-nitrophenol esters and α-napthol esters of acetate, butyrate, caproate, caprylate, caprate, laurate, myristate, palmitate and stearate were obtained from Sigma (St. Louis, MO). 14C-1-alkyl-N-Octadecyl-N-[(palmitoyloxy)-ethyl]-N,N-dimethyl ammonium chloride (DSDMAC monoester) was synthesized by Procter and Gamble with a specific activity of 29.1 mCi/mg and a radiochemical purity >94.8% (D.R. Walley, unpublished data).
Activated sludges
Protein levels and patterns in activated sludge samples
Esterase activity was characterized in extracellular, freeze–thaw, and particulate fractions prepared from activated sludge samples collected from three wastewater treatment plants (WWTPs). The three WWTPs received different proportions of domestic and industrial wastewater. One WWTP, Colerain Heights, was a small residential facility that received 100% domestic wastewater. The second WWTP, Sycamore, was a medium-sized residential facility with >95% of the wastewater from domestic sources. The
DISCUSSION
Experiments with model substrates and an ester containing quaternary ammonium surfactant demonstrated that the esterase activity in three activated sludge plants was localized in the particulate flocs of the sludge. This lack of significant extracellular activity might not be expected for activated sludge communities that actively metabolize polymeric substrates, but it is consistent with a selective pressure for bacteria to keep their enzymes in close proximity so that they can directly
CONCLUSIONS
This work indicates that gel electrophoresis and the use of model substrates can provide valuable quantitative information on the distribution and activity of esterase enzymes in activated sludge. In general, esterase activity in activated sludge exhibits a high level of functional redundancy and is closely associated with the particulate floc or cellular material. No extracellular esterase activity was detected against any of the substrates we tested, and esterase activity in freeze–thaw
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