Characterization and Distribution of Esterase Activity in Activated Sludge

Abstract

The location and activity of esterase enzymes in activated sludge from three municipal wastewater treatment plants were characterized using model substrates and denaturing and non-denaturing polyacrylamide gel electrophoresis (PAGE) of particulate, freeze–thaw (primarily periplasmic enzymes and those associated with outer cell surfaces) and extracellular fractions of activated sludge bacteria. Particulate and freeze–thaw fractions had a similar spectrum of substrate specificity and contained significant levels of protein and esterase activity against model substrates, C₂–C₁₈ monoesters of p-nitrophenol and C₂–C₈ diesters of fluorescein. Esterase activity was highest with substrates that had short alkyl chains (C₄) and decreased as the chain lengths increased beyond C₈. Extracellular fractions contained very low levels of protein (<0.1 mg/l) and showed no esterase activity against any of the model substrates tested. Multiple bands were observed upon analysis of particulate and freeze–thaw fractions by non-denaturing PAGE in combination with activity staining using various α-naphthol ester substrates (C₂–C₈). Our results indicate that esterase enzymes in activated sludge are fairly diverse from a structural standpoint but exhibit a high level of functional redundancy, with different enzymes catalyzing the same reactions in different sludges. Extracellular esterase activity was totally absent for the substrates we tested and the esterase activity that we observed was closely linked to a particulate floc or cellular material.

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.