Evaluation of extracellular, high-affinity β-N-acetylglucosaminidase measurements from freshwater lakes: An enzyme assay to estimate protistan grazing on bacteria and picocyanobacteria

Abstract

Protistan community grazing rates upon both bacterioplankton and autotrophic picoplankton were estimated using fluorescently-labeled prey and by measurement of extracellular hydrolysis of 4-methylumbelliferyl (MUF) β-N-acetylglucosaminide in a eutrophic reservoir and an oligo-mesotrophic lake during phytoplankton blooms. In addition, enzyme methods were optimized in bacterivorous flagellate cultures by two enzyme assays, based on fluorometric detection of protistan digestive activity, which were compared and calibrated independently against flagellate bacterivory. Enzymatic hydrolyses of MUF β-N,N′,N″-triacetylchitotriose and MUF β-N-acetylglucosaminide were measured in cell-free (sonicated) and whole-cell (unsonicated) samples. The hydrolysis of both substrates, using the whole-cell enzyme assay at in situ pH, was correlated significantly with total grazing rate of Bodo saltans. Thus the whole-cell enzyme assay with MUF β-N-acetylglucosaminide was used for freshwater samples. High-affinity (K _m < 1 μmol 1⁻¹) and low-affinity (K _m > 100 μmol 1⁻¹) enzymes were distinguished kinetically in most samples from both systems studied. Activities (V _max ) of the high-affinity enzyme varied from 0.24 to 1.43 nmol 1⁻¹ h⁻¹. Protistan community grazing on bacterioplankton was in the range of 0.15–1.36 μg C 1⁻¹ h⁻¹. both for lake and reservoir, the differences being observed in grazing on picocyanobacteria (lake, 0.03-0.22 μg C 1⁻¹ h⁻¹. reservoir, 0.35–1.56 μg C 1⁻¹) h⁻¹. The enzyme activities were correlated significantly with the protistan grazing both on bacterioplakton (r _s = 0.62, P < 0.001) and total procaryotic picoplankton (the sum of organic carbon grazed from bacteria and picocyanobacteria, r _s = 0.73, P < 0.001) in the eutrophic reservoir. Weaker relationships (r _s = 0.42) with a lower slope were found for the oligo-mesotrophic lake. Ingestion rate studies are time-consuming and the digestive enzyme assay with MUF β-N-acetylglucosaminide presents a rapid alternative for estimating total protistan prokaryotic picoplanktivory in freshwaters.