Non-destructive investigation of extracellular enzyme activities and kinetics in intact freshwater biofilms with mineral beads as carriers.

Current procedures for fluorometric detection of extracellular hydrolytic enzyme activities in intact aquatic biofilms are very laborious and insufficiently standardized. To facilitate the direct determination of a multitude of enzymatic parameters without biofilm disintegration, a new approach was followed. Beads made of different mineral materials were subjected to biofilm growth in various aquatic environments. After biofilm coating, the beads were singly placed in microplate wells, containing the required liquid analytical medium and a fluorogenic substrate. Based on fluorometric detection of the enzymatically generated reaction products, enzyme activities and kinetics were determined. Mean enzymatic activities of ceramic bead-attached biofilms grown in a natural stream followed the decreasing sequence L-alanine aminopeptidase > L-leucine aminopeptidase > phosphomonoesterase > ß-glucosidase > phosphodiesterase > α-glucosidase > sulfatase. After one week of exposure, the relative standard deviations of enzyme activities ranged from 21 to 67%. Sintered glass bead-associated biofilms displayed the lowest standard deviations ranging from 19 to 34% in all experiments. This material proved to be suitable for short-time experiments in stagnant media. Ceramic beads were stable during more than three weeks of exposure in a natural stream. Biofilm formation was inhomogeneous or poorly visible on glass and lava beads accompanied by high variations of enzyme activities. The applicability of the method to study enzyme inhibition reactions was successfully proven by the determination of inhibition effects of caffeine on biofilm-associated phosphodiesterase.Key points• Optimized method to determine enzymatic parameters in aquatic biofilms• Direct investigation of bead-bound biofilms without biofilm disintegration• Fluorometric detection offers high sensitivity and sample throughput.

Inhibition of the activated sludge-associated enzyme phosphatase by transition metal oxyanions.

Organic esters of phosphoric acid and other organophosphorous compounds are enzymatically hydrolyzed during wastewater treatment by microbial phosphoesterases, especially by phosphomonoesterase (phosphatase). For physiological reasons, the enzyme is inhibited by its main inorganic reaction product, ortho-phosphate. It is known that oxyanions of transition metals, resembling the molecular topology of ortho-phosphate, e.g. vanadate and tungstate, are more potent inhibitors for microbial alkaline phosphatase than phosphate. To proof this effect for activated sludge, a multitude of samples from a communal wastewater treatment plant was exposed at pH values from 7.00 to 8.50 to tungstate, vanadate, and molybdate. Inhibition effects were determined by a sensitive fluorimetric microplate assay and characteristic parameters (IC50 and IC20 concentrations) were deduced from modelled dose-response functions. Mean inhibitor concentrations (in brackets: ranges) causing 50% inactivation (IC50) at pH 7.50 were 2.5 (1.3-4.1) µM tungstate, 2.9 (1.6-5.5) µM vanadate, and 41.4 (33.6-56.7) µM molybdate. Vanadate and tungstate concentrations between 0.6 and 0.7 µM provoked a 20% (IC20) inhibition. The inhibition efficiency of tungstate and molybdate decreased with increasing pH, whereas vanadate reacted pH independently. These results underline the necessity to consider enzyme inhibition assessing the limitations and potentials of biological wastewater treatment processes.

A 1-year study of the activities of seven hydrolases in a communal wastewater treatment plant: trends and correlations.

The activities of seven hydrolytic enzymes (L-alanine aminopeptidase, esterase, α-and ß-glucosidase, phosphomonoesterase, phosphodiesterase, sulfatase) were monitored during 1 year in parallel and serial treatment units of the biological stage of a communal wastewater treatment plant. The spatial homogeneity of enzyme activities was high (coefficients of variation <10 % for the entire treatment stage). A significant difference between aerated and stirred tanks was not observed. Temperature seemed not to exert a direct influence. Long periods with comparably constant activities were interrupted by a few strong, short-time rises. The mean enzyme activities followed the sequence sulfatase < α-glucosidase < phosphodiesterase ≈ ß-glucosidase≈esterase < phosphomonoesterase < L-alanine aminopeptidase. The enzyme activities correlated among themselves at different levels. Very strong (r > 0.8) and highly significant (p < 0.01) correlations between the activities of both glucosidases, both phosphoesterases, and between phosphomonoesterase and both glucosidases were ascertained, pointing to the importance of substrate specificity and similarity of metabolic functions. Moderate and strong activity correlations with various wastewater constituents and with process parameters, e.g., concentrations, loads and eliminated amounts of phosphorous, TOC concentrations and loads of the plant effluent, dry matter content of activated sludge, and sludge volume, were found. The esterase activity was least correlated with other enzymes and often showed deviating dependencies on process parameters, raising questions concerning its appropriateness as a sum parameter for enzymatic and heterotrophic activity.

Fluorimetric microplate assay for the determination of extracellular alkaline phosphatase kinetics and inhibition kinetics in activated sludge.

The microbial enzyme alkaline phosphatase contributes to the removal of organic phosphorus compounds from wastewaters. To cope with regulatory threshold values for permitted maximum phosphor concentrations in treated wastewaters, a high activity of this enzyme in the biological treatment stage, e.g., the activated sludge process, is required. To investigate the reaction dynamics of this enzyme, to analyze substrate selectivities, and to identify potential inhibitors, the determination of enzyme kinetics is necessary. A method based on the application of the synthetic fluorogenic substrate 4-methylumbelliferyl phosphate is proven for soils, but not for activated sludges. Here, we adapt this procedure to the latter. The adapted method offers the additional benefit to determine inhibition kinetics. In contrast to conventional photometric assays, no particle removal, e.g., of sludge pellets, is required enabling the analysis of the whole sludge suspension as well as of specific sludge fractions. The high sensitivity of fluorescence detection allows the selection of a wide substrate concentration range for sound modeling of kinetic functions.•Fluorescence array technique for fast and sensitive analysis of high sample numbers•No need for particle separation - analysis of the whole (diluted) sludge suspension•Simultaneous determination of standard and inhibition kinetics.