Abstract
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 > β-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
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Data are available from the authors upon reasonable request.
References
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Acknowledgements
The authors acknowledge the assistance and fruitful discussions by Dr. Reinhard Bierl, Lara Schmitgen, and the research meeting group members of the Department of Hydrology, University of Trier. We thank Björn Klaeß and Dr. Oscar Baeza-Urrea, Department of Geology, University of Trier, for taking the SEM images. We would also like to thank Mrs. Zwartenkot for proofreading. Finally, we would like to thank the editor and two anonymous reviewers whose comments have substantially improved the manuscript quality. Experimental work received financial support by the research fund of the University of Trier.
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KF, TS, and MR conceived and designed this methodological study as well as its application. KF and TS supervised the whole research project. The execution and validation of the experiments were done by MR. MW gave assistance in sampling and for the initial lab procedure. MR performed formal data analysis and visualization of data and wrote the first draft of the manuscript. All authors read and approved the final manuscript.
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Riese, M., Schuetz, T., Wacht, M. et al. Non-destructive investigation of extracellular enzyme activities and kinetics in intact freshwater biofilms with mineral beads as carriers. Appl Microbiol Biotechnol 106, 425–440 (2022). https://doi.org/10.1007/s00253-021-11712-1
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DOI: https://doi.org/10.1007/s00253-021-11712-1