ACTA UNIVERSITATIS PALACKIANAE OLOMUCENSIS FACULTAS RERUM NATURALIUM (1999) BIOLOGICA 37

ASSESSMENT OF EXTRACELLULAR ENZYME ACTIVITIES OF a- AND b-GLUCOSIDASE IN SEDIMENTS OF A SMALL LOWLAND STREAM (SITKA STREAM, CZECH REPUBLIC)
Martin Rulk & Robert Spil

Department of Ecology Hydrobiological Division, Faculty of Science, Palack University, lechtitel 11, 783 71 Olomouc, Czech Republic (e-mail: rulik@prfholnt.upol.cz; spacil@prfholnt.upol.cz) Received September 10, 1999; accepted October 24, 1999 Key words: Extracellular enzyme activity, -glucosidase, -glucosidase, hyporheic zone, interstitial water, bacteria, organic matter decomposition

Abstract Activity of -glucosidase and -glucosidase was measured fluorometrically in surface water, interstitial water and bed sediments of a small lowland stream. A very low level of hydrolytic activity was observed in the water column and interstitial water, as compared with the gravel-sand sediment. The values 1 1 of -glucosidase activity varied between 0.0021.2 mol.l .h for water samples and between 0.663.4 1 1 mol.g DM.h for sediment samples. The values of -glucosidase activity varied between 0.0021.7 1 1 1 1 mol .l .h for water samples and between 1.09.1 mol.g DM.h for sediment samples. Ratio between activity of -glucosidase and -glucosidase was found to be higher in the surface water than those from interstitial water and sediments. This supports the assumption that decomposition of cellulose mainly of allochthonous origin should dominate in the sediments of the Sitka stream.

Introduction Within a hyporheic zone, large quantities of dissolved and particulate organic matter are available to the heterotrophic bacteria (Crocker & Meyer 1987, Leichtfried 1988). This material is both allochthonous and autochthonous in origin. Most of the organic compounds have polymeric structure and they are too large to be readily assimilated. In order to be available for microbial metabolism, polymeric compounds must be transformed into smaller molecules through enzymatic depolymerization (Chrst 1990, Marxsen & Witzel 1991). The final step in the hydrolysis of starch and cellulose is the cleavage of maltose and cellobiose by -glucosidase and -glucosidase,

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respectively, to constituent monomers. Estimation of their activity in both the hyporheic sediments and interstitial water may be useful in order to characterize the structure, origin and spatio-temporal distribution of the assimilated organic compounds. The -glucosidase activity (GlcA) and -glucosidase activity (GlcA) ratio (GlcA/GlcA) seems to be a good indicator of the fate of organic matter originating from primary production (Vrba 1992). Hence, in accordance with our previous results (Rulk et al. 1999) we expect that this ratio should be low, indicating higher activity of -glucosidase and thus lead to a predominance of cellulose as a carbon source for heterotrophic bacteria. In this paper the first results of measurements of -glucosidase activity and glucosidase activity in surface water, interstitial water and bed sediments of a small lowland stream (Sitka) are presented with respect to the assumption above.

Materials and methods Sampling site Investigations were conducted in the Sitka, a small, unpolluted lowland stream above the city of Olomouc, in the Czech Republic, during the 1999 summer season. Stream width ranges between 4 & 6 m and the substrate is composed of gravel-sand. A pool-riffle-pool sequence of ca 20 m in longitudinal profile occurs at the sampling site. The stream is described in further detail by Rulk & Hekera (1998). Sampling Surface water was collected from the midstream lowering a borosilicate bottle slowly down through the water column. Interstitial porewater samples were collected with a vacuum handpump from piezometers installed at sediment depth of 2530 cm (Rulk et al., in print). Piezometers were placed at two different locations along a hydrologic flowpath through sediments (downwelling zone and upwelling zone). Samples were stored (4 C) in borosilicate bottles and processed within 2 h after collection. Sediment cores were taken with a plastic cylinder pressed into the substrate (010 cm in depth). The core was removed after supporting the lower end of the cylinder with a rubber stopper and the sediment was homogenized immediately in the field. We only considered sediment particle sizes < 1 mm, since most of the biofilm is associated with this fraction (Leichtfried 1988). Sediment samples were transported to the laboratory in plastic boxes, and processed no later than 2 hours after sampling. Measurement of enzyme activity Extracellular activities of -glucosidase (EC 3.2.1.20) and -glucosidase (EC 3.2.1.21) were estimated fluorimetrically, using 4-methylumbelliferyl (MUF) substrate analogues, cleavage of which yields the fluorogenic subunit methylumbelliferone (Hoppe 1983, Chrst & Krambeck 1986, Mnster et al. 1989). The substrates MUF-D-glucoside (MUF--Glc) and MUF--D-glucoside (MUF--Glc)(Sigma Chemie),

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respectively and MUF were first dissolved in a small volume of ethylene glycol monomethylether (EGME), then in deionized water, and stock solutions were frozen o at 20 C. After thawing, they were diluted to the appropriate concentrations. Enzyme kinetic parameters Vmax and Km were calculated by non-linear regression analysis using Enzfitter program for the PC, version 1.05. Experimental procedure 1. Surface and interstitial water. For all assays, three replicates of 4.5 ml of water 1 sample, 0.5 ml of substrate solution (final conc. 100 mol.l ) were mixed and incubated for 36 hours at in situ temperature. Blanks (3 replicates) were inactivated with HgCl2 (100 l) prior to the start of the assay and processed in parallel. Incubations were stopped by adding 100 l of HgCl2. Just before the measurement of fluorescence, 100 l of alkaline solution (2 M NaOH + 0.4 M EDTA) was added to the tubes to convert MUF to its more fluorescent anionic form. The fluorescence was measured in a FLUOROSCAN fluorimeter at at 465 nm under 365 nm excitation in a 96 PS microplate (ELISA) using Ascent PC software. Activity was measured at a single, 1 saturating substrate concentration (apparent activity, 100 mol litre final concentration). To measure saturation kinetics of enzymatic hydrolysis, we used set substrate 1 concentrations (5, 10, 30, 50, 100, 150, 200 mol litre final concentration). 2. Stream sediments. Three 1 ml replicates of the homogenized sediment (approx. 1 g wet mass), together with 4.5 ml of previously-filtered (Whatman GF/C) and boiled (30 min) interstitial water, were placed into sterile polypropylene tubes and installed in a shaker. Blanks (3 replicates) were inactivated with HgCl2 (200 l) prior to the start of the assay. Individual, saturated substrates were added to the samples to final concentrations of 100 and 300 mol litre1, respectively. The saturation kinetics were measured by using a set of substrate concentrations (5, 10, 30, 50, 100, 200, 300 and 500 mol liter1 final concentration). Samples were incubated for 1 hour at ambient room temperature. Previous trials had shown that it was impracticable to use longer incubation time because of the very high levels of enzyme activity in the sediment. Incubations were stopped by adding 200 l of HgCl2. The samples were centrifuged (5000 G for 15 min), and supernatant was transferred to clean glass tubes and the fluorescence was measured as described above. For each experiment, the fluorometer was calibrated by a set of concentrations of MUF, mixing 4.0 ml of the water sample with 0.5 ml of the MUF solution, and 100 l of alkaline solution (NaOH + EDTA) (Vrba et al. 1992). Substrate hydrolysis in sediments was expressed per gramme of dry mass (DM, 105 C overnight) sediment.

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Preliminary results and discussion

-glucosidase activity Maximum GlcA (Vmax) was found to be much lower in both surface and interstitial waters than in sediments (Table 1). Km values were higher in the sediments as well. Vmax has shown no marked difference between surface water and interstitial water and Km was also very similar (Table 1). Generally, kinetic of -glucosidase fits well the Michaelis-Menten equation and substrate saturation is about substrate concentration of 30 mol for the surface- and interstitial water and about 150 mol for sediment 1 1 samples. The values of GlcA varied between 0.0021.2 mol.l .h for water samples 1 1 and between 0.663.4 mol.g DM.h for sediment samples.
Table 1: Kinetic parameters of extracellular -Glc activity in surface water and stream-bed sediments
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-glucosidase activity Maximum GlcA (Vmax) was found to be much lower in both surface and interstitial waters than in sediments (Table 1). Km values were higher in the sediments as well. Although Vmax has shown no marked difference between surface water and interstitial water, Km was two times higher (Table 1). This observation of lower affinity of enzymes in the interstitial water compared to surface water suggests that there may be presence a mixture of enzymes with different afinity to substrate. The values of GlcA varied 1 1 1 1 between 0.0021.7 mol.l .h for water samples and between 1.09.1 mol.g DM.h for sediment samples.
Table 2: Kinetic parameters of extracellular -Glc activity in surface water and stream-bed sediments
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102

Extracellular enzymatic activity in stream-, interstitial water and sediment A very low level of hydrolytic activity for both MUF- -Glc and MUF--Glc was observed in the water column and interstitial water, as compared with the gravel-sand sediment (Fig. 1.). Relatively low extracellular activities of both enzymes found in the interstitial water suggest that most enzyme activity within the sediment is associated with the sediment surfaces and that levels of enzymes in the porewater is maintained by production of sediment biofilm (cf Marxsen & Fiebig 1993).

Fig. 1: Comparison of MUF-a-glucoside (MUF-a-Glc) and MUF-b-glucoside (MUF-b-Glc) hydrolysis in Sitka bed-sediments (020 cm depth), interstitial water and surface water. Experiments were conducted at initial saturation concentrations

Range of activities of -glucosidase and -glucosidase in river sediments published by other authors from various habitats markedly varied (Table 3). These differences are primarilly due to different methods and experiments used for determination of extracellular enzymatic activity. Nevertheless, our data obtained from gravel-sand sediments corresponded to that listed in Table 3. The GlcA/GlcA ratio found in the surface water (0.85) was higher than the ratio for interstitial water (0.5) and sediments (0.55). This supports our assumption that decomposition of cellulose mainly of allochthonous origin should dominate in the sediments of the Sitka stream (Rulk et al. 1999). Of the two extracellular enzymes studied here, -1,4, glucosidase specifically catalyzes the hydrolysis of -linked dissacharides of glucose (cellobiose), while -1,4,glucosidase hydrolyses 1,4--glucosidic linkages (maltose and starch). The predominance of cellulose rather than maltose and starch as a carbon source can be inferred from the higher activity of -glucosidase than

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-glucosidase. Activities of both enzymes reflect, at least in part, the use of autochthonous (algal) or allochthonous (plant) carbon material by heterotrophic bacteria. Although the possibility of a direct relationship with the allochthonous organic carbon was not studied between extracellular activities and stream DOC, results of measurement of UV absorbances indicate that interstitial DOC is composed mainly of pedogenic, refractory material of allochthonous origin (unpublished data). As expected, activity of -glucosidase in the surface water corresponded to algal primary production in stream water. However, relatively high -glucosidase activity found within sediments is surprizing. Explanation of this fact is not yet clear.
Table 3: Comparison of enzymatic activities (GlcA, GlcA) in river bed sediments from various studies (1M cm2 h1 ; 2M ml1 sed.h1; 3M g1 DM h1 ; 4M l1 h1)
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Acknowledgements The authors would like to thank Petra Sedlkov and Alena Habigerov for their assistance with laboratory work. We are very indebted to Jaroslav Vrba for his suggestions and valuable comments to an earlier version of this paper and to Dr. Douglas Fraser, Sheffield Hallam University, for revising the English text. This research was supported by the Czech Grant agency grant 206/99/P030.

References
BATTIN, T. J. (1997): Assessment of fluorescein diacetace hydrolysis as a measure of total esterase activity in natural stream sediment biofilms. The Science of Total Environment 198: 5160. CROCKER, M. T., MEYER, J. L. (1987): Interstitial dissolved organic carbon in sediments of a southern Appalachian headwater stream. J. N. Am. Benthol. Soc. 6,3: 159167. HOPPE, H.G. (1983): Significance of exoenzymatic activities in the ecology of brackish water: measurements by means of methylumbelliferyl substrates. Mar. Ecol. Prog. Ser. 11: 299308. CHRST, R. J.(1990): Microbial ectoenzymes in aquatic environments. pp. 4778. In: Overbeck, J., Chrst, R. J. (eds.): Aquatic microbial ecology. Springer-Verlag, New York, 190 pp.

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CHRST, R.J., KRAMBECK,H.J. (1986): Fluorescence correction for measurement of enzyme activity in natural waters using methylumbelliferyl substrates. Arch. Hydrobiol. 106: 7990. JONES, S. E., LOCK, M. A. (1989): Hydrolytic extracellular enzyme activity in heterotrophic biofilms from two contrasting streams. Freshw. Biol. 22: 289296. LEICHTFRIED, M. (1988): Bacterial substrates in gravel beds of a second order alpine stream (Project Ritrodat-Lunz, Austria). Verh. Internat. Verein. Limnol. 23: 13251332. MARXSEN, J., FIEBIG, D. M. (1993): Use of perfused cores for evaluating extracellular enzyme activity in stream-bed sediments. Microbiol. Ecol. 13: 112. MARXSEN, J., WITZEL, K. P. (1991): Significance of extracellular enzymes of organic matter degradation and nutrient regeneration in small streams. pp. 270285. In: Chrst, R. J. (ed.): Microbial enzymes in aquatic environments. Springer Vlg. New York. MNSTER, U., EINIO, P., NURMINEN, J. (1989): Evaluation of the measurements of extracellular enzyme activities in a polyhumic lake by means of studies with 4-methylumbelliferyl-substrates. Arch.Hydrobiol. 115: 321338. RULK, M., P, L., HLAVOV, E. (in print): Methane in the hyporheic zone of small lowland stream (Sitka, Czech Republic). Limnologica. RULK, M., HEKERA, P.(1998): Occurence of both acetic and lactic acids in subsurface water of the bed sediments comparison of two different localities. pp. 7787. In: Bretschko, G., Heleic, J. (eds.): Advances in river bottom ecology. Backhuys Publishers, Leiden, The Netherlands, 1998. RULK, M., SPIL, R., P, L., ZAVELOV, P., KOUTN, J., HLAVOV, E., POPELKA, J., MEDKOV, J., HEKERA, P. (1999): Role nch sediment v kolobhu organickho uhlku [Role of river sediments in organic carbon cycling]. In: Sbornk pednek z konference Sedimenty vodnch tokov a ndr, Bratislava 1999. VRBA, J. (1992): Seasonal extracellular enzyme activities in decomposition of polymeric organic matter in a reservoir. Arch. Hydrobiol. Beih. 37: 3342.

Acta Univ. Palacki. Olomuc. Fac. rer. nat. (1999) Biol. 37, 99105

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