Rapid potential rates of extracellular enzymatic hydrolysis in Arctic sediments

The extracellular enzymatic hydrolysis rates of three fluorescently-labeled polysaccharides (pullulan, laminarin, and xylan) were measured in the upper -11 cm of sediment cores collected near Svalbard in the Arctic Ocean. The three polysaccharides differ in molecular weight (200,000, -6,000, and -8,000 Da for pullulan, laminarin, and xylan, respectively), as well as in monomer composition, linkage position, and anomeric configuration, and are most probably hydrolyzed by distinctly different enzymes. Potential hydrolysis rates of pullulan and laminarin were rapid throughout the sediment cores (average rates of 52 cuts per nmol glucose cm ’ hm’ for pullulan, 38 cuts per nmol glucose cm-’ h ’ for laminarin) and were comparable to rates measured in sediment cores from more temperate sites. Xylan potential hydrolysis rates were considerably slower at all Svalbard stations (average of 3 cuts per nmol xylose cm-’ h I). Rapid microbial remineralization of particulate organic carbon requires high levels of extracellular enzyme activity; the high potential hydrolysis rates of tluorescently-labeled pullulan and laminarin in Svalbard sediments demonstrate that at least some types of extracellular enzymes can function rapidly in permanently cold environments. Sedimentary microbial communities in deep-ocean sediments have the capability to rapidly metabolize an influx of fresh particulate organic matter (Lochte and Turley 1988; Gooday and Turley 1990; Poremba 1994). Investigations of extracellular enzymatic activities, however, have suggested that potential rates of enzymatic hydrolysis are markedly slower in deep-ocean sediments than in shallower and more temperate sites (Boetius 1995; Poremba and Hoppe 1995). Although enzymatic activities are not greatly affected by pressure (Helmke and Weyland 1986; Meyer-Reil and Koster 1992; Poremba 1995) temperature optima of extracellular enzymes isolated from marine bacteria from the deep and polar oceans frequently have been found to be far above ambient growth temperatures (Reichardt 1987; Helmke and Weyland 1991). These studies seem to present a paradox: rapid microbial utilization of particulate organic matter implies rapid hydrolysis of organic macromolecules, because substrates ~600 Da must be hydrolyzed enzymatically outside the cell prior to transport into the periplasmic space (Weiss et al. 1991), yet there is little evidence that extracellular enzymes function rapidly in deep and cold environments. One possible explanation lies in the fact that the specificities and activities of macromolecule-hydrolyzing enzymes of marine bacteria are still only poorly understood. Bacteria have the capability of producing a wide range of enzymes with precise substrate specificities. In marine systems, however, a limited range of low-molecular-weight substrates Acknowledgments I thank B. B. Jorgensen for inviting me to join the Svalbard project, D. Canfield for his work as chief scientist, and the captain, crew, and scientific party of the RV Jan Mayen for a stimulating and successful cruise. I also thank the scientists and staff of the Max-Planck Institute for Marine Microbiology for their hospitality during the summer of 1995. Y.-A. Vetter, J. Hedges, and an anonymous reviewer provided insightful comments about the manuscript. Funding for this work was provided by the National Science Foundation, the Petroleum Research Fund, and the Max Planck Society. have been used as proxies for organic macromolecules to measure extracellular enzyme activities in seawater and sediments. Methylumbelliferyl (MUF) substrates, for example, consist of a MUF fluorophore attached to a monosaccharide or amino acid. Hydrolysis of a MUF substrate leads to an increase in fluorescence signal from the MUF fluorophore (e.g. Hoppe 1983; Somville 1984; Meyer-Reil 1987; Boetius 1996). The MUF substrates, although convenient to use, are poor proxies for many high-molecular-weight substrates such as polysaccharides (Helmke and Weyland 199 I ). In addition, hydrolysis of MUF substrates measures activities of periplasmic as well as extracellular enzymes (Martinez and Azam 1993), so their relevance as measures of extracellular enzymatic activity is doubtful. A number of laboratory studies have used specific polysaccharide substrates, not proxies, to measure the hydrolytic capabilities of isolated species of bacteria (Norkrans and Stehn 1978; Reichardt 1988; Helmke and Weyland 1991). Isolation of bacteria, however, necessarily selects a sub-population from the total community originally present in the sediments; culture conditions that favor specific bacteria may not accurately reflect the microbial community responsible for macromolecule hydrolysis in sediments. In order to investigate the hydrolytic potential of microbial communities in permanently cold sediments, a new technique, based on fluorescently-labeled (FLA) polysaccharides (Arnosti 1995, 1996), was used to measure rates of extracellular enzymatic hydrolysis in sediment cores. Because FLA polysaccharides are far too large to be transported intact across bacterial membranes, enzymatic hydrolysis of these substrates must occur outside of the microbial cell. Polysaccharides were selected as the target macromolecular substrates because they are major components of marine organic matter such as phytoplankton (Parsons et al. 1961). Three specific polysaccharide substrates (pullulan, laminarin, and xylan) were used because they occur in marine algae and(or) their enzyme activities have been measured in marine bacteria. Pullulanase can function as a debranching enzyme of starch (White and Kennedy 1988) and starch is a