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40529-AC2
An EPR Investigation of Surface Associations in Marine Sediments and Their Consequences for Enzyme Activities and Organic Matter Degradation

Carol Arnosti, University of North Carolina (Chapel Hill)

Our investigations this year extended our results from last year's laboratory investigations of model systems (now published in Marine Chemistry) to measuring the activities of enzymes associated with surfaces naturally present in marine systems: marine snow, as well as mineral particles associated with resuspended surface sediments.

The marine snow investigation was aimed at determining the extent to which enzymes produced by natural bacterial populations associate with marine snow particles, and quantifying possible contributions of bacterial enzymes to the dissolved phase. Marine snow consists macroscopic aggregates that often form in the euphotic zone at the decline of a phytoplankton bloom (Alldredge and Silver, 1988). Hydrolytic activities of marine snow-attached bacteria are frequently reported to exceed those of their free-living counterparts (Karner and Herndl, 1992; Smith et al., 1992; Rath and Herndl, 1994; Ploug et al., 1999; Grossart and Ploug, 2001; Grossart et al., 2003; 2007; Zoppini et al., 2005). All of these studies, however, measured hydrolytic activities on aggregates using a small range of low molecular weight substrate proxies, typically MUF-α- and –β-glucose and leu-MCA, after Hoppe (1983). Although these measurements simplify comparisons across a range of studies, they cannot distinguish between the activities of periplasmic and extracellular enzymes (Martinez and Azam, 1993) and do not measure the activity of endo-acting (mid-chain cleaving) enzymes used by microbes to hydrolyze high molecular weight substrates. Our study is the first to use macromolecular substrates to measure hydrolytic activities in marine snow aggregates, the first detailed investigation of the activities of polysaccharide hydrolases in marine snow aggregates, and the first systematic comparison of the extent to which specific extracellular enzymes attach to aggregates vs remain free in the water column.

Hydrolysis was measured in aggregates formed from waters collected at two near-shore sites off St George Island, in the Gulf of Mexico. These locations were chosen since naturally-formed aggregates are frequently observed at the sediment-water interface. For these experiments, water collected at the sites was rotated on a roller table (Shanks & Edmondson, 1989). Hydrolysis rates were measured in aggregates collected from the tanks and resuspended in autoclaved seawater, as well as in aggregate-free seawater collected from the tanks.

All six polysaccharides investigated (laminarin, xylan, pullulan, arabinogalactan, fucoidan, and chondroitin sulfate) were hydrolyzed very readily in aggregates, but only four of the six (laminarin, xylan, pullulan, and chondroitin sulfate) were hydrolyzed in ambient (aggregate-free) waters. These observations show that extracellular enzymes differ in their production and/or association with laboratory-made aggregates. Since most cells were incorporated into the aggregates, hydrolysis in ambient waters strongly suggests a spatial disconnect from microbial cells, demonstrating that enzymes not attached to cells, aggregates, or macroscopic particles can also play a significant role in the hydrolysis of high molecular weight substrates. The fact that two of the six polysaccharides were only hydrolyzed in aggregates demonstrates that specific substrates may be available to microbial communities only when those substrates and/or organisms are attached to surfaces such as marine snow particles. Results of this investigation are now in press at Environmental Microbiology.

A second line of investigation involved comparing the effects of resuspended sediment particles (primarily clays) on enzyme activity in the water column. The rationale is that in dynamic marine environments, there is frequent particle exchange between the water column and unconsolidated surface sediment. Our previous laboratory investigations (see last year's report) demonstrated that surface-attached substrates were readily hydrolyzed by surface attached enzymes. This initial investigation extended this work to natural clay particles collected from cores obtained in the Delaware Bay. Enzyme activities (hydrolysis of laminarin, xylan, pullulan, arabinogalactan, fucoidan, and chondroitin sulfate) were compared in surface water samples and in overlying water containing visible particles collected from sediment cores (water column depth: 15 m). Pullulan was hydrolyzed in particle-containing (‘core') water, but not in surface waters, and rates of arabinogalactan and fucoidan hydrolysis in core water were on average more than double those of surface water. Given that the rate and spectrum of enzyme activities usually decreases with depth in the water column (Arnosti et al., 2005a; Steen et al., submitted; unpubl. data), higher rates of hydrolysis measured in the core water relative to surface water are likely due to the presence of particle associated enzymes and/or organisms. The complete data set from this investigation is currently being written up for submission in the near future.

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