Coupling microbial ecology to the biogeochemical cycling of dissolved organic matter in the oligotrophic ocean.

Dissolved organic matter (DOM) is one of the largest and most dynamic carbon reservoirs on the planet, and serves as the center at the marine microbial food web. Spectral analyses show that a good portion of DOM is comprised of a closely related family of polysaccharides with a remarkably uniform composition that is conserved across all major ocean basins. DOM polysaccharides collected at Station ALOHA (Fig. 1) are chemically and spectrally indistinguishable from DOM collected in the North Atlantic Ocean (off Bermuda). The presence of such a ubiquitous, specific, and abundant component of DOM provides us with a chemically well defined and biogeochemically important substrate that we can target for microbial-DOM studies.

The metabolic and biogeochemical pathways responsible for DOM cycling are “black boxes” in the context of the biology and chemistry of marine carbon cycling. In collaboration with Ed DeLong’s group at MITand with funding from the Gordon and Betty Moore Foundation we are working to isolate, cultivate and characterize microbes that drive DOM polysaccharide cycling, and to develop model systems to explore the physiology, ecology and biogeochemistry of DOM turnover in nutrient-poor waters. DOM polysaccharides are isolated (Figs. 2&3), purified, characterized (Fig. 4), and used as substrates in high throughput culture screens (Fig. 5), which are profiled using genomic and transcriptomic approaches (Figs. 6, 7). Our approach leverages the microbiology to elucidate details of DOM-degradation biochemistry, and conversely uses DOM chemical characterization and manipulation to test hypotheses microbial diversity and metabolism in open ocean oligotrophic gyres.

Elemental, isotopic, spectral, and chromatographic data along with analytical protocols can be found within the subpage link at upper left

Figure 1. Our work is based at Station ALOHA, the study site of the Hawaii Ocean Time-series program and the Natural Energy Laboratory on the big island of Hawaii. (Figure from the HOT website).

Figure 2. We use ultrafiltration to selective capture DOM polysaccharides for our studies. In the technique, seawater is pressurized and passed through a filter with nanometer-sized pores (grey sheet in figure). Organic molecules (red ellipses) with hydrodynamic diameters greater than the filter pore size are retained on one side of the membrane, while salt, low molecular weight DOM, and water (blue circles) permeate through the filter. Once the sample has been concentrated to < 1-2 L, residual salts are removed by serial dilution with ultra-pure water followed by filtration.

Figure 3. When the sample is fully processed, we obtain several grams of a brilliant white, cotton-like material that we can use for detailed chemical analyses and for microbial culture experiments.

Figure 4. 1H-, 13C-, 31P, and 15N-NMR analyses show most of the DOM is a complex polysaccharide that is rich in N-acetyl amino and 6-deoxy sugars. The polysaccharide is unlike other carbohydrates isolated from terrestrial systems, and full structure has so far defied efforts at full characterization. Here a 2D NMR HSQC spectra of the polysaccharide highlights the presence of amino sugars at 22 (13C) x 2.0 (1H) ppm and 6-deoxy sugars (rhamnose and fucose) at 16 (13C) x 1.2 (1H) ppm.

Figure 5. We use dilution-to-extinction to isolate bacteria that are able to hydrolyze DOM polysaccharides. In this technique, a water sample from Station ALOHA with typically ~106 L-1 bacterial cells is diluted with sterile filtered seawater until there are only a few cells mL-1. The sample is then transferred into a large number of well plates and each well is supplemented with DOM polysaccharide. Bacteria capable of degrading DOM polysaccharide continue to grow, while wells with bacteria that cannot use DOM run out of substrate and test negative for cell growth. Figure courtesy of Oscar Sosa.

Figure 6. Extracellular hydrolysis of DOM polysaccharides by heterotophic bacteria. DOM polysaccharides incorporate carbon, nitrogen, and phosphorus, all of which are in demand by heterotrophic bacteria inhabiting oligotrophic ecosystems. Polysaccharides are too large to be transported directly across cell membranes and metabolized within the cell, and therefore must be processed by extracellular hydrolytic enzymes before assimilation. We use transcriptomics to assemble the metabolic pathways up-regulated by bacteria once exposed to purified DOM polysaccharides. Figure courtesy of Oscar Sosa.

Figure 7. Isolates are selected for growth against DOM polysaccharides (bottom). Genes encoding DOM polysaccharide metabolic pathways are expressed (top, left), extracted and transposed via fosmids into E. coli (top, right). E. coli that are able to metabolize DOM polysaccharides are then sequenced to identify genes encoding DOM hydrolytic enzymes. Figure courtesy of Oscar Sosa.