ALBERT

Description: Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution May 1993

Description: The goal of this thesis was to develop and apply an integrated chemical and microbial approach to study the effects of chemical structure on the rates and patterns of carbohydrate degradation by anaerobic marine bacteria. Polysaccharides produced by five species of marine plankton, Dunaliella tertiolecta, Emiliania huxleyi, Stephanopixis palmeri, a Phaeocystis sp., and Synechococcus WH7335, were surveyed using one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR). Two carbohydrates from Synechococcus WH7335 were characterized in detail. Synechococcus produced an α(1,4) glucose polysaccharide with α(1,6) branches, which probably functions as an energy reserve. The nominal molecular weight of the polysaccharide was ~5000 daltons. Large quantities of a glycerolpolysaccharide, which was tentatively identified as a teichoic acid similar in structure to teichoic acids found in cell walls of gram-positive bacteria, were also produced by Synechococcus WH7335. This is the first report of teichoic acids in cyanobacteria. Enrichment of bacteria from anoxic marine sediments on specific carbohydrates yielded reproducible model systems with which to study the degradation of chemically well-defined substrates. Headspace gases (C02, H2, CH4, H2S) were monitored by gas chromatography, and carbohydrate substrates and intermediates were separated and quantified via gel-permeation chromatography and high-pressure liquid chromatography. The transfer of carbon from substrates through to end products was followed quantitatively. Nuclear magnetic resonance spectroscopy was used to check for selective structural alterations (such as preferential cleavage of specific linkage types or positions) of the substrates. A series of enrichment experiments showed that mixed cultures of marine bacteria distinguish even between small, very closely-related substrates which do not require extracellular hydrolysis prior to uptake. A galactose-β(1,3)-arabinose dimer was degraded at half the rate of seven other similar disaccharides and three larger oligosaccharides. A further series of degradation experiments with polysaccharides (pullulan, laminarin) showed that they are degraded by bacteria at virtually the same rate as structurally related substrates in the molecular weight range of 300-600 daltons. Degradation of the branched glucan and the teichoic acid-type polysaccharide from Synechococcus WH7335 was also very rapid. The time-course of bacterial hydrolysis of pullulan was examined with gel permeation chromatography and NMR to provide the first molecular-level evidence in marine systems of the bacterial extracellular transformation of high molecular weight organic matter to lower molecular weight organic matter. NMR spectra provided evidence that the pullulan was hydrolyzed by pullulanase, an endo-acting extracellular enzyme which preferentially hydrolyzes α(1,6) linkages. This is the first experimental evidence of pullulanase activity among marine mesophilic bacteria. The culture results suggest that enzymatic hydrolysis of macromolecular carbohydrates to transportable pieces is not the slow step in bacterial degradation of at least some types of polysaccharides. The results from the oligosaccharide experiments suggest that certain heteropolysaccharides may not be degraded as quickly. Chemical structure can be more important than molecular weight in determining degradation rates of carbohydrates. Varying rates of organic polymer degradation in anoxic sediments may be largely determined by the sensitivity of bacterial enzymatic and transport systems to structural features.

Description: Funding for this work was provided by a graduate fellowship from the Office of Naval Research, and grants from the WHOI Ocean Ventures Fund and the Department of Energy's Ocean Margins Program (DE-FG02-92ER61428).