Identity and dynamics of the microbial community responsible for carbon monoxide oxidation in marine environments

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 September 2003As colored dissolved organic matter in seawater absorbs UV solar radiation, a variety of simple chemical species are produced, including carbon monoxide (CO). The ocean surface water is saturated with respect to CO, and is thus a source of CO to the atmosphere. CO reacts with and removes free-radical compounds, and may itself contribute to the 'greenhouse' gas content of the atmosphere. An important sink for CO in seawater is the biological oxidation of CO to CO2 by marine microorganisms. The objectives of this study are to identify component members of the microbial community responsible for the oxidation of CO in coastal marine environments through a combination of recent microbiological and molecular approaches, and to estimate their contributions to total in situ CO bio-oxidation. We utilize an enrichment method that involves cultivation of bacteria on membrane filters, subsequent incubation with radiolabeled CO, and the use of autoradiography to screen colonies with the desired phenotype. Cell-specific CO-oxidation activity is determined for selected purified strains with a time-series 14CO-oxidation method. Molecular phylogeny based on 16S-rDNA gene sequence information within the context of the large and growing 168 database determines the phylogenetic relatedness and identity of marine CO-oxidizing bacteria that result from our cultivation program. The CO oxidizing organisms isolated in this study with greatest activity are closely related to the Roseobacter and Paracoccus genera of the alpha-proteobacteria, collectively known as the "marine alpha group". Other microorganisms found to oxidize CO at environmentally relevant rates are members of beta- and gamma-proteobacteria, and one in the Cytophaga-Flavobacterium-Bacteroides group. A collective CO-oxidation activity was calculated from physiological measurements of purified isolates and abundance estimates of CO-oxidizing marine alpha group organisms. Relative proportions of CO-oxidizing Roseobacter and Paracoccus cells were resolved microscopically by microautoradiography in combination with DAPI and fluorescent-labeled oligonucleotide probes (Substrate Tracking AutoRadiography - Fluorescent In Situ Hybridization (STAR-FISH)). Marine alpha group organisms were a major component of total cell numbers (45.7%) at the time of sampling (March 2003), and CO-oxidizing members of the marine alpha group contributed up to 40.7% of total CO oxidation occurring in coastal waters.Financial support was provided by the WHOI Education office and the wonderful people who work there, NSF grant #OCE-0136876, and the Coastal Ocean Institute and Rinehart Coastal Research Center grant #BI10918

Biological CO oxidation in the Sargasso Sea and in Vineyard Sound, Massachusetts

Author Posting. © The Author(s), 2005. This is the author's version of the work. It is posted here by permission of American Society of Limnology and Oceanography for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 50 (2005): 1205-1212.In situ dissolved carbon monoxide (CO) in oligotrophic waters follows a diel cycle varying from 0.3 to 0.5 nmol L-1 before dawn to 2.5 to 3 nmol L-1 in early afternoon, when photo-production of CO exceeds biological CO oxidation and other sinks. Coastal waters may contain up to 15 nmol L-1 [CO] in the daytime. Assays to measure the rate of CO bio-oxidation typically involve the addition of labeled CO to sealed samples, resulting in CO concentrations that are above ambient levels during incubation (up to 9 nmol L-1 CO). We find that biological oxidation of CO obeys first-order kinetics when incubated with up to 4 nmol L-1 [CO] in coastal water samples and up to between 4 and 10.8 nmol L-1 in oligotrophic waters. At higher [CO], kinetic behavior transitions to zero-order or saturation kinetics. CO–oxidation rate coefficients obtained in dark incubations were not representative of the entire diurnal period, as others have assumed. Biological CO–oxidation rate coefficients kco measured in dark incubations of Sargasso Sea surface water in summer were 0.020 ± 0.002 h-1 (mean ± standard deviation) and an order of magnitude greater than those measured in situ during daylight hours (0.002 ± 0.001 h-1). Dark and in situ rate coefficients in early spring were 0.006 ± 0.004 h-1 and 0.003 ± 0.001 h-1, respectively. In dark incubations of Vineyard Sound water, kco was 0.127 ± 0.038 h-1. The apparent half-saturation constant Kapp for CO ranged from 2.04 to 5.44 nmol L-1 CO in both environments.This research was supported by National Science Foundation grants OCE-98-11208 and OCE-01-36876, and the Reinhard Coastal Research Center and Coastal Ocean Institute grant BI-10918