Edward J. Carpenter

Chapter 4 – Nitrogen Fixation in the Marine Environment

Publisher Summary This chapter first summarizes the recent advances and insights relative to nitrogen (N2) fixation in benthic marine environments, move on to the water column, then summarizes controls on N2 fixation and comments on the broader, biogeochemical impacts of N2 fixation. In benthic environments, ranging from the rhizosphere of shallow water macrophyte communities such as Zostera, Thalassia, and Spartina hundreds of different diazotrophic strains have been isolated. These diazotrophs make significant contributions to the nitrogen economy of their respective plant communities. In the pelagic zone, iron and phosphorus availability is a major constraint on N2 fixation. In the north Atlantic, positive nitrogen anomalies in the subeuphotic zone, resulting from Aeolian dust deposition of Fe corresponds to areas in which P limits growth of diazotrophs. There is precious little information on the pathways of fixed N from pelagic diazotrophs to other members of the plankton and nekton. Viral lysis may be a major pathway as is the leakage of fixed N from healthy Trichodesmium cells. The spectrum of organisms recognized to contribute to N2 fixation in the sea has been greatly expanded, and there has been a virtual revolution in understanding of its quantitative significance, the interplay between N2 fixation and the C cycle and the major controls on this process.

Phytoplankton ecology of a barrier island estuary: Great South Bay, New York

The phytoplankton ecology of Great South Bay, New York, was studied over a 1-year period. The study area, a large barrier island estuary (coastal lagoon with estuarine circulation), was characterized by high levels of inorganic nutrients, high turbidity and a shallow euphotic zone (<2 m). Net annual primary production by phytoplankton was high—450 g C m−2 year−1—and accounted for approximately 85% of the total ecosystem primary production. Chlorophyll a-specific productivity was dependent on mean photic zone light intensity in areas of the bay <1 m in depth from September 1979 through June 1980; 65–95% of the total light extinction in those areas was attibutable to suspended solids. Nitrogenous nutrient concentration did not limit phytoplankton productivity. Diatom and dinoflagellate cell densities varied greatly over time, while cryptomonad and chlorophyte species were abundant throughtout the year. Chlorophytes of 2–4 μm (‘small forms’) were numerically dominant, and contributed approximately half of the total phytoplankton biomass. Dilution of bay water by intruding ocean water appeared to control the spatial distribution of chlorophyll a on the south side of the bay; in other areas, growth appeared to exceed the rate of dilution by flushing. Waters entrained in eelgrass beds were significantly higher in salinity and mean photic zone light intensity, and had lower phytoplankton standing stock and depth-integrated primary production than control areas; waters in the sediment plume of active clamdigging boats were statistically similar to control areas with respect to water quality and phytoplankton community characteristics.

AN EMPIRICAL PROTOCOL FOR WHOLE-CELL IMMUNOFLUORESCENCE OF MARINE PHYTOPLANKTON1

An immunofluorescence protocol for intracellular antigens of phytoplankton was developed empirically. Paraformaldehyde (4%) was used to fix samples for 6 h at 4° C; fixed samples can be stored in cold (–20°C) methanol for at least 1 month. We used Triton X‐100, Nonidet P‐40, and dimethyl sulfoxide to permeabilize the cells for antibody penetration across the cell wall and plasma membrane. After immunolabeling at room temperature, the samples were mounted on slides and could be stored at 4° C for up to 6 months without significant decay of the fluorescence. The staining was examined with an epifluorescence microscope; semiquantitative (percentage of cells with positive staining) and quantitative (staining intensity measured with an imaging system) analyses were performed. Reproducible staining was achieved for nuclear, chloroplastic, cytoplasmic, and cell surface antigens in species of Chlorophyceae, Prymnesiophyceae, Bacillariophyceae, and Dinophyceae including some field‐collected samples. This protocol is advantageous to previously published protocols in that it allows relatively simple and long‐term storage of fixed samples and allows staining of multiple antigens for the same sample. Although improvement on cell permeabilization is required for some species, the present protocol could prove useful for physiological or ecological studies when frequent sampling is required and immediate processing of the samples is not possible.

GROWTH CHARACTERISTICS OF MARINE PHYTOPLANKTON DETERMINED BY CELL CYCLE PROTEINS: THE CELL CYCLE OF ETHMODISCUS REX (BACILLARIOPHYCEAE) IN THE SOUTHWESTERN NORTH ATLANTIC OCEAN AND CARIBBEAN SEA1

Ethmodiscus spp. is an important contributor to oceanic tropical‐ooze sediments and thus might be an important transport vehicle of carbon from the ocean surface to sediments. The knowledge of its cell cycle and growth rate, which is still lacking, is necessary to evaluate the importance of Ethmodiscus in nutrient cycling and to solve the discrepancy between its high sedimentary abundance and rarity in the plankton. We used immunofluorescence of a cell cycle protein, prolqerating cell nuclear antigen (PCNA), and DNA‐specific staining to study the progression of the cell cycle and roughly estimate the growth rate for E. rex (Rattray) Wiseman and Hendey in the southwestern North Atlantic Ocean and Caribbean Sea in June 1994 and January 1995.

Effects of Desiccation and Rehydration on Nitrogen Fixation by Epiphylls in a Tropical Rainforest

Nitrogen fixation rates by epiphyllous microorganisms are affected by desiccation. Rates from leaf samples which had been dried for 12 h were 0.66 ng N/10 cm2/h. In contrast, rates from leaves which had been kept continuously wet were 18.69 ng N/10 cm2/h. Leaf samples which had been rehydrated for 2 and 4 h showed intermediate fixation rates (4.26 and 9.76 ng N/10 cm2/h, respectively). Epiphyllous bryophytes maintain moist conditions on the leaf surface and thus create a microenvironment suitable for prolonged fixation by the microorganisms.

ULTRASTRUCTURE AND IMMUNOLOCALIZATION OF PHYCOBILIPROTEINS AND RIBULOSE 1,5‐BISPHOSPHATE CARBOXYLASE/OXYGENASE IN THE MARINE CYANOBACTERIUM TRICHODESMIUM THIEBAUTII1

Trichodesmium thiebautii Gomont, a marine planktonic diazotrophic cyanobacterium, has an unusual subcellular arrangement. To identify subcellular structures related to photosynthesis, antibodies against phycoerythrin, phycocyanin, and ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) were used together with an immuno‐gold labeling technique and electron microscopy. Thylakoid membranes, identified by transmission electron microscopy and phycobiliprotein labeling, were arranged as a loose network throughout all cells. Rubisco showed a particularly intense localization in medium electron‐dense polyhedral bodies, therefore identified as carboxysomes. The average density of the carboxysomal Rubisco label was about five times higher than that in the cytoplasm. The carboxysomes (4–11 per cell section) were scattered throughout the cytoplasm. These data, together with those obtained from double immunolabeling experiments using nitrogenase (Fe‐protein) and Rubisco antibodies, revealed that Trichodesmium contains both N2‐ and CO2‐fixing proteins within the same cell. This is in contrast to the previous concept of a spatial segregation of the two processes in Trichodesmium and demonstrate that nitrogenase‐containing cells are not comparable to heterocysts in this context.

Trichodesmium : Ultrastructure and Protein Localization

Colonial morphology, associated organisms, and ultrastructural organization of Trichodesmium thiebautii cells were investigated in detail using light, scanning and transmission electron microscopy. The ultrastructure of Trichodesmium is quite different from that of other cyanobacteria. T.thiebautii can be differentiated from T. erythraeum on the basis of colonial morphology and the subcellular arrangement of inclusions. The main differentiating features are highlighted. A variety of associated organisms were observed in the colonies including two additional cyanobacteria. The presence and distribution of proteins involved in carbon and nitrogen metabolism and their regulation in T. thiebautii are also briefly discussed.

Preface to Second Edition

During the nine years since the publication of the first edition of this book, there has been substantial progress on the treatment of well-set problems of nonlinear solid mechanics. The main purposes of this second edition are to update the first edition by giving a coherent account of some of the new developments, to correct errors, and to refine the exposition. Much of the text has been rewritten, reorganized, and extended. The philosophy underlying my approach is exactly that given in the following (slightly modified) Preface to the First Edition. In particular, I continue to adhere to my policy of eschewing discussions relying on technical aspects of theories of nonlinear partial differential equations (although I give extensive references to pertinent work employing such methods). Thus I intend that this edition, like the first, be accessible to a wide circle of readers having the traditional prerequisites given in Sec. 1.2. I welcome corrections and comments, which can be sent to my electronic mail address: ssa@math.umd.edu. In due time, corrections will be placed on my web page: http://www.ipst.umd.edu/Faculty/antman.htm. I am grateful to the following persons for corrections and helpful comments about the first edition: J. M. Ball, D. Bourne, S. Eberfeld, T. Frohman, T. J. Healey, K. A. Hoffman, J. Horváth, O. Lakkis, J. H. Maddocks, H.-W. Nienhuys, R. Rogers, M. Schagerl, F. Schuricht, J. G. Simmonds, Xiaobo Tan, R. Tucker, Roh Suan Tung, J. P. Wilber, L. von Wolfersdorf, and S.-C. Yip. I thank the National Science Foundation for its continued support and the Army Research Office for its recent support.