John McN. Sieburth

Coulometric TCO2 analyses for marine studies; an introduction

Progress in the introduction of coulometry for the analysis of total carbon dioxide (TCO2) in marine waters is described. An extractor—stripper removes CO2 that is measured coulometrically by the quantity of electricity (coulombs) used to electrogenerate OH− ions for the titration of the acid formed by the reaction of CO2 and ethanolamine. The equivalence point is detected photometrically with thymolphthalein as the indicator, and Faradays Law relates coulombs to equivalents of titrant generated and CO2 determined so that there are no standard curves needed or titrants to standardize or store. Accuracy was determined by adding gelatin capsules containing 100–1500 μg C of pure CaCO3 into the stripper, and accuracies of better than ± 1 μg C were achieved. The best precision for natural seawater (± 1 standard error) of ± 0.5 μmol l−1 was found for 17 samples of Bermuda coastal waters having a mean TCO2 of 2007.2 μmol l−1 (0.05% CV). Sources of error and precautions are discussed. This method, which has been used successfully at sea, can be used to study a variety of marine phenomena involving TCO2.

IN-SITU MORPHOLOGY AND OCCURRENCE OF EUCARYOTIC PHOTOTROPHS OF BACTERIAL SIZE IN THE PICOPLANKTON OF ESTUARINE AND OCEANIC WATERS1

Concentrates of the picoplankton (0.2–2.0 μm) sized fraction from the euphotic zone of estuarine and oceanic waters were examined by transmission electron microscopy. In addition to the numerous phototrophic procaryotes (chroococcoid cyanobacteria) previously reported, small phototrophic eucaryotes were observed in 20 of 25 samples examined. Micromonas pusilla (Butcher) Manton and Parks, a 1 × 1.5 μm flagellate, was abundant in estuarine samples in summer. Similar sized cells of non‐flagellated chlorophytes, either Nannochloris Naumann or Chlorella Beijerinck, were observed sporadically in many samples. The most ubiquitous microalga was a scaled, non‐flagellated prasinophyte that occurred at 9 of 15 different locations on 15 of 20 sampling dates in water samples from Iceland to the Caribbean Sea, This tiny alga (0.5 to 1.0 μm in diam.) is probably the smallest known photo‐trophic eucaryote and has not heretofore been described. Enrichment cultures using conventional techniques on several cruises yielded only the Chlorella‐type of green alga, as well as numerous isolates of unicellular chroococcoid cyanobacteria.

Heterotrophic Bacteria and Bacterivorous Protozoa in Oceanic Macroaggregates

Oceanic macroaggregates (marine snow and Rhizosolenia mats) sampled from the Sargasso Sea are associated with bacterial and protozoan populations up to four orders of magnitude greater than those present in samples from the surrounding water. Filamentous, curved, and spiral bacteria constituted a higher proportion of the bacteria associated with the particles than were found among bacteria in the surrounding water. Protozoan populations were dominated numerically by heterotrophic microflagellates, but ciliates and amoebas were also observed. Macroaggregates are highly enriched heterotrophic microenvironments in the oceans and may be significant for the cycling of particulate organic matter in planktonic food chains.

Ultrastructure and ecology of Aureococcus anophagefferens gen. et sp. nov. (Chrysophyceae): the dominant picoplankter during a bloom in Narragansett Bay, Rhode Island, summer 1985

Observations of a marked cessation of feeding in filter feeding animals maintained in flowing Narragansett Bay seawater in June 1985 drew our attention to a bloom of a golden alga 2 μm in diameter at unprecedented populations of 109 cells. L−1. This picoplankter lacked morphological features useful in discriminating it from other similar sized forms with either phase contrast or epifluorescence light microscopy. Natural populations of picoplankton, obtained from the height of the bloom until its decline, were examined in thin section with transmission electron microscopy. A cell with a single chloroplast, nucleus, and mitochondrion and an unusual exocellular polysaccharide‐like layer was apparently the bloom alga. The ultrastructure of this alga is consistent with that of the Chrysophyceae, and a new genus and species, Aureococcus anophagefferens is described.

Dissolved carbohydrates in seawater. I, A precise spectrophotometric analysis for monosaccharides

A relatively precise and rapid method for the analysis of total dissolved monosaccharides at the concentrations that occur in seawater is described which uses 1-ml quantities for each analytical and control replicate. The alditols remain unchanged while the pentoses and hexoses are reduced to the alditol form by borohydride. The total alditols are then oxidized with periodate to form two moles of formaldehyde per mole of monosaccharide and the formaldehyde is determined spectrophotometrically with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). Unlike other spectrophotometric methods, similar curves are obtained for equimolar concentrations of different carbohydrates while the differences on a weight basis are only due to the small difference in molecular weight between pentoses and hexoses. Winter—Spring samples from surface waters of lower Narragansett Bay ranged from 122 to 226 μg l−1 with a mean of 159 μg l−1.

Dissolved carbohydrates in seawater. II, A spectrophotometric procedure for total carbohydrate analysis and polysaccharide estimation

A procedure is described which adds a hydrolysis step to the Johnson and Sieburth 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) assay for total dissolved monosaccharides. The advantages of the monosaccharide test are retained in the total carbohydrate test, giving nearly equivalent responses for a variety of combined carbohydrates. A total sample of 50 ml is adequate for both total carbohydrate and monosaccharide assays which permit the estimation of polysaccharide by difference. Values for Narragansett Bay and adjacent waters ranged from 452 to 2023 μg l−1 for total dissolved carbohydrate, 272 to 1353 μg l−1 for polysaccharide, and 153 to 814 μg l−1 for monosaccharide, which accounted for 6–18%, 4–13%, and 2–5% of the total dissolved carbon, respectively. We suggest that this is a sensitive and precise procedure which will be useful for investigating the distribution of dissolved carbohydrates in seawater and factors which affect its production, distribution and utilization.

Studies on algal substances in the sea. III. The production of extracellular organic matter by littoral marine algae

In both natural sunlight and in the dark Fucus vesiculosus L. gives no consistent or appreciable accumulation of dissolved organic matter in a closed system. On the addition of a bacteriostatic concentration of Thiomersal or the use of autoclaved sea water there was an accumulation of organic matter but the limitations of a closed system were not entirely overcome. An open system with 2 to 3 dilutions/h of fresh membrane-filtered (0.45 μ) sea water gave consistent and appreciable quantities of extracellular organic carbon which ranged from 4.4 mg C100g/h for Chondrus crispus Stackh. to 54.2 mg C100g/h for fruiting Ascophyllum nodosum (L.) Le Jol. Exudation in Fucus, which was coupled directly to photosynthesis, increased with solar radiation and was absent in the dark but unlike Fucus, Laminaria digitata (Huds.) Lamour. and Laminaria agardhii Kjellm. gave exudation in the dark. Reducing the salinity of the sea water caused an apparent reduction in the rate of exudation of organic matter by Fucus vesiculosus. Exudation also occurred in littoral algae on emersion during low tide. Desiccation caused a 68–89 % loss of moisture in surface fronds of Fucus vesiculosus and there was an organic carbon loss upon re-immersion in sea water of from 52–380 mg C100g. Complete clumps of algae on exposed rocks lost some 39 mg C100g. A light natural rainfall was more effective than artificial rain water in extraction and gave rates of exudation ranging from 7.5 and 46.4 mg C100g/mm of rain for Chondrus crispus and Fucus vesiculosus, respectively. Freezing at −15°C had little more effect than emersion, but algae held at −30°C for 4 h lost approximately 3 % of their organic matter upon thawing in sea water. Colorimetric assays were used to determine the nature of the exudates. Carbohydrate material appears to be most abundant followed by equal amounts of nitrogenous and polyphenolic material. Nitrogenous materials were exuded in greater concentrations during conditions which appeared injurious to the plants, including excessive desiccation, heavy rain, and extremely low temperature. A carbon balance for Fucus vesiculosus during spring conditions indicates that of the 1098 mg C100g fixed during the 6 h daily illuminated immersion period respiration accounts for some 276 mg C100g while exudation accounts for a total of 337 mg C100g during a 24 h period. This suggests that approximately 30 % of the total carbon or 40 % of the net carbon fixed daily is exuded by this plant. Since beds of Fucus can exceed 1000 g C/m2 and fix at least 16.5 g C/m2/day, extracellular organic matter production from such beds may be equivalent to 5–7 g C/m2/day.

Dissolved Organic Matter and Heterotrophic Microneuston in the Surface Microlayers of the North Atlantic

Dissolved organic carbon, carbohydrates, and adenosine triphosphate in the size fractions 0.2 to 3 micrometers and 3 to 1000 micrometers are significantly enriched in the upper 150-micrometer surface layer compared to subsurface water, mean enrichment factors being 1.6, 2.0, 2.5, and 3.1, respectively. When calculated as a 0.1-micrometer microlayer of wet surfactants, the mean concentration of organic matter was 2.9 grams per liter, of which carbohydrates accounted for 28 percent. The data for plant pigments and particulate adenosine triphosphate indicated that bacterioneuston was enriched at seven of nine stations while phagotrophic protists were enriched at five stations. Instances of enrichment and inhibition were verified by cultural data for bacteria and amoebas. The observations indicate that the surface microlayers are largely heterotrophic microcosms, which can be as rich as laboratory cultures, and that an appreciable part of the dissolved organic carbon is carbohydrate of phytoplankton origin, released and brought to the surface by migrating and excreting phagotrophic protists.

Studies on algal substances in the sea. II. The formation of Gelbstoff (humic material) by exudates of phaeophyta

Abstract Preliminary observations on the exudation from whole plants of the brown seaweed Fucus vesiculosus L. have confirmed the importance of this process in the ecology of inshore waters. There was an exudation of as much as 500–800 mg of phenolic and 1.0–1.5 g of carbohydrate materials per kg dry weight of alga maintained in the laboratory for 24 h. Qualitative studies on standing exudates from three of the most important Norwegian seaweeds ( Laminaria hyperborea (Gunn.) Fosl. [ cloustonii ], Ascophyllum nodosum (L.) Le Jol., and Fucus vesiculosus ) show that exudates form Gelbstoff material which is inseparable from that present in sea water. Observations based on paper chromatography, dialysis, Sephadex fractionation and recombination of the fractions obtained, led to the elaboration of the following hypothesis for Gelbstoff formation in algal exudates. The brown seaweeds exude a few simple precursors of (potential) phenolic nature. In the alkaline medium of sea water these form polyphenols which rapidly react with proteinaceous and carbohydrate material of either algal or other origin. The resulting phenolic complexes make up a considerable part of marine Gelbstoff. It is suggested that the larger molecular complexes of Gelbstoff are continually precipitating to form organic aggregates. The reactive phenols are toxic to some larval forms. Similar transformations of phenolic precursors to soluble Gelbstoff complexes and the subsequent formation of coloured precipitates was found to take place in bog water and in a sulphate paper mill effluent. It is therefore suggested that a common mechanism involving simple phenols, proteinaceous, and carbohydrate matter may be responsible for the formation of both terrestrial and marine humic substances (Gelbstoff).

Studies on algal substances in the sea. I. Gelbstoff (humic material) in terrestrial and marine waters

Abstract Nylon columns were used to concentrate the dissolved yellow colouring matter of terrestrial and marine waters. A concentration factor of 10 000 was easily obtained with a recovery of approximately 70 %. The method allowed the ready isolation of a reference ‘Gelbstoff’ of marine origin which was used to estimate Gelbstoff concentrations in bog (17 mg/1), river (~ 1 mg/1) and sea water (0.8–0.003 mg/1). Considerable seasonal and geographical variation in the Gelbstoff concentration of sea water was observed. Methods based on spectra and differential spectra were found inadequate for characterization of the different types of yellow material from terrestrial and sea water; they appear to be derived from polyphenols. A two dimensional paper chromatographic system was developed which allowed a fair separation of Gelbstoff from different sources. Marine Gelbstoff could be distinguished from both river and bog water pigments. A considerable fraction of the terrestrial Gelbstoff was found to precipitate rapidly in contact with sea water. Marine Gelbstoff also seems to be in a state of flux as its content and characteristics are variable and it is not detected during the dark winter months of January, February and March. Part of the yellow colour of sea water is due to particulate organic matter which is largely amorphous and shows polyphenol staining. Precipitating Gelbstoff may be a major source of these organic aggregates.