Chemical characterization and quantification of the brown algal storage compound laminarin

In search of a meaningful stress indicator for Fucus vesiculosus we found that the often used quantitative determination procedures for the polysaccharide laminarin (beta-1,3-glucan) result in different kind of problems, uncertainties and limitations. This chemical long-term storage form of carbon enables perennial brown algae in seasonally fluctuating ecosystems to uncouple growth from photosynthesis. Because of this high ecological relevance a reliable and precise method for determination and quantification of laminarin is needed. Therefore, a simple, cold water extraction method coupled to a new quantitative liquid chromatography-mass spectrometrical method (LC-MS) was developed. Laminarin was determined in nine out of twelve brown algal species, and its expected typical molar mass distribution of 2000-7000 Da was confirmed. Furthermore, laminarin consisted of a complex mixture of different chemical forms, since fifteen chemical laminarin species with distinct molecular weights were measured in nine species of brown algae. Laminarin concentrations in the algal tissues ranged from 0.03 to 0.86% dry weight (DW). The direct chemical characterization and quantification of laminarin by LC-MS represents a powerful method to verify the biochemical and ecological importance of laminarin for brown algae. Single individuals of Laminaria hyperborea, L. digitata, Saccharina latissima, F. serratus, F. vesiculosus, F. spiralis, Himanthalia elongata, Cystoseira tamariscifolia, Pelvetia canaliculata, Ascophyllum nodosum, Halidrys siliquosa and Dictyota dichotoma were collected in fall (18.11.2013) during spring low tide from the shore of Finavarra, Co. Clare, west coast of Ireland (53° 09' 25'' N, 09° 06' 58'' W). After sampling, the different algae were immediately transported to the lab, lyophilized and sent to the University of Rostock. Laminarin was extracted with cold ultrapure water from the algal samples. Before extraction they were ground to < 1 mm grain size with an analytical mill (Ika MF 10 Basic). The algal material (approx. 1.5 g DW) was extracted in ultrapure water (8 mL) on a shaker (250 rpm) for 5 h. After the addition of surplus ultrapure water (4 mL) and shaking manually, 1 mL of the sample was filter centrifuged (45 µm) at 14,000 rpm (Hettich Mikro 22 R). The slightly viscous supernatant was free of suspended material and converted into a microvial (300 µL) for further analysis. The extracts were analyzed using liquid chromatography-mass spectrometry (LC-MS) analysis (LTQ Velos Pro ion trap spectrometer with Accela HPLC, Thermo Scientific). Laminarin species were separated on a KinetexTM column (2.6 µm C18, 150 x 3 mm). The mobile phase was 90 % ultrapure water and 10 % acetonitrile, run isocratically at a flow rate of 0.2 mL min-1. MS was working in ESI negative ion mode in a mass range of 100 - 4000 amu. Glucose contents were determined after extraction using high-performance liquid chromatography (HPLC). Extracted samples were analyzed in an HPLC (SmartLine, Knauer GmbH) equipped with a SUPELCOGELTM Ca column (30 x 7,8 mm without preColumn) and RI-detector (S2300 PDA S2800). Water was used as eluent at a flow rate of 0.8 mL min-1 at 75 °C. Glucose was quantified by comparison of the retention time and peak area with standard solutions using ChromGate software. Mannitol was extracted from three subsamples of 10-20 mg powdered alga material (L. hyperborea, L. digitata, S. latissima, F. serratus, F. vesiculosus, F. spiralis, H. elongata, P. canaliculata, A. nodosum, H. siliquosa) and quantified, following the HPLC method described by Karsten et al. (1991). For analyzing carbon and nitrogen contents, dried algal material was ground to powder and three subsamples of 2 mg from each alga thalli were loaded and packed into tin cartridges (6×6×12 mm). The packages were combusted at 950 °C and the absolute contents of C and N were automatically quantified in an elemental analyzer (Elementar Vario EL III, Germany) using acetanilide as standard according to Verardo et al. (1990).

C1-C8 hydrocarbons in DSDP Leg 64 Holes sediments

Sediments from the Baja California Continental Margin Transect - Sites 474 and 476 - showed small amounts of C2-C8 hydrocarbons and functionalized compounds (alkenes) typical of organic-rich, Recent, cold (<30°C) marine sediments. In contrast, some samples from Sites 477, 478, 479, and Hole 481A in the Guaymas Basin, an active spreading center, showed the characteristics of thermally generated hydrocarbons. These include an increase (sometimes exponential) in amount and diversity of C2-C8 hydrocarbons and a decrease in alkenes in more thermally mature sediments. The results indicate that the injection of basaltic sills has minimal effect on C2-C8 hydrocarbon generation except in the immediate vicinity of the sill. The absence of light hydrocarbons close to the hottest sills suggests that the compounds distill away as they are formed in these areas of very active hydrothermal circulation. A sample of young sediment exposed to very high temperatures (>300°C) from deeper thermal sources at the hottest site, 477, showed a very limited hydrocarbon distribution, including primarily ethane, benzene, and toluene, together with smaller amounts of propane and butane.

Pore water acetate and hydrogen, helium and methane gas concentrations in ODP Leg 204 sediments

Acetate and hydrogen concentrations in pore fluids were measured in samples taken at seven sites from southern Hydrate Ridge (SHR) offshore Oregon, USA. Acetate concentrations ranged from 3.17 to 2515 µM. The maximum acetate concentrations occurred at Site 1251, which was drilled on a slope basin to the east of SHR at depths just above the bottom-simulating reflector (BSR) that marks the boundary of gas hydrate stability. Acetate maxima and localized high acetate concentrations occurred at the BSR at all sites and frequently corresponded with areas of gas hydrate accumulation, suggesting an empirical relationship. Acetate concentrations were typically at a minimum near the seafloor and above the sulfate/methane interface, where sulfate-reducing bacteria may consume acetate. Hydrogen concentrations in pressure core samples ranged from 16.45 to 1036 parts per million by volume (ppmv). In some cases, hydrogen and acetate concentrations were elevated concurrently, suggesting a positive correlation. However, sampling of hydrogen was limited in comparison to acetate, so any relationships between the two analytes, if present, were difficult to discern.

Feeding of Sagitta setosa in the Black Sea

Gut dissection of fixed individuals from samples collected during Cruise 6 of R/V Vityaz-2 in April-May 1984 was used to study feeding of Sagitta setosa in the layers of daytime plankton accumulation at the lower boundary of the oxycline. The principal food was copepodite stage V of Calanus and females of Calanus and Pseudocalanus. Analysis of daytime and night data with reference to length of migratory alterations of Sagitta populations and gut passage time indicates that they feed actively in the layers of day¬time plankton accumulations. Total food consumption during time spent in the subsurface layers ranged from 0.025-0.097 cal/indiv. in 12 h, equivalent to 37-143% of their metabolic energy expenditure. Over the course of 12 h Sagitta population consumes 0.3-5% and 0.5-6% of population of stage V copepodites and females of Calanus and Pseudocalanus females, respectively.

Ingestion and clearance rates of Copepods

Feeding activity, selective grazing and the potential grazing impact of two dominant grazers of the Polar Frontal Zone, Calanus simillimus and Rhincalanus gigas, and of copepods < 2 mm were investigated with incubation experiments in the course of an iron fertilized diatom bloom in November 2000. All grazers were already actively feeding in the low chlorophyll waters prior to the onset of the bloom. C. simillimus maintained constant clearance rates and fed predominantly on diatoms. R. gigas and the small copepods strongly increased clearance and ingestion of diatoms in response to their enhanced availability. All grazers preyed on microzooplankton, most steadily on ciliates, confirming the view that pure herbivory appears to be the exception rather than the rule in copepod feeding. The grazers exhibited differences in feeding behavior based on selectivity indices. C. simillimus and R. gigas showed prey switching from dinoflagellates to diatoms in response to the phytoplankton bloom. All grazers most efficiently grazed on large diatoms leading to differences in daily losses for large and small species, e.g. Corethron sp. or Thalassionema nitzschioides. Species-specific diatom mortality rates due to grazing suggest that the high feeding activity of C. simillimus prior to and during the bloom played a role in shaping diatom population dynamics

(Table 1) Organic carbon contents and vitrinite reflection in sediments from DSDP Holes 63-467, 63-471, and 64-481A

A number of C25 and C30 highly branched isoprenoid (HBI) sulphur compounds (E.G., thiolanes, 1-oxo-thiolanes, thiophenes, and benzo[b]thiophenes) with 2,6,10,14-tetramethyl-7-(3-methylpentyl)pentadecane and 2,6,10,14,18-pentamethyl-7-(3-methylpentyl)nonadecane carbon skeletons were identified in sediments, ranging from Holocene to Upper Cretaceous. These identifications are based on mass spectral characterisation, desulphurisation, and, in some cases, by comparison of mass spectral and relative retention time data with those of authentic standards. The presence of unsaturated C25 and C30 HBI thiolanes in a Recent sediment from the Black Sea (age 3-6 ka) strongly supports their formation during early diagenesis. The co-occurrence of HBI polyenes (C25 and C30) and unsaturated HBI thiolanes (C25 and C30) possessing two double bonds less than the corresponding HBI polyenes, in this Recent sediment, testifies to the formation of unsaturated HBI thiolanes by a reaction of inorganic sulphur species with double bonds of the HBI polyenes. Furthermore, a diagenetic scheme for HBI sulphur compounds is proposed based on the identification of HBI sulphur compounds in sediment samples with different maturity levels.