From Ecological Stoichiometry to Biochemical Composition: Variation in N and P Supply Alters Key Biosynthetic Rates in Marine Phytoplankton

One of the major challenges in ecological stoichiometry is to establish how environmental changes in resource availability may affect both the biochemical composition of organisms and the species composition of communities. This is a pressing issue in many coastal waters, where anthropogenic activities have caused large changes in riverine nutrient inputs. Here we investigate variation in the biochemical composition and synthesis of amino acids, fatty acids (FA), and carbohydrates in mixed phytoplankton communities sampled from the North Sea. The communities were cultured in chemostats supplied with different concentrations of dissolved inorganic nitrogen (DIN) and phosphorus (DIP) to establish four different types of resource limitations. Diatoms dominated under N-limited, N+P limited and P-limited conditions. Cyanobacteria became dominant in one of the N-limited chemostats and green algae dominated in the one P-limited chemostat and under light-limited conditions. Changes in nutrient availability directly affected amino acid content, which was lowest under N and N+P limitation, higher under P-limitation and highest when light was the limiting factor. Storage carbohydrate content showed the opposite trend and storage FA content seemed to be co-dependent on community composition. The synthesis of essential amino acids was affected under N and N+P limitation, as the transformation from non-essential to essential amino acids decreased at DIN:DIP ≤ 6. The simple community structure and clearly identifiable nutrient limitations confirm and clarify previous field findings in the North Sea. Our results show that different phytoplankton groups are capable of adapting their key biosynthetic rates and hence their biochemical composition to different degrees when experiencing shifts in nutrient availability. This will have implications for phytoplankton growth, community structure, and the nutritional quality of phytoplankton as food for higher trophic levels

Response of the benthic food web to short- and long-term nutrient 1 enrichment in saltmarsh mudflats

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of Inter-Research for personal use, not for redistribution. The definitive version was published in Marine Ecology Progress Series 474 (2013): 27-41, doi:10.3354/meps10090.We examined the responses of biota at or near the base of the benthic food web to nutrient enrichment in salt marsh mudflats in Plum Island estuary (Massachusetts, USA). To simulate eutrophication, nitrate and phosphate loading rates were increased 10- to 15-fold in creeks fertilized for 2 mo (i.e. short-term enrichment) or 6 yr (chronic enrichment). We found that benthic invertebrate community structure was not altered by nutrient enrichment, although the abundance of epifaunal, but not infaunal, grazers increased. Short-term enrichment had no effect on the food web, but significant changes were detected with chronic enrichment. Grazing experiments with 15N-enriched bacteria and 13C-enriched benthic algae revealed higher per capita ingestion rates of benthic microalgae by nematodes, copepods and hydrobiid snails in the creek with chronic nutrient enrichment where isotope composition also indicated that algae increased in dietary importance. The fraction of bacterial biomass grazed was not affected by nutrient enrichment; however, the fraction of benthic algal biomass grazed increased by 235% with chronic enrichment. This higher grazing pressure was partly the result of dietary changes (increases in per capita feeding rate or a change in selection) but was mostly due to an increased abundance of the grazing consumer with the highest biomass, the snail Nassarius obsoletus. This increased top-down control partially masked the bottom-up effects of nutrient enrichment on algal biomass and helps explain the slow and inconsistent response of microalgal biomass to chronic nutrient enrichment previously observed in this estuary. Our research shows that eutrophication may subtly affect benthic food webs before large, sustained increases in algal biomass are observed.Pierre-Yves Pascal conducted this research while being supported by a 563 postdoctoral fellowship funded by the Department of Energy Office of Biological and 564 Environmental Research Award DE-FG02-05ER64070 and the Louisiana State University 565 College of Science. This material is based upon work supported by the National Science 566 Foundation under Grant Nos. 0213767 and 9726921

The cell envelope structure of cable bacteria

Cable bacteria are long, multicellular micro-organisms that are capable of transporting electrons from cell to cell along the longitudinal axis of their centimeter-long filaments. The conductive structures that mediate this long-distance electron transport are thought to be located in the cell envelope. Therefore, this study examines in detail the architecture of the cell envelope of cable bacterium filaments by combining different sample preparation methods (chemical fixation, resin-embedding, and cryo-fixation) with a portfolio of imaging techniques (scanning electron microscopy, transmission electron microscopy and tomography, focused ion beam scanning electron microscopy, and atomic force microscopy). We systematically imaged intact filaments with varying diameters. In addition, we investigated the periplasmic fiber sheath that remains after the cytoplasm and membranes were removed by chemical extraction. Based on these investigations, we present a quantitative structural model of a cable bacterium. Cable bacteria build their cell envelope by a parallel concatenation of ridge compartments that have a standard size. Larger diameter filaments simply incorporate more parallel ridge compartments. Each ridge compartment contains a similar to 50 nm diameter fiber in the periplasmic space. These fibers are continuous across cell-to-cell junctions, which display a conspicuous cartwheel structure that is likely made by invaginations of the outer cell membrane around the periplasmic fibers. The continuity of the periplasmic fibers across cells makes them a prime candidate for the sought-after electron conducting structure in cable bacteria

The Cell Envelope Structure of Cable Bacteria

Cable bacteria are long, multicellular micro-organisms that are capable of transporting electrons from cell to cell along the longitudinal axis of their centimeter-long filaments. The conductive structures that mediate this long-distance electron transport are thought to be located in the cell envelope. Therefore, this study examines in detail the architecture of the cell envelope of cable bacterium filaments by combining different sample preparation methods (chemical fixation, resin-embedding, and cryo-fixation) with a portfolio of imaging techniques (scanning electron microscopy, transmission electron microscopy and tomography, focused ion beam scanning electron microscopy, and atomic force microscopy). We systematically imaged intact filaments with varying diameters. In addition, we investigated the periplasmic fiber sheath that remains after the cytoplasm and membranes were removed by chemical extraction. Based on these investigations, we present a quantitative structural model of a cable bacterium. Cable bacteria build their cell envelope by a parallel concatenation of ridge compartments that have a standard size. Larger diameter filaments simply incorporate more parallel ridge compartments. Each ridge compartment contains a ~50 nm diameter fiber in the periplasmic space. These fibers are continuous across cell-to-cell junctions, which display a conspicuous cartwheel structure that is likely made by invaginations of the outer cell membrane around the periplasmic fibers. The continuity of the periplasmic fibers across cells makes them a prime candidate for the sought-after electron conducting structure in cable bacteria

Linking Microbial Community Function to Phylogeny of Sulfate-Reducing Deltaproteobacteria in Marine Sediments by Combining Stable Isotope Probing with Magnetic-Bead Capture Hybridization of 16S rRNA▿

We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine 13C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotope-labeled [13C]glucose, [13C]propionate, or [13C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that 13C labeling can be much lower, to the extent that δ13C ratios can be studied even at their natural abundances

Comparison of rRNA and Polar-Lipid-Derived Fatty Acid Biomarkers for Assessment of (13)C-Substrate Incorporation by Microorganisms in Marine Sediments

We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in (13)C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of (13)C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the (13)C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the (13)C-labeled compounds. Similar results were obtained with the (13)C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the (13)C analysis of the captured SSU rRNA

Data from: Nutrient deficiencies and the restriction of compensatory mechanisms in copepods

1.The flexible regulation of feeding behaviour and nutrient metabolism is a prerequisite for consumers to grow and survive under variable food conditions. Thus, it is essential to understand the ecological trade-offs that restrict regulatory mechanisms in consumers to evaluate the consequences of nutrient limitations for trophic interactions. 2.Here, we assessed behavioural and physiological adjustments to nutrient deficiencies in copepods and examined whether energy limitation, food digestibility or co-limitation with a second nutrient restricted compensatory mechanisms. 3.A combination of 13C-labelling and compound-specific stable isotope measurements revealed that copepods compensated nitrogen deficiencies by raising retention efficiencies of amino acids. The costs of higher retention efficiencies were reflected in the doubling of structural fatty acids, probably required for morphological adaptations of the gut. A depletion of highly unsaturated fatty acids in storage lipids and their selective retention suggested that these fatty acids became co-limiting and restricted a further increase in amino acids retention efficiencies. 4.Copepods feeding on phosphorus-limited algae showed a marked increase of ingestion rates but were not fully able to compensate dietary deficiencies. The increase of ingestion rates was thereby not restricted by higher foraging costs because energy storage in copepods increased. Instead thicker cell walls of nutrient limited algae indicated that algal digestion resistance restricted the extent of surplus feeding. 5.The strongly nutrient-specific response of copepods had large implications for recycling rates, growth efficiencies and the potential top-down control at the plant-animal interface. Compensatory mechanisms to mitigate nutrient deficiencies are, therefore, an essential aspect of trophic interactions and have the potential to alter the structure of food-web

Trophic role of large benthic sulfur bacteria in mangrove sediment

Large filamentous sulfur-oxidizing bacteria belonging to the Beggiatoacae family can cover large portions of shallow marine sediments surrounding mangroves in Guadeloupe (French West Indies). In order to assess the importance of Beggiatoa mats as an infaunal food source, observations were conducted of the area within mats and at increasing distances from mats. We used natural isotopic compositions and a C-13 enrichment study. Both revealed an ingestion of bacterial mats by associated meiofauna, dominated by rotifers and to a smaller extent by small polychaetes and nematodes. Compared to adjacent sites, sediment covered by bacterial mats presented a higher abundance of diatoms, whereas the total biomass of bacteria did not vary. This constant bacterial abundance suggests that the proportion of organic matter represented by sulfur bacteria is limited compared to the fraction of total bacteria. There was no significant difference in infaunal abundance in mats, suggesting that the availability of this chemosynthetic food resource had a limited local effect. Grazers presented a delta C-13 value increasing with distance from the mat. However, isotopic composition of phospholipid-derived fatty acids specific for diatoms and bacteria revealed that this change is related to modifications of delta C-13 dietary components rather than to changes in diet composition. These complementary methods revealed that the occurrence of sulfur-oxidizing bacterial mats does not necessarily affect grazer abundance and importance of bacteria in their diet. Despite its wide occurrence, Beggiatoa mats would consequently have a minor influence on the structure of the mangrove food web

Microbial biomass in compost during colonization of Agaricus bisporus

Agaricus bisporus mushrooms are commercially produced on a microbe rich compost. Here, fungal and bacterial biomass was quantified in compost with and without colonization by A. bisporus. Chitin content, indicative of total fungal biomass, increased during a 26-day period from 576 to 779 nmol N-acetylglucosamine g−1 compost in the absence of A. bisporus (negative control). A similar increase was found in the presence of this mushroom forming fungus. The fungal phospholipid-derived fatty acid (PLFA) marker C18:2ω6, indicative of the living fraction of the fungal biomass, decreased from 575 to 280 nmol g−1 compost in the negative control. In contrast, it increased to 1200 nmol g−1 compost in the presence of A. bisporus. Laccase activity was absent throughout culturing in the negative control, while it correlated with the fungal PLFA marker in the presence of A. bisporus. PLFA was also used to quantify living bacterial biomass. In the negative control, the bacterial markers remained constant at 3000–3200 nmol PLFA g−1 compost. In contrast, they decreased to 850 nmol g−1 compost during vegetative growth of A. bisporus, implying that bacterial biomass decreased from 17.7 to 4.7 mg g−1 compost. The relative amount of the Gram positive associated PLFA markers a15:0 and a17:0 and the Gram negative PLFA associated markers cy17:0 and cy19:0 increased and decreased, respectively, suggesting that Gram negative bacteria are more suppressed by A. bisporus. Together, these data indicate that fungal biomass can make up 6.8% of the compost after A. bisporus colonization, 57% of which being dead. Moreover, results show that A. bisporus impacts biomass and composition of bacteria in compost