Quantifying Oxygen Management and Temperature and Light Dependencies of Nitrogen Fixation by Crocosphaera watsonii.

Crocosphaera is a major dinitrogen (N2)-fixing microorganism, providing bioavailable nitrogen (N) to marine ecosystems. The N2-fixing enzyme nitrogenase is deactivated by oxygen (O2), which is abundant in marine environments. Using a cellular scale model of Crocosphaera sp. and laboratory data, we quantify the role of three O2 management strategies by Crocosphaera sp.: size adjustment, reduced O2 diffusivity, and respiratory protection. Our model predicts that Crocosphaera cells increase their size under high O2 Using transmission electron microscopy, we show that starch granules and thylakoid membranes are located near the cytoplasmic membranes, forming a barrier for O2 The model indicates a critical role for respiration in protecting the rate of N2 fixation. Moreover, the rise in respiration rates and the decline in ambient O2 with temperature strengthen this mechanism in warmer water, providing a physiological rationale for the observed niche of Crocosphaera at temperatures exceeding 20°C. Our new measurements of the sensitivity to light intensity show that the rate of N2 fixation reaches saturation at a lower light intensity (∼100 µmol m-2 s-1) than photosynthesis and that both are similarly inhibited by light intensities of >500 µmol m-2 s-1 This suggests an explanation for the maximum population of Crocosphaera occurring slightly below the ocean surface.IMPORTANCECrocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean.

Roadmaps and Detours: Active Chlorophyll- a Assessments of Primary Productivity Across Marine and Freshwater Systems.

Assessing phytoplankton productivity over space and time remains a core goal for oceanographers and limnologists. Fast Repetition Rate fluorometry (FRRf) provides a potential means to realize this goal with unprecedented resolution and scale yet has not become the "go-to" method despite high expectations. A major obstacle is difficulty converting electron transfer rates to equivalent rates of C-fixation most relevant for studies of biogeochemical C-fluxes. Such difficulty stems from methodological inconsistencies and our limited understanding of how the electron requirement for C-fixation (Φe,C) is influenced by the environment and by differences in the composition and physiology of phytoplankton assemblages. We outline a "roadmap" for limiting methodological bias and to develop a more mechanistic understanding of the ecophysiology underlying Φe,C. We 1) re-evaluate core physiological processes governing how microalgae invest photosynthetic electron transport-derived energy and reductant into stored carbon versus alternative sinks. Then, we 2) outline steps to facilitate broader uptake and exploitation of FRRf, which could transform our knowledge of aquatic primary productivity. We argue it is time to 3) revise our historic methodological focus on carbon as the currency of choice, to 4) better appreciate that electron transport fundamentally drives ecosystem biogeochemistry, modulates cell-to-cell interactions, and ultimately modifies community biomass and structure.

Fast reactivation of photosynthesis in arctic phytoplankton during the polar night1.

Arctic microalgae experience long periods of continuous darkness during the polar night, when they are unable to photosynthesize. Despite numerous studies on overwintering strategies, such as utilization of stored energy products, formation of resting stages, reduction of metabolic rates and heterotrophic lifestyles, there have been few attempts to assess the in situ physiological state and restoration of the photosynthetic apparatus upon re-illumination. In this study, we found diverse and active marine phytoplankton communities during the polar night at 78°N. Furthermore, we observed rapid changes (≤20 min) in the efficiency of photosynthetic electron transport upon re-illumination. High photosynthetic capacity and net primary production were established after 24 h of re-illumination. Our results suggest that some Arctic autotrophs maintain fully functional photosystem II and downstream electron acceptors during the polar night even though the low in situ net primary production levels measured in January prove that light was not sufficient to support any measurable primary production. Due to low temperatures resulting in low respiratory rates as well as the absence of photodamage during the polar night, maintenance of basic photosynthetic machinery may actually pose relatively low metabolic costs for algal cells. This could allow Arctic microalgae to endure the polar night without the formation of dormant stages, enabling them to recover and take advantage of light immediately upon the suns return during the winter-spring transition.

Diel regulation of photosynthetic activity in the oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501.

The oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501 exhibits large diel changes in abundance of both Photosystem II (PSII) and Photosystem I (PSI). To understand the mechanisms underlying these dynamics, we assessed photosynthetic parameters, photosystem abundance and composition, and chlorophyll-protein biosynthesis over a diel cycle. Our data show that the decline in PSII activity and abundance observed during the dark period was related to a light-induced modification of PSII, which, in combination with the suppressed synthesis of membrane proteins, resulted in monomerization and gradual disassembly of a large portion of PSII core complexes. In the remaining population of assembled PSII monomeric complexes, we detected the non-functional version of the D1 protein, rD1, which was absent in PSII during the light phase. During the dark period, we also observed a significant decoupling of phycobilisomes from PSII and a decline in the chlorophyll a quota, which matched the complete loss of functional PSIIs and a substantial decrease in PSI abundance. However, the remaining PSI complexes maintained their photochemical activity. Thus, during the nocturnal period of nitrogen fixation C. watsonii operates a suite of regulatory mechanisms for efficient utilization/recycling of cellular resources and protection of the nitrogenase enzyme.

Differential effects of changes in spectral irradiance on photoacclimation, primary productivity and growth in Rhodomonas salina (Cryptophyceae) and Skeletonema costatum (Bacillariophyceae) in simulated blackwater environments.

The underwater light field in blackwater environments is strongly skewed toward the red end of the electromagnetic spectrum due to blue light absorption by colored dissolved organic matter (CDOM). Exposure of phytoplankton to full spectrum irradiance occurs only when cells are mixed up to the surface. We studied the potential effects of mixing-induced changes in spectral irradiance on photoacclimation, primary productivity and growth in cultures of the cryptophyte Rhodomonas salina and the diatom Skeletonema costatum. We found that these taxa have very different photoacclimation strategies. While S. costatum showed classical complementary chromatic adaption, R. salina showed inverse chromatic adaptation, a strategy previously unknown in the cryptophytes. Transfer of R. salina to periodic full spectrum light (PFSL) significantly enhanced growth rate (µ) by 1.8 times and primary productivity from 0.88 to 1.35 mg C · (mg Chl-1 ) · h-1 . Overall, R. salina was less dependent on PFSL than was S. costatum, showing higher µ and net primary productivity rates. In the high-CDOM simulation, carbon metabolism of the diatom was impaired, leading to suppression of growth rate, short-term 14 C uptake and net primary production. Upon transfer to PFSL, µ of the diatom increased by up to 3-fold and carbon fixation from 2.4 to 6.0 mg C · (mg Chl-1 ) · h-1 . Thus, a lack of PFSL differentially impairs primarily CO2 -fixation and/or carbon metabolism, which, in turn, may determine which phytoplankton dominate the community in blackwater habitats and may therefore influence the structure and function of these ecosystems.

Carbon use efficiencies and allocation strategies in Prochlorococcus marinus strain PCC 9511 during nitrogen-limited growth.

We studied cell properties including carbon allocation dynamics in the globally abundant and important cyanobacterium Prochlorococcus marinus strain PCC 9511 grown at three different growth rates in nitrogen-limited continuous cultures. With increasing nitrogen limitation, cellular divinyl chlorophyll a and the functional absorption cross section of Photosystem II decreased, although maximal photosynthetic efficiency of PSII remained unaltered across all N-limited growth rates. Chl-specific gross and net carbon primary production were also invariant with nutrient-limited growth rate, but only 20% of Chl-specific gross carbon primary production was retained in the biomass across all growth rates. In nitrogen-replete cells, 60% of the assimilated carbon was incorporated into the protein pool while only 30% was incorporated into carbohydrates. As N limitation increased, new carbon became evenly distributed between these two pools. While many of these physiological traits are similar to those measured in other algae, there are also distinct differences, particularly the lower overall efficiency of carbon utilization. The latter provides new information needed for understanding and estimating primary production, particularly in the nutrient-limited tropical oceans where P. marinus dominates phytoplankton community composition.

Freshwater ice as habitat: partitioning of phytoplankton and bacteria between ice and water in central European reservoirs.

Abundant phytoplankton and bacteria were identified by high-throughput 16S rRNA tag Illumina sequencing of samples from water and ice phases collected during winter at commercial fish ponds and a sand pit lake within the UNESCO Trebon Basin Biosphere Reserve, Czech Republic. Bacterial reads were dominated by Proteobacteria and Bacteroidetes. Despite dominance by members of just two phyla, UniFrac principal coordinates analysis of the bacterial community separated the water community of Klec fish pond, as well as the ice-associated community of Klec-Sand Pit from other samples. Both phytoplankton and cyanobacteria were represented with hundreds of sequence reads per sample, a finding corroborated by microscopy. In particular, ice from Klec-Sand Pit contained high contributions from photoautotrophs accounting for 25% of total reads with reads dominated by single operational taxonomic units (OTUs) of the cyanobacterium Planktothrix sp. and two filamentous diatoms. Dominant OTUs recovered from ice were largely absent (< 0.01%) from underlying water suggestive of low floristic similarity of phytoplankton partitioned between these phases. Photosynthetic characterization of phototrophs resident in water and ice analysed by variable chlorophyll a fluorescence showed that communities from both phases were photosynthetically active, thus supporting ice as viable habitat for phytoplankton in freshwater lakes and reservoirs.

Predicting the electron requirement for carbon fixation in seas and oceans.

Marine phytoplankton account for about 50% of all global net primary productivity (NPP). Active fluorometry, mainly Fast Repetition Rate fluorometry (FRRf), has been advocated as means of providing high resolution estimates of NPP. However, not measuring CO2-fixation directly, FRRf instead provides photosynthetic quantum efficiency estimates from which electron transfer rates (ETR) and ultimately CO2-fixation rates can be derived. Consequently, conversions of ETRs to CO2-fixation requires knowledge of the electron requirement for carbon fixation (Φe,C, ETR/CO2 uptake rate) and its dependence on environmental gradients. Such knowledge is critical for large scale implementation of active fluorescence to better characterise CO2-uptake. Here we examine the variability of experimentally determined Φe,C values in relation to key environmental variables with the aim of developing new working algorithms for the calculation of Φe,C from environmental variables. Coincident FRRf and (14)C-uptake and environmental data from 14 studies covering 12 marine regions were analysed via a meta-analytical, non-parametric, multivariate approach. Combining all studies, Φe,C varied between 1.15 and 54.2 mol e(-) (mol C)(-1) with a mean of 10.9 ± 6.91 mol e(-) mol C)(-1). Although variability of Φe,C was related to environmental gradients at global scales, region-specific analyses provided far improved predictive capability. However, use of regional Φ e,C algorithms requires objective means of defining regions of interest, which remains challenging. Considering individual studies and specific small-scale regions, temperature, nutrient and light availability were correlated with Φ e,C albeit to varying degrees and depending on the study/region and the composition of the extant phytoplankton community. At the level of large biogeographic regions and distinct water masses, Φ e,C was related to nutrient availability, chlorophyll, as well as temperature and/or salinity in most regions, while light availability was also important in Baltic Sea and shelf waters. The novel Φ e,C algorithms provide a major step forward for widespread fluorometry-based NPP estimates and highlight the need for further studying the natural variability of Φe,C to verify and develop algorithms with improved accuracy.

Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy system.

Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter. Here, we describe an instrument that uses a laser trap based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy, thus avoiding interference from the surrounding medium. Quantitative measurements of optical power give data in the form of photons emitted per photon of exposure for an individual phytoplankton cell. Residence times for individual phytoplankton in the instrument can be as long as several minutes with no substantial change in their fluorescence excitation spectra. The laser trap was found to generate two-photon fluorescence from the organisms so a modification was made to release the trap momentarily during data acquisition. Typical signal levels for an individual cell are in the range of 10(6) photons/s of fluorescence using a monochromated 75 W Xe arc lamp excitation source with a 2% transmission neutral density filter.

Spectral fluorometric characterization of phytoplankton community composition using the Algae Online Analyser.

The utility of a multiple-fixed-wavelength spectral fluorometer, the Algae Online Analyser (AOA), as a means of quantifying phytoplankton biomass and community composition was tested using natural communities from two southeastern United States estuaries, North Inlet, South Carolina, and the Neuse River Estuary, North Carolina. Estimates of biomass (as chlorophyll a) were correlated with HPLC values and variations (usually over-estimates) were consistent with effects of light intensity and nutrient availability on fluorescence quenching. AOA estimates of taxonomic structure were consistent with those from HPLC-derived marker pigments by ChemTax, with both methods indicating domination by chromophytes and green algae in North Inlet and chromophytes and cyanobacteria in the Neuse. We recommend frequent calibration by discrete sample collection, and calibration with species representative of the region of interest. Overall, the AOA appears to be a useful tool for monitoring of phytoplankton community composition, especially as an early warning system for the detection of harmful algal blooms.