Margaret R. Mulholland

Marine phytoplankton temperature versus growth responses from polar to tropical waters – outcome of a scientific community-wide study

“It takes a village to finish (marine) science these days” Paraphrased from Curtis Huttenhower (the Human Microbiome project) The rapidity and complexity of climate change and its potential effects on ocean biota are challenging how ocean scientists conduct research. One way in which we can begin to better tackle these challenges is to conduct community-wide scientific studies. This study provides physiological datasets fundamental to understanding functional responses of phytoplankton growth rates to temperature. While physiological experiments are not new, our experiments were conducted in many laboratories using agreed upon protocols and 25 strains of eukaryotic and prokaryotic phytoplankton isolated across a wide range of marine environments from polar to tropical, and from nearshore waters to the open ocean. This community-wide approach provides both comprehensive and internally consistent datasets produced over considerably shorter time scales than conventional individual and often uncoordinated lab efforts. Such datasets can be used to parameterise global ocean model projections of environmental change and to provide initial insights into the magnitude of regional biogeographic change in ocean biota in the coming decades. Here, we compare our datasets with a compilation of literature data on phytoplankton growth responses to temperature. A comparison with prior published data suggests that the optimal temperatures of individual species and, to a lesser degree, thermal niches were similar across studies. However, a comparison of the maximum growth rate across studies revealed significant departures between this and previously collected datasets, which may be due to differences in the cultured isolates, temporal changes in the clonal isolates in cultures, and/or differences in culture conditions. Such methodological differences mean that using particular trait measurements from the prior literature might introduce unknown errors and bias into modelling projections. Using our community-wide approach we can reduce such protocol-driven variability in culture studies, and can begin to address more complex issues such as the effect of multiple environmental drivers on ocean biota.

NUTRIENT CONTROLS ON NITROGEN UPTAKE AND METABOLISM BY NATURAL POPULATIONS AND CULTURES OF TRICHODESMIUM (CYANOBACTERIA)

The effects of inorganic nutrient (ammonium [NH4+] and nitrate [NO3−]) and amino acid (glutamate [glu] and glutamine [gln]) additions on rates of N2 fixation, N uptake, glutamine synthetase (GS) activity, and concentrations of intracellular pools of gln and glu were examined in natural and cultured populations of Trichodesmium. Additions of 1 μM glu, gln, NO3−, or NH4+ did not affect short‐term rates of N2 fixation. This may be an important factor that allows for continued N2 fixation in oligotrophic areas where recycling processes are active. N2 fixation rates decreased when nutrients were supplied at higher concentrations (e.g. 10 μM). Uptake of combined N (NH4+, NO3−, and amino acids) by Trichodesmium was stimulated by increased concentrations. For NO3−, proportional increases in NO3− uptake and decreases in N2 fixation were observed when additions were made to cultures before the onset of the light period. GS activity did not change much in response to the addition of NH4+, NO3−, glu, or gln. GS is necessary for N metabolism, and the bulk of this enzyme pool may be conserved. Intracellular pools of glu and gln varied in response to 10 μM additions of NH4+, glu, or gln. Cells incubated with NH4+ became depleted in intracellular glu and enriched with intracellular gln. The increase in the gln/glu ratio corresponded to a decrease in the rate of N2 fixation. Although the gln/glu ratio decreased in cells exposed to the amino acids, there was only a corresponding decrease in N2 fixation after the gln addition. The results presented here suggest that combined N concentrations on the order of 1 μM do not affect rates of N2 fixation and metabolism, although higher concentrations (e.g. 10 μM) can. Moreover, these effects are exerted through products of NH4+ assimilation rather than exogenous N, as has been suggested for other species. These results may help explain how cultures of Trichodesmium are able to simultaneously fix N2 and take up NH4+ and how natural populations continue to fix N2 once combined N concentrations increase within a bloom.

Ocean urea fertilization for carbon credits poses high ecological risks

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed.

Dinitrogen Fixation in Aphotic Oxygenated Marine Environments

We measured N2 fixation rates from oceanic zones that have traditionally been ignored as sources of biological N2 fixation; the aphotic, fully oxygenated, nitrate (NO−3)-rich, waters of the oligotrophic Levantine Basin (LB) and the Gulf of Aqaba (GA). N2 fixation rates measured from pelagic aphotic waters to depths up to 720 m, during the mixed and stratified periods, ranged from 0.01 nmol N L−1 d−1 to 0.38 nmol N L−1 d−1. N2 fixation rates correlated significantly with bacterial productivity and heterotrophic diazotrophs were identified from aphotic as well as photic depths. Dissolved free amino acid amendments to whole water from the GA enhanced bacterial productivity by 2–3.5 fold and N2 fixation rates by ~2-fold in samples collected from aphotic depths while in amendments to water from photic depths bacterial productivity increased 2–6 fold while N2 fixation rates increased by a factor of 2 to 4 illustrating that both BP and heterotrophic N2 fixation were carbon limited. Experimental manipulations of aphotic waters from the LB demonstrated a significant positive correlation between transparent exopolymeric particle (TEP) concentrations and N2 fixation rates. This suggests that sinking organic material and high carbon (C): nitrogen (N) micro-environments (such as TEP-based aggregates or marine snow) could support high heterotrophic N2 fixation rates in oxygenated surface waters and in the aphotic zones. Indeed, our calculations show that aphotic N2 fixation accounted for 37 to 75% of the total daily integrated N2 fixation rates at both locations in the Mediterranean and Red Seas with rates equal or greater to those measured from the photic layers. Moreover, our results indicate that that while N2 fixation may be limited in the surface waters, aphotic, pelagic N2 fixation may contribute significantly to new N inputs in other oligotrophic basins, yet it is currently not included in regional or global N budgets.

Is dinitrogen fixation significant in the Levantine Basin, East Mediterranean Sea?

We report N(2) fixation rates measured from two stations monitored monthly off the Mediterranean coast of Israel during 2006 and 2007, and along a transect from Israel to Crete in September 2008. Analyses of time-series data revealed expression of nifH genes from diazotrophs in nifH clusters I and II, including cyanobacterial bloom-formers Trichodesmium and diatom-Richelia intracellularis associations. However, nifH gene abundance and rates of N(2) fixation were very low in all size fractions measured (> 0.7 µm). Volumetric (15) N uptake ranged from below detection (∼ 36% of > 300 samples) to a high of 0.3 nmol N l(-1) d(-1) and did not vary distinctly with depth or season. Areal N(2) fixation averaged ∼ 1 to 4 µmol N m(-2) d(-1) and contributed only ∼ 1% and 2% of new production and ∼ 0.25% and 0.5% of primary production for the mixed (winter) and stratified (spring-fall) periods respectively. N(2) fixation rates along the 2008 east-west transect were also extremely low (0-0.04 nmol N l(-1) d(-1), integrated average 2.6 µmol N m(-2) d(-1) ) with 37% of samples below detection and no discernable difference between stations. We demonstrate that diazotrophy and N(2) fixation contribute only a minor amount of new N to the P impoverished eastern Mediterranean Sea.

Effluent organic nitrogen (EON): bioavailability and photochemical and salinity-mediated release.

The goal of this study was to investigate three potential ways that the soluble organic nitrogen (N) fraction of wastewater treatment plant (WWTP) effluents, termed effluent organic N (EON), could contribute to coastal eutrophication--direct biological removal, photochemical release of labile compounds, and salinity-mediated release of ammonium (NH4+). Effluents from two WWTPs were used in the experiments. For the bioassays, EON was added to water from four salinities (approximately 0 to 30) collected from the James River (VA) in August 2008, and then concentrations of N and phosphorus compounds were measured periodically over 48 h. Bioassay results, based on changes in DON concentrations, indicate that some fraction of the EON was removed and that the degree of EON removal varied between effluents and with salinity. Further, we caution that bioassay results should be interpreted within a broad context of detailed information on chemical characterization. EON from both WWTPs was also photoreactive, with labile NH4+ and dissolved primary amines released during exposure to sunlight. We also present the first data that demonstrate that when EON is exposed to higher salinities, increasing amounts of NH4+ are released, further facilitating EON use as effluent transits from freshwater through estuaries to the coast.

Diversity, distribution, and expression of diazotroph nifH genes in oxygen-deficient waters of the Arabian Sea

The Arabian Sea oxygen minimum zone (OMZ), the largest suboxic region in the worlds oceans, is responsible for up to half of the global mesopelagic fixed nitrogen (N) loss from the ocean via denitrification and anammox. Dinitrogen (N(2)) fixation is usually attributed to cyanobacteria in the surface ocean. Model prediction and physiological inhibition of N(2) fixation by oxygen, however, suggest that N(2) fixation should be enhanced near the oxygen-deficient zone (ODZ) of the Arabian Sea. N(2) fixation and cyanobacterial nifH genes (the gene encoding dinitrogenase reductase) have been reported in surface waters overlying the Arabian Sea ODZ. Here, water samples from depths above and within the Arabian Sea ODZ were examined to explore the distribution, diversity, and expression of nifH genes. In surface waters, nifH DNA and cDNA sequences related to Trichodesmium, a diazotroph known to occur and fix N(2) in the Arabian Sea, were detected. Proteobacterial nifH phylotypes (DNA but not cDNA) were also detected in surface waters. Proteobacterial nifH DNA and cDNA sequences, as well as nifH DNA and cDNA sequences related to strictly anaerobic N -fixers, were obtained from oxygen-deficient depths. This first report of nifH gene expression in subsurface low-oxygen waters suggests that there is potential for active N(2) fixation by several phylogenetically and potentially metabolically diverse microorganisms in pelagic OMZs.

Reactivity and chemical characterization of effluent organic nitrogen from wastewater treatment plants determined by Fourier transform ion cyclotron resonance mass spectrometry

In advanced wastewater treatment plants that achieve high levels of nitrogen (N) removal, up to one-third of the N in effluent is organic, herein referred to as effluent organic N (EON). While we know that inorganic N is highly labile, it is unclear what fraction of EON is bioavailable. In this study, we demonstrate the utility of a method that can be used to examine the reactivity of EON in natural receiving waters to better understand both the ecosystem response and the potential bioavailability of EON. The technique is suitable for analyzing polar organic matter in natural waters; electrospray ionization coupled with Fourier transform mass spectrometry. Bioassays were performed on samples collected at the end of the biological process from two wastewater treatment plants achieving advanced N removal. The samples were concentrated, and then added to natural water samples collected from the oligohaline James River, a major tributary of the Chesapeake Bay. Our results demonstrate that while the lignin-like fraction of the effluent dissolved organic matter (some of which contains N) was conserved, a large portion of aliphatic and aromatic compounds containing N was removed (79-100%) during incubations, while other compounds were produced. Furthermore, the two effluents exhibited differences in the degree of degradation and type of degradation, which can be related both to the various processes employed in the two WWTPs and the dramatic differences in the type of influent they received. These findings suggest that EON is highly reactive in the natural environment and that simple assays examining net consumption or production of bulk dissolved organic N pools are inadequate for assessing the bioavailability of EON.

INTERACTIVE EFFECTS OF IRRADIANCE AND CO2 ON CO2 FIXATION AND N2 FIXATION IN THE DIAZOTROPH TRICHODESMIUM ERYTHRAEUM (CYANOBACTERIA)(1).

The diazotrophic cyanobacteria Trichodesmium spp. contribute approximately half of the known marine dinitrogen (N2) fixation. Rapidly changing environmental factors such as the rising atmospheric partial pressure of carbon dioxide (pCO2) and shallower mixed layers (higher light intensities) are likely to affect N2‐fixation rates in the future ocean. Several studies have documented that N2 fixation in laboratory cultures of T. erythraeum increased when pCO2 was doubled from present‐day atmospheric concentrations (∼380 ppm) to projected future levels (∼750 ppm). We examined the interactive effects of light and pCO2 on two strains of T. erythraeum Ehrenb. (GBRTRLI101 and IMS101) in laboratory semicontinuous cultures. Elevated pCO2 stimulated gross N2‐fixation rates in cultures growing at 38 μmol quanta · m−2 · s−1 (GBRTRLI101 and IMS101) and 100 μmol quanta · m−2 · s−1 (IMS101), but this effect was reduced in both strains growing at 220 μmol quanta · m−2 · s−1. Conversely, CO2‐fixation rates increased significantly (P < 0.05) in response to high pCO2 under mid‐ and high irradiances only. These data imply that the stimulatory effect of elevated pCO2 on CO2 fixation and N2 fixation by T. erythraeum is correlated with light. The ratio of gross:net N2 fixation was also correlated with light and trichome length in IMS101. Our study suggests that elevated pCO2 may have a strong positive effect on Trichodesmium gross N2 fixation in intermediate and bottom layers of the euphotic zone, but perhaps not in light‐saturated surface layers. Climate change models must consider the interactive effects of multiple environmental variables on phytoplankton and the biogeochemical cycles they mediate.

INTENSE GRAZING AND PREY‐DEPENDENT GROWTH OF PFIESTERIA PISCICIDA (DINOPHYCEAE)1

Grazing and growth of Pfiesteria piscicida (Pfiest) were investigated using batch and cyclostat cultures with Rhodomonas sp. (Rhod) as prey. Observed maximum growth rates (1.4 d−1) and population densities (2 × 105 cells·mL−1) corresponded to values predicted by Monod functions (1.76 d−1; 1.4 × 105 cells·mL−1). In batch cultures under a range of prey‐to‐predator ratios (0.1:1 to 180:1) and prey concentrations (1000–71,000 cells·mL−1), Rhodomonas sp. was always depleted rapidly and P. piscicida concentrations increased briefly. The rate of Rhodomonas sp. depletion and the magnitude of P. piscicida population maxima depended on the prey‐to‐predator ratio and prey concentration. Starvation resulted in cell cycle arrest at G1 and G2+M and ultimately the demise of both P. piscicida and Rhodomonas sp. populations, demonstrating the dependence of P. piscicida on the supply of appropriate prey. The depletion of Rhodomonas sp. populations could be attributed directly to grazing, because P. piscicida did not exert detectable inhibitory effects on the growth of Rhodomonas sp. but grazed intensely, with maximum grazing rates>10 Rhod·Pfiest−1·d−1 and with no apparent threshold prey abundance for grazing. The results suggest that 1) the abundance of appropriate prey may be an important factor regulating P. piscicida abundance in nature, 2) P. piscicida may control prey population, and 3) high growth and grazing potentials of P. piscicida along with cell cycle arrest may confer survival advantages.