ALBERT

Description: Motivation: Studies of the biochemical functions and activities of uncultivated microorganisms in the environment require analysis of DNA sequences for phylogenetic characterization and for the development of sequence-based assays for the detection of microorganisms. The numbers of sequences for genes that are indicators of environmentally important functions such as nitrogen (N 2 ) fixation have been rapidly growing over the past few decades. Obtaining these sequences from the National Center for Biotechnology Information’s GenBank database is problematic because of annotation errors, nomenclature variation and paralogues; moreover, GenBank’s structure and tools are not conducive to searching solely by function. For some genes, such as the nifH gene commonly used to assess community potential for N 2 fixation, manual collection and curation are becoming intractable because of the large number of sequences in GenBank and the large number of highly similar paralogues. If analysis is to keep pace with sequence discovery, an automated retrieval and curation system is necessary. Results: ARBitrator uses a two-step process composed of a broad collection of potential homologues followed by screening with a best hit strategy to conserved domains. 34 420 nifH sequences were identified in GenBank as of November 20, 2012. The false-positive rate is ~0.033%. ARBitrator rapidly updates a public nifH sequence database, and we show that it can be adapted for other genes. Availability and implementation: Java source and executable code are freely available to non-commercial users at http://pmc.ucsc.edu/~wwwzehr/research/database/ . Contact: zehrj@ucsc.edu Supplementary information: Supplementary information is available at Bioinformatics online.

Description: Nitrogen (N) is the major limiting nutrient for phytoplankton growth and productivity in large parts of the world's oceans. Differential preferences for specific N substrates may be important in controlling phytoplankton community composition. To date, there is limited information on how specific N substrates influence the composition of naturally occurring microbial communities. We investigated the effect of nitrate ( ), ammonium ( ), and urea on microbial and phytoplankton community composition (cell abundances and 16S rRNA gene profiling) and functioning (photosynthetic activity, carbon fixation rates) in the oligotrophic waters of the North Pacific Ocean. All N substrates tested significantly stimulated phytoplankton growth and productivity. Urea resulted in the greatest (〉300%) increases in chlorophyll a (〈0.06 μ g L −1 and ∼0.19 μ g L −1 in the control and urea addition, respectively) and productivity (〈0.4 μ mol C L −1 d −1 and ∼1.4 μ mol C L −1 d −1 in the control and urea addition, respectively) at two experimental stations, largely due to increased abundances of Prochlorococcus (Cyanobacteria). Two abundant clades of Prochlorococcus , High Light I and II, demonstrated similar responses to urea, suggesting this substrate is likely an important N source for natural Prochlorococcus populations. In contrast, the heterotrophic community composition changed most in response to . Finally, the time and magnitude of response to N amendments varied with geographic location, likely due to differences in microbial community composition and their nutrient status. Our results provide support for the hypothesis that changes in N supply would likely favor specific populations of phytoplankton in different oceanic regions and thus, affect both biogeochemical cycles and ecological processes.

Description: One challenge in field-based marine microbial ecology is to achieve sufficient spatial resolution to obtain representative information about microbial distributions and biogeochemical processes. The challenges are exacerbated when conducting rate measurements of biological processes due to potential perturbations during sampling and incubation. Here we present the first application of a robotic microlaboratory, the 4 L-submersible incubation device (SID), for conducting in situ measurements of the rates of biological nitrogen (N 2 ) fixation (BNF). The free-drifting autonomous instrument obtains samples from the water column that are incubated in situ after the addition of 15 N 2 tracer. After each of up to four consecutive incubation experiments, the 4-L sample is filtered and chemically preserved. Measured BNF rates from two deployments of the SID in the oligotrophic North Pacific ranged from 0.8 to 2.8 nmol N L –1 day –1 , values comparable with simultaneous rate measurements obtained using traditional conductivity–temperature–depth (CTD)–rosette sampling followed by on-deck or in situ incubation. Future deployments of the SID will help to better resolve spatial variability of oceanic BNF, particularly in areas where recovery of seawater samples by CTD compromises their integrity, e.g. anoxic habitats.

Description: Marine N2 fixing microorganisms, termed diazotrophs, are a key functional group in marine pelagic ecosystems. The biological fixation of dinitrogen (N2) to bioavailable nitrogen provides an important new source of nitrogen for pelagic marine ecosystems and influences primary productivity and organic matter export to the deep ocean. As one of a series of efforts to collect biomass and rates specific to different phytoplankton functional groups, we have constructed a database on diazotrophic organisms in the global pelagic upper ocean by compiling about 12 000 direct field measurements of cyanobacterial diazotroph abundances (based on microscopic cell counts or qPCR assays targeting the nifH genes) and N2 fixation rates. Biomass conversion factors are estimated based on cell sizes to convert abundance data to diazotrophic biomass. The database is limited spatially, lacking large regions of the ocean especially in the Indian Ocean. The data are approximately log-normal distributed, and large variances exist in most sub-databases with non-zero values differing 5 to 8 orders of magnitude. Reporting the geometric mean and the range of one geometric standard error below and above the geometric mean, the pelagic N2 fixation rate in the global ocean is estimated to be 62 (52–73) Tg N yr−1 and the pelagic diazotrophic biomass in the global ocean is estimated to be 2.1 (1.4–3.1) Tg C from cell counts and to 89 (43–150) Tg C from nifH-based abundances. Reporting the arithmetic mean and one standard error instead, these three global estimates are 140 ± 9.2 Tg N yr−1, 18 ± 1.8 Tg C and 590 ± 70 Tg C, respectively. Uncertainties related to biomass conversion factors can change the estimate of geometric mean pelagic diazotrophic biomass in the global ocean by about ±70%. It was recently established that the most commonly applied method used to measure N2 fixation has underestimated the true rates. As a result, one can expect that future rate measurements will shift the mean N2 fixation rate upward and may result in significantly higher estimates for the global N2 fixation. The evolving database can nevertheless be used to study spatial and temporal distributions and variations of marine N2 fixation, to validate geochemical estimates and to parameterize and validate biogeochemical models, keeping in mind that future rate measurements may rise in the future. The database is stored in PANGAEA (doi:10.1594/PANGAEA.774851).

Description: Marine N2 fixing microorganisms, termed diazotrophs, are a key functional group in marine pelagic ecosystems. The biological fixation of dinitrogen (N2) to bioavailable nitrogen provides an important new source of nitrogen for pelagic marine ecosystems and influences primary productivity and organic matter export to the deep ocean. As one of a series of efforts to collect biomass and rates specific to different phytoplankton functional groups, we have constructed a database on diazotrophic organisms in the global pelagic upper ocean by compiling about 12 000 direct field measurements of cyanobacterial diazotroph abundances (based on microscopic cell counts or qPCR assays targeting the nifH genes) and N2 fixation rates. Biomass conversion factors are estimated based on cell sizes to convert abundance data to diazotrophic biomass. The database is limited spatially, lacking large regions of the ocean especially in the Indian Ocean. The data are approximately log-normal distributed, and large variances exist in most sub-databases with non-zero values differing 5 to 8 orders of magnitude. Lower mean N2 fixation rate was found in the North Atlantic Ocean than the Pacific Ocean. Reporting the geometric mean and the range of one geometric standard error below and above the geometric mean, the pelagic N2 fixation rate in the global ocean is estimated to be 62 (53–73) Tg N yr−1 and the pelagic diazotrophic biomass in the global ocean is estimated to be 4.7 (2.3–9.6) Tg C from cell counts and to 89 (40–200) Tg C from nifH-based abundances. Uncertainties related to biomass conversion factors can change the estimate of geometric mean pelagic diazotrophic biomass in the global ocean by about ±70%. This evolving database can be used to study spatial and temporal distributions and variations of marine N2 fixation, to validate geochemical estimates and to parameterize and validate biogeochemical models. The database is stored in PANGAEA (http://doi.pangaea.de/10.1594/PANGAEA.774851).

Description: The VAHINE mesocosm experiment, conducted in the low-nutrient low-chlorophyll waters of the Noumea Lagoon (coastal New Caledonia) was designed to trace the incorporation of nitrogen (N) fixed by diazotrophs into the food web, using large volume (50 m3) mesocosms. This experiment provided a unique opportunity to study the succession of different N2-fixing microorganisms (diazotrophs) and calculate in situ net growth and loss rates in response to fertilization with dissolved inorganic phosphate (DIP) over a 23 day period, using quantitative polymerase chain reaction (qPCR) assays. Inside the mesocosms, the diazotroph community assemblage was dominated by the heterocyst-forming Richelia associated with Rhizosolenia (Het-1) in the first half of the experiment, and unicellularcyanobacterial Group C (UCYN-C) became the dominant diazotroph in the second half of the experiment. Decreasing DIP concentrations following the fertilization event and increasing temperatures were significantly correlated with increasing abundances of UCYN-C. Maximum net growth rates for UCYN-C were calculated to be between 1.23 ± 0.07 and 2.16 ± 0.07 d−1 which are among the highest growth rates reported for diazotrophs. Outside the mesocosms in the Noumea Lagoon, UCYN-C abundances remained low, despite increasing temperatures, suggesting that the microbial community response to the DIP fertilization created conditions favorable for UCYN-C growth inside the mesocosms. Maximum net growth and loss rates for nine diazotroph phylotypes throughout the 23 day experiment were variable between mesocosms, and repeated fluctuations between periods of net growth and loss were commonly observed. The field population of diazotrophs in the Noumea Lagoon, was dominated by Het-1 over the course of the study period. However, eight additional diazotroph phylotypes were present in the lagoon at lower abundances, indicating a diverse field population of diazotrophs. Two ecotypes of the Braarudosphaera bigelowii symbiont unicellular cyanobacterial group A (UCYN-A) were present simultaneously in the lagoon, with the recently described B. bigelowii/UCYN-A2 association present at higher abundances than the B. bigelowii/UCYN-A1 association.

Description: N2 fixation rates were measured daily in large (~ 50 m3) mesocosms deployed in the tropical South West Pacific coastal ocean (New Caledonia) to investigate the spatial and temporal dynamics of diazotrophy and the fate of diazotroph-derived nitrogen (DDN) in a low nutrient, low chlorophyll ecosystem. The mesocosms were intentionally fertilized with ~ 0.8 μM dissolved inorganic phosphorus (DIP) to stimulate diazotrophy. Bulk N2 fixation rates were replicable between the three mesocosms, averaged 18.5 ± 1.1 nmol N L−1 d−1 over the 23 days, and increased by a factor of two during the second half of the experiment (days 15 to 23) to reach 27.3 ± 1.0 nmol N L−1 d−1. These rates are higher than the upper range reported for the global ocean, indicating that the waters surrounding New Caledonia are particularly favourable for N2 fixation. During the 23 days of the experiment, N2 fixation rates were positively correlated with seawater temperature, primary production, bacterial production, standing stocks of particulate organic carbon, nitrogen and phosphorus, and alkaline phosphatase activity, and negatively correlated with DIP concentrations, DIP turnover time, nitrate, and dissolved organic nitrogen and phosphorus concentrations. The fate of DDN was investigated during the bloom of the unicellular diazotroph, UCYN-C, that occurred during the second half of the experiment. Quantification of diazotrophs in the sediment traps indicates that ~ 10 % of UCYN-C from the water column were exported daily to the traps, representing as much as 22.4 ± 5.5 % of the total POC exported at the height of the UCYN-C bloom. This export was mainly due to the aggregation of small (5.7 ± 0.8 μm) UCYN-C cells into large (100–500 μm) aggregates. During the same time period, a DDN transfer experiment based on high-resolution nanometer scale secondary ion mass spectrometry (nanoSIMS) coupled with 15N2 isotopic labelling revealed that 16 ± 6 % of the DDN was released to the dissolved pool and 21 ± 4 % was transferred to non-diazotrophic plankton, mainly picoplankton (18 ± 4 %) followed by diatoms (3 ± 2 %) within 24 h of incubation. This is consistent with the observed dramatic increase in picoplankton and diatom abundances, primary production, bacterial production and standing stocks of particulate organic carbon, nitrogen and phosphorus during the second half of the experiment in the mesocosms. These results offer insights into the fate of DDN during a bloom of UCYN-C in low nutrient, low chlorophyll ecosystems.

Description: The VAHINE mesocosm experiment was designed to trigger a diazotroph bloom and to follow the subsequent transfer of diazotroph derived nitrogen (DDN) in the rest of the foodweb. Three mesocosms (50 m3) located inside the Nouméa lagoon (New Caledonia, South West Pacific) were enriched with dissolved inorganic phosphate (DIP) in order to promote N2 fixation in these Low Nutrient Low Chlorophyll (LNLC) waters. Initial diazotrophic community were dominated by diatom diazotroph associations (DDAs), mainly by Rhizosolenia/Richelia intracellularis, and by Trichodesmium which fueled enough DDN to sustain the growth of other diverse diatom species and Synechococcus populations, that were well adapted to limiting DIP levels. After DIP fertilization (1 mM) on day 4, an initial lag time of 10 days was necessary for the mesocosm ecosystems to start building up biomass. Yet changes in community structure were already observed during this first period, with a significant drop of both Synechococcus and diatom populations, while Prochlorococcus benefited from DIP-addition. At the end of this first period, corresponding to when most added DIP was consumed, the diazotroph community changed drastically and became dominated by UCYN-C populations, which were accompanied by a monospecific bloom of the diatom Cylindrotheca closterium. During the second period, biomass increased sharply together with primary production and N2 fixation fluxes near tripled. Diatom populations, as well as Synechococcus and nano-phytoeukaryotes showed a re-increase towards the end of the experiment, showing efficient transfer of DDN to non diazotrophic phytoplankton.