In Situ Diazotroph Population Dynamics Under Different Resource Ratios in the North Pacific Subtropical Gyre.

Major advances in understanding the diversity, distribution, and activity of marine N2-fixing microorganisms (diazotrophs) have been made in the past decades, however, large gaps in knowledge remain about the environmental controls on growth and mortality rates. In order to measure diazotroph net growth rates and microzooplankton grazing rates on diazotrophs, nutrient perturbation experiments and dilution grazing experiments were conducted using free-floating in situ incubation arrays in the vicinity of Station ALOHA in March 2016. Net growth rates for targeted diazotroph taxa as well as Prochlorococcus, Synechococcus and photosynthetic picoeukaryotes were determined under high (H) and low (L) nitrate:phosphate (NP) ratio conditions at four depths in the photic zone (25, 45, 75, and 100 m) using quantitative PCR and flow cytometry. Changes in the prokaryote community composition in response to HNP and LNP treatments were characterized using 16S rRNA variable region tag sequencing. Microzooplankton grazing rates on diazotrophs were measured using a modified dilution technique at two depths in the photic zone (15 and 125 m). Net growth rates for most of the targeted diazotrophs after 48 h were not stimulated as expected by LNP conditions, rather enhanced growth rates were often measured in HNP treatments. Interestingly, net growth rates of the uncultivated prymnesiophyte symbiont UCYN-A1 were stimulated in HNP treatments at 75 and 100 m, suggesting that N used for growth was acquired through continuing to fix N2 in the presence of nitrate. Net growth rates for UCYN-A1, UCYN-C, Crocosphaera sp. (UCYN-B) and the diatom symbiont Richelia (associated with Rhizosolenia) were uniformly high at 45 m (up to 1.6 ± 0.5 d-1), implying that all were growing optimally at the onset of the experiment at that depth. Differences in microzooplankton grazing rates on UCYN-A1 and UCYN-C in 15 m waters indicate that the grazer assemblage preyed preferentially on UCYN-A1. Deeper in the water column (125 m), both diazotrophs were grazed at substantial rates, suggesting grazing pressure may increase with depth in the photic zone. Constraining in situ diazotroph growth and mortality rates are important steps for improving parameterization for diazotrophs in global ecosystem models

Diverse diazotrophs are present on sinking particles in the North Pacific Subtropical Gyre.

Sinking particles transport carbon and nutrients from the surface ocean into the deep sea and are considered hot spots for bacterial diversity and activity. In the oligotrophic oceans, nitrogen (N2)-fixing organisms (diazotrophs) are an important source of new N but the extent to which these organisms are present and exported on sinking particles is not well known. Sinking particles were collected every 6 h over a 2-day period using net traps deployed at 150 m in the North Pacific Subtropical Gyre. The bacterial community and composition of diazotrophs associated with individual and bulk sinking particles was assessed using 16S rRNA and nifH gene amplicon sequencing. The bacterial community composition in bulk particles remained remarkably consistent throughout time and space while large variations of individually picked particles were observed. This difference suggests that unique biogeochemical conditions within individual particles may offer distinct ecological niches for specialized bacterial taxa. Compared to surrounding seawater, particle samples were enriched in different size classes of globally significant N2-fixing cyanobacteria including Trichodesmium, symbionts of diatoms, and the unicellular cyanobacteria Crocosphaera and UCYN-A. The particles also contained nifH gene sequences of diverse non-cyanobacterial diazotrophs suggesting that particles could be loci for N2 fixation by heterotrophic bacteria. The results demonstrate that diverse diazotrophs were present on particles and that new N may thereby be directly exported from surface waters on sinking particles

Consistency in microbiomes in cultures of Alexandrium species isolated from brackish and marine waters

Phytoplankton and bacteria interactions have a significant role in aquatic ecosystem functioning. Associations can range from mutualistic to parasitic, shaping biogeochemical cycles and having a direct influence on phytoplankton growth. How variations in phenotype and sampling location, affect the phytoplankton microbiome is largely unknown. A high-resolution characterization of the bacterial community in cultures of the dinoflagellate Alexandrium was performed on strains isolated from different geographical locations and at varying anthropogenic impact levels. Microbiomes of Baltic Sea Alexandrium ostenfeldii isolates were dominated by Betaproteobacteria and were consistent over phenotypic and genotypic Alexandrium strain variation, resulting in identification of an A. ostenfeldii core microbiome. Comparisons with in situ bacterial communities showed that taxa found in this A. ostenfeldii core were specifically associated to dinoflagellate dynamics in the Baltic Sea. Microbiomes of Alexandrium tamarense and minutum, isolated from the Mediterranean Sea, differed from those of A. ostenfeldii in bacterial diversity and composition but displayed high consistency, and a core set of bacterial taxa was identified. This indicates that Alexandrium isolates with diverse phenotypes host predictable, species-specific, core microbiomes reflecting the abiotic conditions from which they were isolated. These findings enable in-depth studies of potential interactions occurring between Alexandrium and specific bacterial taxa

Short-term variability in the activity and composition of the diazotroph community in a coastal upwelling system

Today we know that diazotrophs are common and active in nitrogen (N) replete regions, however the factors controlling their distribution remain elusive. Previous studies in upwelling regions revealed that the composition of diazotrophs responded to changes in hydrodynamic forcing over seasonal scales. Here we used high-frequency observations collected during a 3-week cruise in the upwelling region off NW Iberia to describe changes in the activity and composition of diazotrophs over shorter temporal scales. The cruise started after a strong upwelling event followed by a few days of relaxation-downwelling, and soon after another upwelling pulse. Higher N2 fixation rates (2.2 ± 0.7 µmol m-3 d-1) were measured during relaxation-downwelling, when surface nitrate concentration was low. During the fertilization associated with the upwelling, N2 fixation dramatically decreased to 0.10 ± 0.09 µmol m-3 d-1. The comparison with nitrate consumption and diffusion confirmed the minor role of N2 fixation (<1%) as a source of new N for primary production. The unicellular cyanobacterium UCYN-A2 was the dominant diazotroph during the cruise. UCYN-A2 abundance was four times higher during relaxation-downwelling (4x104 copies L-1) compared to upwelling conditions (0.2x104 copies L-1), when the unusual Epsilonproteobacteria increased their relative abundance. These results indicate that diazotrophs can respond rapidly to changes in the environment, and point out to the availability of N as a key factor controlling the activity, composition and distribution of diazotrophs in eutrophic regions

Nitrogenase Gene Amplicons from Global Marine Surface Waters Are Dominated by Genes of Non-Cyanobacteria

Cyanobacteria are thought to be the main N2-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant

A denitrifying community associated with a major, marine nitrogen fixer

The diazotrophic cyanobacterium, Trichodesmium, is an integral component of the marine nitrogen cycle and contributes significant amounts of new nitrogen to oligotrophic, tropical/subtropical ocean surface waters. Trichodesmium forms macroscopic, fusiform (tufts), spherical (puffs) and raft-like colonies that provide a pseudobenthic habitat for a host of other organisms including marine invertebrates, microeukaryotes and numerous other microbes. The diversity and activity of denitrifying bacteria found in association with the colonies was interrogated using a series of molecular-based methodologies targeting the gene encoding the terminal step in the denitrification pathway, nitrous oxide reductase (nosZ). Trichodesmium spp. sampled from geographically isolated ocean provinces (the Atlantic Ocean, the Red Sea and the Indian Ocean) were shown to harbor highly similar, taxonomically related communities of denitrifiers whose members are affiliated with the Roseobacter clade within the Rhodobacteraceae (Alphaproteobacteria). These organisms were actively expressing nosZ in samples taken from the mid-Atlantic Ocean and Red Sea implying that Trichodesmium colonies are potential sites of nitrous oxide consumption and perhaps earlier steps in the denitrification pathway also. It is proposed that coupled nitrification of newly fixed N is the most likely source of nitrogen oxides supporting nitrous oxide cycling within Trichodesmium colonies

Doubling of marine dinitrogen-fixation rates based on direct measurements

Biological dinitrogen fixation provides the largest input of nitrogen to the oceans, therefore exerting important control on the ocean’s nitrogen inventory and primary productivity. Nitrogen-isotope data fromocean sediments suggest that the marine-nitrogen inventory has been balanced for the past 3,000 years (ref. 4). Producing a balanced marine-nitrogenbudget based on direct measurements has proved difficult, however, with nitrogen loss exceeding the gain from dinitrogen fixation by approximately 200 TgNyr-1 (refs 5, 6). Here we present data from the Atlantic Ocean and show that the most widely used method of measuring oceanic N2-fixation rates underestimates the contribution of N2-fixing microorganisms (diazotrophs) relative to a newly developed method. Using molecular techniques to quantify the abundance of specific clades of diazotrophs in parallel with rates of 15N2 incorporation into particulate organic matter, we suggest that the difference between N2-fixation rates measured with the established method and those measured with the new method8 can be related to the composition of the diazotrophic community. Our data show that in areas dominated by Trichodesmium, the established method underestimatesN2-fixation rates by an averageof 62%. We also find that the newly developed method yields N2-fixation rates more than six times higher than those from the established method when unicellular, symbiotic cyanobacteria and c-proteobacteria dominate the diazotrophic community. On the basis of average areal rates measured over the Atlantic Ocean, we calculated basin-wide N2-fixation rates of 14+/-1TgNyr-1 and 24+/-1TgNyr-1 for the established and new methods, respectively. If our findings can be extrapolated to other ocean basins, this suggests that the global marine N2-fixation rate derived from direct measurements may increase from 103+/-8TgNyr-1 to 177+/-8TgNyr-1, and that the contribution of N2 fixers other than Trichodesmium is much more significant than was previously thought

Functional responses of key marine bacteria to environmental change - toward genetic counselling for coastal waters

Coastal ecosystems deteriorate globally due to human-induced stress factors, like nutrient loading and pollution. Bacteria are critical to marine ecosystems, e.g., by regulating nutrient cycles, synthesizing vitamins, or degrading pollutants, thereby providing essential ecosystem services ultimately affecting economic activities. Yet, until now bacteria are overlooked both as mediators and indicators of ecosystem health, mainly due to methodological limitations in assessing bacterial ecosystem functions. However, these limitations are largely overcome by the advances in molecular biology and bioinformatics methods for characterizing the genetics that underlie functional traits of key bacterial populations - "key" in providing important ecosystem services, being abundant, or by possessing high metabolic rates. It is therefore timely to analyze and define the functional responses of bacteria to human-induced effects on coastal ecosystem health. We posit that categorizing the responses of key marine bacterial populations to changes in environmental conditions through modern microbial oceanography methods will allow establishing the nascent field of genetic counselling for our coastal waters. This requires systematic field studies of linkages between functional traits of key bacterial populations and their ecosystem functions in coastal seas, complemented with systematic experimental analyses of the responses to different stressors. Research and training in environmental management along with dissemination of results and dialogue with societal actors are equally important to ensure the role of bacteria is understood as fundamentally important for coastal ecosystems. Using the responses of microorganisms as a tool to develop genetic counselling for coastal ecosystems can ultimately allow for integrating bacteria as indicators of environmental change.We thank for the many inspiring discussions with Ulla Li Zweifel and Åke Hagström on taking advantage of microbial genetic blueprints for informing on the health status of natural waters. We gratefully acknowledge Martin Brusin for contributing drawings of Figure 1. Research on this subject was supported by the marine strategic research program EcoChange to J. P and the BONUS BLUEPRINT project, which has received funding from BONUS, the Joint Baltic Sea Research and Development Program (Art 185), and Swedish, German and Danish research councils to JP, AA, ML and LR.Peer reviewe

Next-generation sequencing (NGS) for assessment of microbial water quality: current progress, challenges, and future opportunities

Water quality is an emergent property of a complex system comprised of interacting microbial populations and introduced microbial and chemical contaminants. Studies leveraging next-generation sequencing (NGS) technologies are providing new insights into the ecology of microbially mediated processes that influence fresh water quality such as algal blooms, contaminant biodegradation, and pathogen dissemination. In addition, sequencing methods targeting small subunit (SSU) rRNA hypervariable regions have allowed identification of signature microbial species that serve as bioindicators for sewage contamination in these environments. Beyond amplicon sequencing, metagenomic and metatranscriptomic analyses of microbial communities in fresh water environments reveal the genetic capabilities and interplay of waterborne microorganisms, shedding light on the mechanisms for production and biodegradation of toxins and other contaminants. This review discusses the challenges and benefits of applying NGS-based methods to water quality research and assessment. We will consider the suitability and biases inherent in the application of NGS as a screening tool for assessment of biological risks and discuss the potential and limitations for direct quantitative interpretation of NGS data. Secondly, we will examine case studies from recent literature where NGS based methods have been applied to topics in water quality assessment, including development of bioindicators for sewage pollution and microbial source tracking, characterizing the distribution of toxin and antibiotic resistance genes in water samples, and investigating mechanisms of biodegradation of harmful pollutants that threaten water quality. Finally, we provide a short review of emerging NGS platforms and their potential applications to the next generation of water quality assessment tools.Singapore-MIT Alliance for Research and Technology. Center for Environmental Sensing and Modelin

Facets of diazotrophy in the oxygen minimum zone waters off Peru

Nitrogen fixation, the biological reduction of dinitrogen gas (N2) to ammonium (NH4+), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N2 fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N2 fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N2 fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N2 fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO2− and PO43− are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N2 fixation may increase in the futu