Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters

Farnelid, Hanna; Harder, Jens; Bentzon-Tilia, Mikkel; Riemann, Lasse

doi:10.1111/1462-2920.12335

Cited by 34 publications

(40 citation statements)

References 60 publications

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“…These were all affiliated with nifH Cluster I (Zehr et al, 2003). Among the noncyanobacterial OTUs, Otu00002 showed 98% nucleotide similarity to gammaproteobacteria γ-2477A11, and Otu00014 was identical with BAL376 which was isolated from surface water in the Baltic Sea (Farnelid et al, 2014), while the rests did not match with known diazotrophs. Otu00001 was identical with UCYN-A1, and Otu00004 showed 98% nucleotide similarity to UCYN-A3.…”

Section: Diazotroph Community Compositionmentioning

confidence: 96%

Linkage Between Dinitrogen Fixation and Primary Production in the Oligotrophic South Pacific Ocean

Shiozaki

Bombar

Riemann

et al. 2018

Global Biogeochemical Cycles

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The import of nitrogen via dinitrogen fixation supports primary production, particularly in the oligotrophic ocean; however, to what extent dinitrogen fixation influences primary production, and the role of specific types of diazotrophs, remains poorly understood. We examined the relationship between primary production and dinitrogen fixation together with diazotroph community structure in the oligotrophic western and eastern South Pacific Ocean and found that dinitrogen fixation was higher than nitrate‐based new production. Primary production increased in the middle of the western subtropical region, where the cyanobacterium Trichodesmium dominated the diazotroph community and accounted for up to 7.8% of the phytoplankton community, and the abundance of other phytoplankton taxa (especially Prochlorococcus) was high. These results suggest that regenerated production was enhanced by nitrogen released from Trichodesmium and that carbon fixation by Trichodesmium also contributed significantly to total primary production. Although volumetric dinitrogen fixation was comparable between the western and eastern subtropical regions, primary production in the western waters was more than twice as high as that in the eastern waters, where UCYN‐A1 (photoheterotroph) and heterotrophic bacteria were the dominant diazotrophs. This suggests that dinitrogen fixed by these diazotrophs contributed relatively little to primary production of the wider community, and there was limited carbon fixation by these diazotrophs. Hence, we document how the community composition of diazotrophs in the field can be reflected in how much nitrogen becomes available to the wider phytoplankton community and in how much autotrophic diazotrophs themselves fix carbon and thereby influences the magnitude of local primary production.

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Section: Diazotroph Community Compositionmentioning

confidence: 96%

Linkage Between Dinitrogen Fixation and Primary Production in the Oligotrophic South Pacific Ocean

Shiozaki

Bombar

Riemann

et al. 2018

Global Biogeochemical Cycles

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“…OTU_18 is a noteworthy example for a heterotrophic diazotroph found in filtrate. This phylotype is closely related to a Pseudomonas stutzeri (BAL376/BAL410) isolated from Baltic Sea surface waters (Farnelid et al, 2014) and is thought to be able to fix N 2 in oxygenated water by forming aggregates. These Pseudomonas types are likely important diazotrophs in the Baltic Sea and Danish coastal waters, and it is notable that we also detected OTU_18 in GF/F filtrate in Roskilde Fjord (Figure 5).…”

Section: Diazotroph Community Composition In Bulk Samples and Filtratmentioning

confidence: 99%

“…However, nitrogenase (nifH) gene sequencing has uncovered an almost ubiquitous distribution of diverse and presumably heterotrophic diazotrophs (Zehr et al, 1998;Farnelid et al, 2011). Notably, these organisms are even found in high-latitude, deep, cold, or coastal waters where cyanobacteria are few or absent (e.g., Fernandez et al, 2011;Blais et al, 2012;Bonnet et al, 2013) and their lifestyles and how they impact biogeochemical element cycling seems fundamentally different from photoautotrophic diazotrophs (Rahav et al, 2013;Farnelid et al, 2014;Moisander et al, 2014;Benavides et al, 2015;Bentzon-Tilia et al, 2015a). This has inspired many investigators to hypothesize that global N inputs by BNF could potentially be much higher than previously thought.…”

Section: Introductionmentioning

confidence: 99%

Filtration via Conventional Glass Fiber Filters in 15N2 Tracer Assays Fails to Capture All Nitrogen-Fixing Prokaryotes

et al. 2018

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Biological dinitrogen fixation (BNF) represents a major input of reduced nitrogen (N) to the oceans. Accurate direct measurements of BNF rates are crucial for reliably determining the biogeochemical significance of diazotrophy at local and global scales. Traditionally, borosilicate glass fiber filters (GF/F, Whatman) with a nominal pore size of 0.7 µm are used to collect suspended particles by filtration after incubations with added 15 N 2 tracer. We carried out BNF experiments in the Baltic Sea, Danish coastal waters, and the Pacific Ocean comparing the retentive characteristics of precombusted GF/F filters with newer Advantec glass fiber filters which have a smaller nominal pore size of 0.3 µm. Where BNF was detected, rates were nearly always higher, and sometimes even exclusively detectable, when using Advantec filters. In the majority of samples across tested habitats, significantly more cells were lost to GF/F filtrate (average = 51%, range = 10-70% of cells) than to Advantec filtrate (average = 40%, range = 10-54%). Using Illumina sequencing of nitrogenase (nifH) gene amplicons, we show that diazotroph communities can markedly differ between bulk water and filtrates from GF/F and Advantec filtrations, suggesting that different diazotrophs can pass through the filter types. In order to reduce the potential underestimations of BNF due to filtration loss of diazotrophs, we recommend using Advantec filters or alternatively silver membranes with 0.2 µm pore size, especially in waters expected to be inhabited by relatively small, unicellular diazotrophs.

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“…Typically, these approaches attempt to mimic the natural niche by reducing nutrients, adding specific compounds, or changing incubation atmosphere, temperature, and time (e.g., see references [20][21][22][23][24][25]. Also, the requirements for a mixed microbial community and signals from this community have been proposed as an explanation for the lack of culturability (1,26).…”

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confidence: 99%

Effects of Gelling Agent and Extracellular Signaling Molecules on the Culturability of Marine Bacteria

Rygaard

Thøgersen

Nielsen

et al. 2017

Appl Environ Microbiol

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Only 1% of marine bacteria are currently culturable using standard laboratory procedures, and this is a major obstacle for our understanding of the biology of marine microorganisms and for the discovery of novel microbial natural products. Therefore, the purpose of this study was to investigate if improved cultivation conditions, including the use of an alternative gelling agent and supplementation with signaling molecules, improve the culturability of bacteria from seawater. Replacing agar with gellan gum improved viable counts 3-to 40-fold, depending on medium composition and incubation conditions, with a maximum of 6.6% culturability relative to direct cell counts. Through V4 amplicon sequencing we found that culturable diversity was also affected by a change in gelling agent, facilitating the growth of orders not culturable on agar-based substrates. Community analyses showed that communities grown on gellan gum substrates were significantly different from communities grown on agar and that they covered a larger fraction of the seawater community. Other factors, such as incubation temperature and time, had less obvious effects on viable counts and culturable diversity. Supplementation with acylated homoserine lactones (AHLs) did not have a positive effect on total viable counts or a strong effect on culturable diversity. However, low concentrations of AHLs increased the relative abundance of sphingobacteria. Hence, with alternative growth substrates, it is possible to significantly increase the number and diversity of cultured marine bacteria.IMPORTANCE Serious challenges to human health, such as the occurrence and spread of antibiotic resistance and an aging human population in need of bioactive pharmaceuticals, have revitalized the search for natural microbial products. The marine environment, representing the largest ecosystem in the biosphere, harbors an immense and virtually untapped microbial diversity producing unique bioactive compounds. However, we are currently able to cultivate only a minute fraction of this diversity. The lack of cultivated microbes is hampering not only bioprospecting efforts but also our general understanding of marine microbes. In this study, we present a means to increase the number and diversity of cultured bacteria from seawater, showing that relatively simple changes to medium components may facilitate the isolation and growth of hitherto unknown bacterial orders.

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Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters

Cited by 34 publications

References 60 publications

Linkage Between Dinitrogen Fixation and Primary Production in the Oligotrophic South Pacific Ocean

Linkage Between Dinitrogen Fixation and Primary Production in the Oligotrophic South Pacific Ocean

Filtration via Conventional Glass Fiber Filters in 15N2 Tracer Assays Fails to Capture All Nitrogen-Fixing Prokaryotes

Effects of Gelling Agent and Extracellular Signaling Molecules on the Culturability of Marine Bacteria

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