Quantification of Aquatic Unicellular Diazotrophs by Immunolabeled Flow Cytometry

doi:10.21203/rs.3.rs-1870373/v1

Download PDF

Research Article

Quantification of Aquatic Unicellular Diazotrophs by Immunolabeled Flow Cytometry

https://doi.org/10.21203/rs.3.rs-1870373/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 22 May, 2023

Read the published version in Biogeochemistry →

You are reading this latest preprint version

Unicellular diazotrophs are recognized as important contributors to the aquatic nitrogen cycle. Yet, quantifying the number of aquatic diazotrophs, especially the unicellular fraction, is highly challenging and relies mostly on microscopical and molecular approaches. In this study a new method was developed to quantify unicellular diazotrophs by combining immunolabeling the nitrogenase enzyme and flow cytometry. The quantification method was initially developed using a diazotrophic monoculture (Vibrio natriegens) and verified by different controls as well as various auxiliary approaches such as N₂ fixation and confocal laser scanning microscopy. It was evident that only 15–20% of the total number of V. natriegens cells have synthesized the nitrogenase enzyme, even though the media was anaerobic, and N limited. This approach was further tested on field samples collected from marine and freshwater environments. It was found that the number of unicellular diazotrophs sampled from the Mediterranean Sea comprised 0.1% of the total bacteria, while 4.7% along the Jordan River. Nevertheless, N₂ fixation per cell was highest in the Mediterranean Sea (~ 90 attmole N cell^− 1 d^− 1), while lower in the lake and the river (1.4 to 0.3 attmole N cell^− 1 d^− 1, respectively). Following the above, it is expected that this direct quantification approach will provide new insights on the number and specific contribution of unicellular diazotrophs to total N₂ fixation in marine and freshwater environments.

Flow cytometry

Immunolocalization

Diazotrophs

N2 fixation

Bacterial abundance

Biological N₂ fixation is a central process in marine and freshwater environments as it supplies bio-available nitrogen and support primary production (Gruber & Galloway, 2008; Zehr & Capone, 2020). Dinitrogen fixation is carried by a specific subgroup of bacteria and archaea known as diazotrophs. These organisms use the nitrogenase enzyme, a two-component complex comprised Mo-Fe protein and Fe-reductase protein (Hoffman et al., 2014). Studies have indicated that aquatic diazotrophs include autotrophic (Zehr, 2011), heterotrophic (Bombar et al., 2016; Riemann et al., 2010) and mixotrophic (Benavides et al., 2020; Feng et al., 2010) metabolism to maintain the energetic requirements of the nitrogenase enzyme. Aquatic diazotrophs can be found in benthic mats, organized in long filamentous chains (up to few hundreds of micrometers), free living planktonic cells, or associated with aggregates (Bergman et al., 2013; Bertics et al., 2010; Riemann et al., 2022).

Despite the biochemical and ecological importance of unicellular diazotrophs, quantifying their abundance is highly challenging, thus missing in most ecological studies. Traditionally, microscopical approaches were used to count diazotrophs, for most part filamentous bacteria such as Trichodesmium sp. and Anabena sp. (Spungin et al., 2016; Zulkefli & Hwang, 2020). During the last two decades various molecular methods were also used to estimate the numbers of smaller, unicellular diazotrophs (Foster et al. 2007; Turk et al. 2011; Moisander et al. 2014). These methods are mostly based on evaluating the numbers of diazotrophs focus on the transcription (DNA) or expression (mRNA) of nifH genes. Similarly, fluorescence in situ hybridization (FISH) of the nifH gene was also applied to image and counted single diazotrophic cells(Krupke et al. 2013). Other studies have moved beyond the nifH gene and immunolabeled the nitrogenase enzyme to visualize aquatic diazotrophs (Currin et al., 1990; Geisler et al., 2019; Lin et al., 1998). Differently than targeting the nifH gene, it was estimated that diazotrophs which synthesized the nitrogenase enzyme are likely to actively fix dinitrogen. However, harnessing the advantages of nitrogenase immunolabeling for unicellular diazotrophs quantification was yet developed.

In this study nitrogenase immunolabeling was compiled with flow cytometry to quantify the number of unicellular diazotrophs in aquatic environments. The quantification method is based on immunolabeling the Mo-Fe subunit of the nitrogenase enzyme by two antibodies conjugated to a green fluorophore. The method was first calibrated with Vibrio natriegens as a representative unicellular diazotrophs and then validated in-situ from several marine and freshwater environments. Complimentary N₂ fixation measurements were undertaken, enabling to identify and quantify the diazotrophic cell-specific activity.

Culturing unicellular diazotroph and non-diazotrophic bacteria

Vibrio natriegens (ATCC 14048) and/or Escherichia coli (ATCC 11303) were acclimated in a gas tight bottles )100 ml( containing Luria Bertani Broth media (LB, Merck Millipore, BD, 0083370) under anaerobic conditions (~ 0.2 mg L^− 1 O₂) at 26°C overnight. The LB media used to grow the V. natriegens also included 1.5% NaCl (Sigma Aldrich, 312525). Cells were further diluted to ~ 5x10⁶ cells ml^− 1 and re-grown to a mid-logarithmic phase with an ~ 2x10⁸ cells ml^− 1 for 1–2 h under the same conditions. Bacterial cells were centrifuged (3500 g for 6 min) to remove the LB and resuspended in artificial brackish water (1 ml) to a final cell concentration of ~ 2x10⁶ cells ml^− 1. The chemical composition of the artificial media is detailed in the supporting information.

Triplicate biological replicates containing either V. natriegens, E. coli or both (1:1) were resuspended in N limited brackish water, enriched with ¹⁵N₂ (99%, Cambridge Isotopes, lot #NLM-363-PK, final concentration 1% v:v). Monocultures or mixed cultures were then incubated for 48 hours under dark and anoxic conditions at 26°C. Additional two bottles from each bacterial type were not enriched with ¹⁵N₂ to determine their natural isotopic ratio. At the conclusion of the incubation, sub samples were analyzed for N₂ fixation rates, colony forming units (CFU), as well as total bacterial abundance (BA) and diazotrophic abundance (DA) as detailed below. In addition, diazotrophs were visualized by capturing immunolabeled subsamples by confocal laser scanning microscopy (CLSM).

Collection of natural samples for diazotrophic quantification

Surface waters were collected from three sampling locations, the Southeastern Mediterranean Sea, Qishon Estuary, and Sea of Galilee Lake (Table S1). Water were incubated in 1 L Nalgene bottles. N₂ fixation rates were determined by enriching the samples with 7.5% of dissolved ¹⁵N₂ stock (Final concentration). Enriched samples were incubated for 48 h at room temperature under 12h light/dark conditions. Additional bottle from each location were not enriched with ¹⁵N₂ to determine their natural isotopic ratio. Subsamples (1.7 ml) were collected at the end of the incubation for BA and DA analysis as well as diazotroph microlocalization.

Analytical methods

Diazotrophs immunolabeling for flow cytometric analysis

Monoculture and natural samples (1.7 ml) were fixed with 50% glutaraldehyde (final concentration, 0.2% Sigma-Aldrich, G7651), flash frozen in liquid nitrogen and stored at -80°C until analyses. Samples were prepared by slow thawing at room temperature (Fig. 1A). Next, ethylenediaminetetraacetic acid) EDTA, Sigma Aldrich, 03690) was added (final concentration of 5 µM) to chelate cations and facilitate aggregates dispersion (Bogler & Bar-Zeev, 2018). Samples were also sonicated in a bath sonicator for 6 min to disassociate cells from the aggregate matrix and one another. Subsamples (1 ml) were transferred and centrifuged for 10 minutes at x4000 g (a subsample was also collected for total bacterial abundance, detailed are provided below). The supernatant was cautiously discarded to maintain the bacterial pellet. Wash solution was prepared by mixing phosphate buffer saline (PBS) and Triton X-100 (T, final concentration of 0.1 %, Sigma Aldrich, X100) define hereafter as PBST. The wash solution was added to perforate the cell envelope. Samples were centrifuged for 10 minutes at x4000 g, while the supernatant was cautiously discarded. This washings-centrifugal cycle was repeated three times to increase the efficiency of cellular perforation. Fresh anti-nitrogenase antibody (3 µg ml^− 1, Agrisera Antibodies AS01 021A) was prepared with PBST and bovine albumin serum (BSA, filtered 0.2 µm, 1 mg ml^− 1, Sigma Aldrich A2153) to minimize unspecific antibody binding (Fig. 1B). Samples were then incubated while slowly rotating (Benchmark Scientific Roto-Therm Plus, H2024) for one hour at room temperature to facilitate binding between the nitrogenase Mo-Fe subunit and the primary antibody. Unbonded antibodies were removed by washing the samples three times with PBST similarly to the above. Washed samples were then incubated at room temperature in the dark for 45 min with the secondary antibody (3 µg ml^− 1, Thermo Fisher Scientific A-11039) conjugated to a green fluorophore (Alexa Fluor™ 488) with Ex spectra of 498 nm and Em of 520 nm (Fig. 1C). Any untagged residues of the secondary antibodies were removed by washing the samples three times with PBST as described above. Immunolabeled samples were suspended with sterile PBS (1 ml) without any additions. Additionally, few controls were prepared following the above procedure and tested to evaluate the specific tagging of diazotrophs: (1) negative control, namely PBST-BSA without any antibodies, to determine whether any autofluorescence could be detected; (2) no addition of the 1st antibody (PBST-BSA with the 2nd antibody only); and (3) no addition of the 2nd antibody (PBST-BSA with the 1st antibody only) to verify if any unspecific adsorption occurred. Additional control was to test unspecific tagging by applying the immunolabeling approach on non-diazotrophic (E. coli) bacteria. Moreover, bacterial abundance was measured after the washing stage from representative samples to evaluate the number of bacterial cell loss (Figure S1).

Counting immunolabeled diazotrophs and total bacteria using flow cytometry

Diazotroph abundance (DA) was determined by detecting and counting immunolabeled subsample (200 µl) using an Attune-Next acoustic flow cytometer (Applied Biosystems). Changes in the abundance of monoculture diazotrophs were determined after diluting the immunolabeled samples (1:100, 1:250, 1:500, 1:1000). Monocultures were analyzed at a flow rate of 100 µl min^− 1, while reduced to 25 µl min^− 1 for natural samples. Stop condition was set to 20,000 counts for all samples. Calibration beads (1 µm, F8815, Invitrogen, Ex: 350 nm Em: 440 nm) were added (final concentration of 1.8x10⁴ beads ml^− 1) every 12 samples to evaluate the size spectrum of the sample.

Following the above, total bacterial abundance (BA) was quantified by staining non-immunolabeled subsamples (200 µl) with SYBR Green I (S7563, Invitrogen, final concentration, 1 nM) (Vaulot and Marie 1999). Samples were incubated for 15 min under dark conditions. Stained samples were measured with an Attune-Next acoustic flow cytometer. MilliQ (sterile) water samples were used to clean the system every five samples, while PBS were tested and subtracted as blanks. The specified lasers, dyes and filters are detailed in Table 1.

Table 1

Analytical specifications of the flow cytometer.
Parameter	Laser (nm)	Excitation (nm)	Emission (nm)	Detector (nm)
Side scattering (SSC)	450 (Blue)	450	-	488 ± 10
SYBR GREEN I	450 (Blue)	497	520 (Green)	520 ± 30
Nitrogenase Immunolocalization	450 (Blue)	500	520 (Green)	520 ± 30

Quantifying cells using colony forming units (CFU) counts

Samples from each individual experiment (n = 3) were collected from the incubation bottles and serial diluted in parallel to the immunolabeled samples (1:100, 1:250, 1:500, 1:1000). Subsamples (25 µl) were plated on an agar plate (1.5%, Bacto Agar, DF0140) with an N limited brackish water media (the recipe is detailed in the supporting information). CFU samples were incubated under anaerobic conditions for 48 hours at 26°C. CFU were determined at the end of the incubation by counting plates with 30–300 colonies.

Microlocalization of diazotrophs using confocal laser scanning microscopy (CLSM)

Immunolabeled samples (50 µl) were stained with 250 µg ml^− 1 of 40 ,6-Diamidino-2- Phenylindole (DAPI, Ex 360 nm and Em 460 nm, Thermo Fisher, D1306) or (SYBR Green I, Ex 497 nm and Em 520 nm) and incubated for 20 minutes under dark conditions. Samples were drop-casted on a glass microscope slide, sealed by a cover slide and nail polish to minimize dehydration. Immunolabeled nitrogenase enzyme was visualized by a CLSM 900 equipped with a 488 nm laser (Power, 0.2% digital gain, ~ 500 V, pinhole, 32 µm). Stained bacteria were visualized with a 405 nm laser (Power, 0.9%, digital gain, ~ 730 V and pinhole, 37 µm). Samples were observed under a x63 lens (63x/1.4 Oil DIC M27, respectively). Non stained samples were used before to identify and subtract autofluorescence. Images were processed using Zeiss ZEN Blue edition (3.5, lite, Germany).

Measuring N₂ fixation rates

Triplicate samples were filtered on a pre-combusted glass microfiber filter (GF/F, Cytivia, 1825025, 450°C, 4.5 h) after 48h incubation in ¹⁵N₂ enriched media. Different volumes of samples (25 ml for lab cultures and 1L for environmental microcosms) were filtered, to ensure sufficient biomass (resulting in an amplitude of ~ 1000 mv) on the filter as their source varied from monocultures to different natural environments. Samples were dried in the oven overnight (60°C) and stored in the desiccator until measurements. Filters were carefully packed in tin capsules, with clean, pre-combusted glass fiber (GF/F) filters used as blanks. The samples were then analyzed using elemental analyzer (EA; Thermo Scientific, Flash 2000 HT) coupled with isotope ratio mass spectrophotometer (IRMS; Thermo Scientific, Delta V Plus). Working with filters and environmental samples over a wide range of concentrations requires caution during the isotopic measurement. The quality control measures taken during the measurement are detailed in the supporting information. Briefly: Three standards (Glutamic Acid USGS 40, Glycine USGS 64, and Caffeine USGS 62) were chosen for calibration, bracketing the expected range for δ¹⁵N of the enriched, as well as natural abundance samples, and ensure accuracy (Figure S2A). Acetanilide (Thermo Scientific, BN240741) was used for linearity test over the measured range (Figure S2B) and for quantitative calibration of peak amplitude vs. µg N in sample’s biomass (Figure S2D). A working range of 10 to 55 µg N per sample was determined to assure precision and avoid linearity effect. Similar ranges of nitrogen (> 10 µg N per filter) were previously determined (White et al., 2020). No drift was measured (slope = 0.01) and good precision was found (± 0.3‰) throughout the analysis (Figure S2C). The natural abundance of ¹⁵N, reflected by the ratio of ¹⁴N/¹⁵N in each culture or environmental sample, was subtracted from that of the corresponding enriched sample to calculate N₂ fixation rates, according to previous reports (Montoya et al., 1996).

Statistical analyses

Statistical tests were ran using XLSTAT (2022.2, New-York). Before analyses, normal distribution of the data was validated using Shapiro Wilk test. The links between BA, DA, CFU and N₂ fixation were evaluated by a Pearson correlation test. For comparing N₂ fixation and BA/DA between samples, Analysis of Variance (ANOVA) was used with post-hoc Tukey test. All the tests were run under the confidence level of 95% (α = 0.05).

Detecting and quantifying a unicellular diazotrophic monoculture

Immunolabeled V. natriegens formed a distinct cluster after analyzing the samples by flow cytometry using a green detector over side scatter (Fig. 2A). In contrast, only few unlabeled cells (< 1000 events) were captured in the same region (Fig. 2B). Similarly, no V. natriegens cells were detected after tagging with the first or the second antibodies only (Fig. 1C-D). Following the above, only conjunction of the two antibodies led to a positive detection of V. natriegens by flow cytometry, excluding negligible autofluorescence or unspecific adsorption of the stain to the cells. Moreover, no immunolabeling by non-diazotroph E. coli cells were detected in the region of interest by the flow cytometer (Fig. 2E). This negative control highlights that only cells that synthesized the nitrogenase enzyme could be tagged by the antibodies and detected as previously reported in other studies (Chelius & Triplett, 2000; Geisler et al., 2019).

Total bacterial abundance was counted in an independent test after tagging a subsample with a nucleic acid stain (SYBR green) only. Tagging bacteria with SYBR green resulted in a distinct cluster that was identified in the same region of interest as described above (Fig. 2F) and similar to previous studies (e.g., Geisler et al., 2019). Complimentary visualization of V. natriegens and E. coli subsamples by CLSM confirmed the results detected by the flow cytometry (Fig. 2, circles).

Linear and significant correlation was detected between the number of immunolabeled V. natriegens and the total number of cells tagged by SYBR green from the same monoculture (Pearson, r = 0.999, p = 0.001, Fig. 2G). That trend line indicates that between 15 to 20% of all V. natriegens bacteria were specifically tagged by immunolabeling, namely the cells that synthesized the nitrogenase enzyme. Correspondingly, a linear correlation was also found between immunolabeled cells and CFU counts that grew on limited nitrogen agar plates under anaerobic conditions for 48h (Fig. 2H). It should be noted that the number of immunolabeled cells detected by flow cytometry was 20–25 times higher than those counted on the agar plates. In addition, a linear relationship was found between the number of V. natriegens that synthesized the nitrogenase enzyme and N₂ fixation rates (Fig. 2I), resulting in a specific N₂ fixation per cell of 1.3 ± 0.3 attomole N cell^− 1.

Lower percentage of free-living diazotrophs that synthesized the nitrogenase enzyme compared to total cell count may indicate that heterotrophic N₂ fixation was partly suppressed even under anerobic conditions and limited concentrations of inorganic nitrogen. This was also confirmed by the low fixation rates per cell. Although the scope of the study was to develop a new quantification method for unicellular diazotrophs, it could be surmised that other constraints that were not measured here such as pH (Luo et al., 2019) and/or carbon liability (Benavides et al., 2020; Rahav et al., 2016) impaired the N₂ fixation rates by heterotrophic bacteria.

Counting Diazotrophic And Non-diazotrophic Mixed Cultures

Two monocultures that included unicellular diazotrophic (V. natriegens) and a non-N₂ fixing (E. coli) bacteria were mixed to test the differentiation capacity of the new immunolabeled—flow cytometry-based approach. Staining the DNA of subsamples with SYBR green for total bacterial count formed a distinct cluster (Fig. 2F). Immunolabeling diazotrophic monoculture, as well as a mixture of V. natriegens and E. coli bacteria, resulted in a clear cluster over the conjugated nitrogenase (green) threshold (Fig. 3A).

Quantifying total bacteria indicated that the numbers of a sole V. natriegens culture or a mixture with E. coli were similar (~ 0.75 x 10⁷ cells ml^− 1) after 48 h of anaerobic incubation in a nitrogen-limited media. Whilst, the number of E. coli cells in the monoculture was lower by 71% (Fig. 3B). Counting the immunolabeled cells indicated that the number of N₂ fixing diazotrophs, namely V. natriegens that synthesized the nitrogenase enzyme, constitute 18% of the total V. natriegens cells and 13% at the mixed culture. Note, no immunolabeled E. coli cells were detected by the flow cytometer (Fig. 3B), ruling out any unspecific links or adsorption of the fluorophore.

Corresponding N₂ fixation rates were found to be significantly higher (1.3 times) in the mixed culture than in the V. natriegens monoculture (Fig. 3C). That difference was even greater (2.5 times) when comparing N₂ fixation rates per cell in the mixed culture to those measured by V. natriegens only (3.3 ± 1.6 and 1.3 ± 0.3 attomole N cell^− 1, respectively). Altogether, it appears that mixing V. natriegens with a non-diazotrophic heterotrophic bacteria such as E. coli spur N₂ fixation rates per cell. These results suggest that in contrast to a monocultures (when all cells fix N₂ and contribute to the N pool), heterotrophic diazotroph in mixed cultures enhance their cellular N₂ fixation rates to compensate for the consumption of NH₄⁺ by non-diazotrophic bacteria (i.e., E. coli).

Evaluating The Abundance Of Unicellular Diazotrophs In Aquatic Environments

Quantification of unicellular diazotrophs from marine and freshwater environments (i.e., SE Mediterranean Sea, Jordan River, and the Sea of Galilee Lake) by immunolabeled flow cytometry resulted in a marked cluster (Fig. 4A). However, this cluster was slightly more scattered than in the monoculture controls. Complimentary imaging of subsamples by CLSM indicated that only a small fraction of the cells collected from the Sea of Galilee Lake were tagged by nitrogenase immunolabeling (Fig. 4A, top circle). Additional CLSM images of immunolabeled bacteria from the Mediterranean Sea and the Jordan River are provided in the supporting information (Figure S4).

The abundance of unicellular diazotrophs was 2 ± 0.2 x10⁷ cells L^− 1 in the Mediterranean Sea, 1 ± 0.7 x10⁷ cells L^− 1 in the Sea of Galilee Lake and 6 ± 0.9 x10⁷ cells L^− 1 in the Jordan River. The percent of unicellular diazotrophs from total bacterial abundance ranged between 0.1% in Mediterranean Sea to 1.2 and 4.7% in the Sea of Galilee Lake and Jordan River, respectively (Fig. 4B). Corresponding N₂ fixation rates were between 0.2 to 1.2 nmole N L^− 1 (Fig. 4C), which are similarly to previous reports (Halm et al., 2009; Marcarelli & Wurtsbaugh, 2006, 2009; Rahav et al., 2022). Note, data on N₂ fixation rates in freshwater environments is highly limited (Marcarelli et al., 2022). Normalizing these rates to the number of diazotrophs detected by immunolabeling flow cytometry (DA) resulted in N₂ fixation per cell, ranging between 88 attomole N cell^− 1 at the Mediterranean Sea to 0.3 attomole N cell^− 1 at the Sea of Galilee Lake (Fig. 4C).

Limited concentrations of dissolved inorganic nitrogen were previously reported to enhance N₂ fixation per cell to provide new bioavailable nitrogen compounds (Zehr et al. 2003; Gruber and Galloway 2008). Therefore, compared to the mesotrophic Jordan River and the Sea of Galilee Lake, measuring the highest N₂ fixation rates per cell in the oligotrophic, nitrogen poor, Mediterranean Sea were expected. Nevertheless, it should be pointed that recent studies have reported that N₂ fixation rates per cell could be somewhat higher after the addition of dissolved inorganic nitrogen (Martínez-Pérez et al. 2018; Mills et al. 2020). These studies, as well as the above indicate that cellular mechanisms controlling N₂ fixation rates are not clear and likely change according to the environmental conditions and the different metabolic (i.e., heterotrophic/autotrophic) pathways used by the diazotrophic community (Mills et al. 2020).

Coupling immunolabeling and flow cytometry can be used as a high throughput approach to quantify the number of unicellular diazotrophs that synthesized the nitrogenase enzyme, thus were likely fixing N₂. This approach can be applied to count unicellular diazotrophs in controlled lab-scale experiments as well as in various aquatic environments. Counting the total and immunolabeled cells of a diazotrophic monoculture indicated that even under anaerobic and N-limiting conditions, only a fraction (15–20%) synthesize the nitrogenase enzyme. That discrepancy was likely due to the experimental conditions (e.g., carbon liability and nutrient concentrations) and yet highlights the importance of counting diazotrophs (even from monocultures under controlled conditions) to determine fundamental aspects such as specific N₂ fixation per cell. Moreover, direct quantification of unicellular diazotrophs from natural environments can provide novel insights related to aquatic nitrogen cycle. For example, the field results indicate N₂ fixation per cell was highest in the oligotrophic Mediterranean Sea compared to the Jordan River and the Sea of Galilee Lake, pointing on their potential significance for total biological N production in N-limited environments.

It should be highlighted that this approach could be further developed: (i) differentiate and specifically count unicellular heterotrophic vs. photoautotrophic diazotrophs. Enhancing the specificity could provide new quantified estimation over the contribution of the different diazotrophic groups to total N₂ fixation; (ii) compare the number of diazotrophs using this immunolabeled flow cytometry approach to molecular, nifH-based approaches that are currently applied at various aquatic environments(Zehr and Capone 2020). Nevertheless, we suggest that adopting this approach could provide information on specific N₂ fixation capacity of freshwater and marine diazotrophs. Moreover, quantifying diazotrophs will likely provide new insights on the contribution of these microorganisms to the aquatic nitrogen cycle.

Acknowledgments

This study was supported by the Israeli Science Foundation (grant number 944\21) to E.B-Z. E.G was supported by the Yohai Ben-Nun (IOLR) and the Negev (BGU) scholarships. This paper is in partial fulfillment of E.G Ph.D. thesis at Ben Gurion University of the Negev.

Supporting information

A full recipe of brackish water is available for read. We also provided tests about washing efficiency of bacteria and quality control of EA-IRMS. Additional images of immunolabeled bacteria are also provided.

Conflict of interest statement

The manuscript is original and have not been submitted elsewhere for consideration. There is no conflict of interest and all co-authors have seen and approved the current version for submission.

Benavides M, Duhamel S, van Wambeke F, Shoemaker KM, Moisander PH, Salamon E, Riemann L, Bonnet, S (2020). Dissolved organic matter stimulates N₂ fixation and nifH gene expression in Trichodesmium. FEMS Microbiology Letters, 367, fnaa034. https://doi.org/10.1093/femsle/fnaa034
Bergman, B, Sandh G, Lin S, Larsson J, Carpenter, EJ (2013). Trichodesmium - a widespread marine cyanobacterium with unusual nitrogen fixation properties. FEMS Microbiology Reviews, 37, 285–302. https://doi.org/10.1111/j.1574-6976.2012.00352.
Bertics VJ, Sohm, JA, Treude, T, Chow CET, Capone, DG, Fuhrman JA, & Ziebis W (2010). Burrowing deeper into benthic nitrogen cycling: The impact of Bioturbation on nitrogen fixation coupled to sulfate reduction. Marine Ecology Progress Series, 409, 1–15. https://doi.org/10.3354/meps08639
Bogler A, Bar-Zeev, E (2018). Membrane distillation biofouling: Impact of feedwater temperature on biofilm characteristics and membrane performance. Environmental Science and Technology, 52, 10019–10029. https://doi.org/10.1021/acs.est.8b02744
Bombar D, Paerl RW, Riemann L (2016). Marine non-cyanobacterial diazotrophs: moving beyond molecular detection. Trends in Microbiology, 24, 916–927. https://doi.org/10.1016/j.tim.2016.07.002
Chelius MK, & Triplett EW (2000). Immunolocalization of Dinitrogenase Reductase Produced by Klebsiella pneumoniae in Association with Zea mays L. Applied and Environmental Microbiology, 66, 783–787. https://doi.org/10.1128/AEM.66.2.783-787.2000
Currin CA, Paerl HW, Suba, GK, Alberte, RS (1990). Immunofluorescence detection and characterization of nitrogen-fixing microorganisms from aquatic environments. Limnol Oceanogr, 35, 59–71. https://doi.org/10.4319/lo.1990.35.1.0059
Feng X, Bandyopadhyay A, Berla B, Page L, Wu B, Pakrasi HB, Tang YJ (2010). Mixotrophic and photoheterotrophic metabolism in Cyanothece sp. ATCC 51142 under continuous light. Microbiology, 156, 2566–2574. https://doi.org/10.1099/mic.0.038232-0
Foster RA, Subramaniam A, Mahaffey C, Carpenter EJ, Capone DG, Zehr JP (2007). Influence of the Amazon River plume on distributions of free-living and symbiotic cyanobacteria in the western tropical north Atlantic. Limnology and Oceanography, 52, 517–532. https://doi.org/10.4319/lo.2007.52.2.0517
Geisler E, Bogler A, Rahav E, Bar-Zeev E (2019). Direct detection of heterotrophic diazotrophs associated with planktonic aggregates. Scientific Reports, 9, 9288. https://doi.org/10.1038/s41598-019-45505-4
Gruber, N., & Galloway, J. N. (2008). An Earth-system perspective of the global nitrogen cycle. Nature, 451, 293–296. https://doi.org/10.1038/nature06592.
Halm H, Musat N, Lam P, Langlois R, Musat F, Peduzzi S, Lavik G, Schubert CJ, Singha B, Laroche J, Kuypers MMM (2009). Co-occurrence of denitrification and nitrogen fixation in a meromictic lake, Lake Cadagno (Switzerland). Environmental Microbiology, 11, 1945–1958. https://doi.org/10.1111/j.1462-2920.2009.01917.
Hoffman BM, Lukoyanov D, Yang ZY, Dean DR, Seefeldt LC (2014). Mechanism of nitrogen fixation by nitrogenase: The next stage. In Chemical Reviews 114, 4041–4062. https://doi.org/10.1021/cr400641.
Krupke A, Musat N, LaRoche J, (2013) In situ identification and N₂ and C fixation rates of uncultivated cyanobacteria populations. Systematic and Applied Microbiology 36, 259–271. https://doi.org/10.1016/j.syapm.2013.02.002
Lin S, Henze S, Lundgren P, Bergman B, & Carpenter EJ (1998). Whole-cell immunolocalization of nitrogenase in marine diazotrophic cyanobacteria, Trichodesmium spp. †. Applied and Environmental Microbiology, 64, 3052–3058. https://doi.org/10.1128/AEM.64.8.3052-3058.1998
Luo YW, Shi D, Kranz SA, Hopkinson BM, Hong H, Shen R, Zhang F (2019). Reduced nitrogenase efficiency dominates response of the globally important nitrogen fixer Trichodesmium to ocean acidification. Nature Communications, 10, 1521. https://doi.org/10.1038/s41467-019-09554-7
Marcarelli AM, Fulweiler RW, Scott JT (2022). Nitrogen fixation: A poorly understood process along the freshwater-marine continuum. Limnology and Oceanography Letters, 7, 1–10. https://doi.org/10.1002/lol2.10220
Marcarelli AM, Wurtsbaugh WA (2006).Temperature and nutrient supply interact to control nitrogen fixation in oligotrophic streams: An experimental examination. Limnology and Oceanography, 51, 2278–2289.
Marcarelli, AM, Wurtsbaugh, WA. (2009). Nitrogen fixation varies spatially and seasonally in linked stream-lake ecosystems. Biogeochemistry, 94, 95–110. https://doi.org/10.1007/s10533-009-9311-2
Martínez-Pérez C, Mohr W, Schwedt A. (2018) Metabolic versatility of a novel N₂-fixing Alphaproteobacterium isolated from a marine oxygen minimum zone. Environmental Microbiology 20, 755–768. https://doi.org/10.1111/1462-2920.14008
Mills MM, Turk-Kubo KA, van Dijken GL, Henke BA, Harding K, Wilson ST, Arrigo KR, Zehr, JP (2020). Unusual marine cyanobacteria/haptophyte symbiosis relies on N₂ fixation even in N-rich environments. ISME Journal, 14, 2395–2406. https://doi.org/10.1038/s41396-020-0691-6
Moisander PH, Serros T, Paerl RW (2014) Gammaproteobacterial diazotrophs and nifH gene expression in surface waters of the South Pacific Ocean. ISME Journal 8, 1962–1973. https://doi.org/10.1038/ismej.2014.49
Montoya JP, Voss M, Kahler P, Capone DG (1996). A Simple, high-precision, high-sensitivity tracer assay for N<background-color:#CCCCFF;subdirection:ltr;>2</background-color:#CCCCFF;subdirection:ltr;> fixation. Applied and Environmental Microbiology, 62, 986–993. https://doi.org/10.1128/aem.62.3.986-993.1996
Rahav E, Giannetto MJ, Bar-Zeev E (2016). Contribution of mono and polysaccharides to heterotrophic N₂ fixation at the eastern Mediterranean coastline. Sceintific Reports, 6, 27858. DOI: 10.1038/srep27858
Rahav E, Herut B, Spungin D, Levi A, Mulholland MR, Berman-Frank I (2022). Heterotrophic bacteria outcompete diazotrophs for orthophosphate in the Mediterranean Sea. Limnology and Oceanography, 67, 159–171. https://doi.org/10.1002/lno.11983
Riemann L, Farnelid H, Steward GFG (2010). Nitrogenase genes in non-cyanobacterial plankton: Prevalence, diversity and regulation in marine waters. Aquatic Microbial Ecology, 61, 235–247. https://doi.org/10.3354/ame01431
Riemann L, Rahav E, Passow U, Grossart HP, de Beer D, Klawonn I, Eichner M, Benavides M, Bar-Zeev E (2022). Planktonic aggregates as hotspots for heterotrophic diazotrophy: The plot thickens. Frontiers in Microbiology, 13, 875050. https://doi.org/10.3389/fmicb.2022.875050
Spungin D, Pfreundt U, Berthelot H, Bonnet S, AlRoumi D, Natale F, Hess WR, Bidle KD, Berman-Frank I (2016). Mechanisms of Trichodesmium demise within the New Caledonian lagoon during the VAHINE mesocosm experiment. Biogeosciences, 13, 4187–4203. https://doi.org/10.5194/bg-13-4187-2016
Turk, KA, Rees AP, Zehr JP, Pereira N, Swift P, Shelley R, Lohan M, Woodward E MS, Gilbert J (2011). Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic. ISME Journal, 5, 1201–1212. https://doi.org/10.1038/ismej.2010.205
White AE, Granger J, Selden C, Gradoville MR, Potts L, Bourbonnais A, Fulweiler RW, Knapp AN, Mohr W, Moisander PH, Tobias CR, Caffin M, Wilson ST, Benavides M, Bonnet S, Mulholland MR, Chang BX (2020). A critical review of the ¹⁵N₂ tracer method to measure diazotrophic production in pelagic ecosystems. Limnology and Oceanography: Methods, 18, 129–147. https://doi.org/10.1002/lom3.10353
Zehr JP (2011). Nitrogen fixation by marine cyanobacteria. Trends in Microbiology, 19, 162–173. https://doi.org/10.1016/j.tim.2010.12.004
Zehr JP, Capone DG (2020). Changing perspectives in marine nitrogen fixation. Science, 368, 1–11. https://doi.org/10.1126/science.aay9514
Zulkefli, NS, Hwang SJ (2020). Heterocyst development and diazotrophic growth of anabaena variabilis under different nitrogen availability. Life, 10, 279. https://doi.org/10.3390/life10110279
Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure a cross-system comparison. Environ Microbiol, 5, 539–554

No competing interests reported.

20220718GeisleretalSI.docx

Download PDF

Journal Publication

published 22 May, 2023

Read the published version in Biogeochemistry →

Editorial decision: Major revision
01 Nov, 2022
Reviews received at journal
20 Sep, 2022
Reviewers agreed at journal
05 Sep, 2022
Reviewers invited by journal
13 Aug, 2022
Editor assigned by journal
20 Jul, 2022
Submission checks completed at journal
19 Jul, 2022
First submitted to journal
18 Jul, 2022

You are reading this latest preprint version

Quantification of Aquatic Unicellular Diazotrophs by Immunolabeled Flow Cytometry

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Culturing unicellular diazotroph and non-diazotrophic bacteria

Collection of natural samples for diazotrophic quantification

Analytical methods

Diazotrophs immunolabeling for flow cytometric analysis

Counting immunolabeled diazotrophs and total bacteria using flow cytometry

Quantifying cells using colony forming units (CFU) counts

Microlocalization of diazotrophs using confocal laser scanning microscopy (CLSM)

Measuring N₂ fixation rates

Statistical analyses

Results And Discussion

Detecting and quantifying a unicellular diazotrophic monoculture

Counting Diazotrophic And Non-diazotrophic Mixed Cultures

Evaluating The Abundance Of Unicellular Diazotrophs In Aquatic Environments

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Quantification of Aquatic Unicellular Diazotrophs by Immunolabeled Flow Cytometry

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Culturing unicellular diazotroph and non-diazotrophic bacteria

Collection of natural samples for diazotrophic quantification

Analytical methods

Diazotrophs immunolabeling for flow cytometric analysis

Counting immunolabeled diazotrophs and total bacteria using flow cytometry

Quantifying cells using colony forming units (CFU) counts

Microlocalization of diazotrophs using confocal laser scanning microscopy (CLSM)

Measuring N2 fixation rates

Statistical analyses

Results And Discussion

Detecting and quantifying a unicellular diazotrophic monoculture

Counting Diazotrophic And Non-diazotrophic Mixed Cultures

Evaluating The Abundance Of Unicellular Diazotrophs In Aquatic Environments

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Measuring N₂ fixation rates