Phosphate limitation intensifies negative effects of ocean acidification on globally important nitrogen fixing cyanobacterium

Laboratory experiments

Culturing

The marine cyanobacterium Trichodesmium erythraeum IMS101 was obtained from the National Center for Marine Algae and Microbiota (Maine, USA) and was grown in Aquil-tricho medium prepared with 0.22 µm-filtered and microwave-sterilized oligotrophic South China Sea surface water⁶. The medium was enriched with various concentrations of chelexed and filter-sterilized NaH₂PO₄ as where indicated, and filter-sterilized vitamins and trace metals buffered with 20 µM EDTA⁶. The cultures were unialgal, and although they were not axenic, sterile trace metal clean techniques were applied for culturing and experimental manipulations. T. erythraeum was pre-adapted to low P condition by semi-continuously culturing at 0.5 μM PO₄³⁻ and at two pCO₂ levels (400 and 750 µatm) for more than one year. To start the chemostat culture, three replicates per treatment were grown in 1-L Nalgene® magnetic culture vessels (Nalgene Nunc International, Rochester, NY, USA), in which the cultures were continuously mixed by bubbling with humidified and 0.22 µm-filtered CO₂–air mixtures and stirring using a suspended magnetic stir bar. The reservoirs contained Aquil-tricho medium with 1.2 μM NaH₂PO₄, which was delivered to the culture vessels using a peristaltic pump (Masterflex® L/S®, USA) at the dilution rate of 0.2 d⁻¹. In all experiments, cultures were grown at ;27 °C and ~80 μmol photons m⁻² s⁻¹ (14 h:10 h light–dark cycle) in an AL-41L4 algae chamber (Percival). The concentration of Chlorophyll a (Chla) was monitored daily in the middle of the photoperiod as an indicator of biomass. When the Chla concentration remained constant for more than one generation, the system was considered to have reached steady-state, and was maintained for at least another four generations prior to sampling for further analysis.

Carbonate chemistry manipulation

pCO₂/pH of seawater media in the culture vessels and in the reservoir was controlled by continuously bubbling with humidified and 0.22 µm-filtered CO₂-air mixtures generated by CO₂ mixers (Ruihua Instrument & Equipment Ltd.). During the experimental period, the pH_T (pH on the total scale) of media was monitored daily using a spectrophotometric method⁴⁶. The dissolved inorganic carbon (DIC) of media was analyzed by acidification and subsequent quantification of released CO₂ with a CO₂ analyzer (LI 7000, Apollo SciTech). Calculations of alkalinity and pCO₂ were made using the CO2Sys program⁴⁷, based on measurements of pH_T and DIC, and the carbonate chemistry of the experiments are shown in Supplementary Table 1.

Chla concentration and cell density and size

Chla concentration was measured daily following Hong et al.⁶. Briefly, T. erythraeum was filtered onto 3 μm polycarbonate membrane filters (Millipore), followed by heating at 65 °C for 6 min in 90% (vol/vol) methanol. After extraction the filter was removed and cell debris were spun down via centrifugation (5 min at 20,000×g) before spectrophotometric analysis. Cell density and the average cell length and width were determined at regular intervals when the chemostat cultures reached steady-state using ImageJ software. Photographs of Trichodesmium were taken using a camera (Canon DS126281, Japan) connected with an inverted microscope (Olympus CKX41, Japan). Total number and length of filaments in 1 mL of culture were measured, and the cell number of ~20 filaments was counted. The average length of cells was obtained by dividing the total length of the 20 filaments by their total cell number. The cell density of the culture was then calculated by dividing the total length of filaments in 1 mL culture by the average cell length. The average cell width was determined by measuring the width of around 1000 cells in each treatment.

Elemental composition

To determine particulate organic C (POC) and N (PON), at the end of the chemostat culturing T. erythraeum cells were collected on pre-combusted 25 mm GF/F filters (Whatman) and stored at −80 °C. Prior to analysis, the filters were dried overnight at 60 °C, treated with fuming HCl for 6 h to remove all inorganic carbon, and dried overnight again at 60 °C. After being packed in tin cups, the samples were subsequently analyzed on a PerkinElmer Series II CHNS/O Analyzer 2400.

Particulate organic P (POP) was measured following Solorzano et al.⁴⁸. Cells were filtered on pre-combusted 25 mm GF/F filters and rinsed twice with 2 mL of 0.17 M Na₂SO₄. The filters were then placed in combusted glass bottles with the addition of 2 mL of 0.017 M MgSO₄, and subsequently evaporated to dryness at 95 °C and baked at 450 °C for 2 h. After cooling, 5 mL of 0.2 M HCl was added to each bottle. The bottle was then tightly capped and heated at 80 °C for 30 min, after which 5 mL Milli-Q H₂O was added. Dissolved phosphate from the digested POP sample was measured colorimetrically following the standard phosphomolybdenum blue method.

C uptake and N₂ fixation rates

Rates of short-term C uptake were determined at the end of the chemostat culturing. 100 µM NaH¹⁴CO₃ (PerkinElmer) was added to 50 mL of cultures in the middle of the photoperiod, which was then incubated for 20 min under the growth conditions. After incubation, the samples were collected onto 3 μm polycarbonate membrane filters (Millipore), which were then washed with 0.22 µm-filtered oligotrophic seawater and placed on the bottom of scintillation vials. The filters were acidified to remove inorganic C by adding 500 µL of 2% HCl. The radioactivity was determined using a Tri-Carb 2800TR Liquid Scintillation Analyzer (PerkinElmer). Rates of N₂ fixation (nitrogenase activity) were measured in the middle of the photoperiod for 2 h by the acetylene reduction assay⁴⁹, using a ratio of 4:1 to convert ethylene production to N₂ fixation.

Soluble reactive phosphate (SRP) analysis

When the chemostat cultures reached a steady-state, SRP concentrations in the culture vessels were measured at regular intervals, using the classic phosphomolybdenum blue (PMB) method with an additional step to enrich PMB on an Oasis HLB cartridge⁵⁰. Briefly, 100 mL of GF/F filtered medium sample was fortified with 2 mL of ascorbic acid (100 g L⁻¹) and 2 mL of mixed reagent (MR, the mixture of 100 mL of 130 g L⁻¹ ammonium molybdate tetrahydrate, 100 mL of 3.5 g L⁻¹ potassium antimony tartrate, and 300 mL of 1:1 diluted H₂SO₄), and then mixed completely. After standing at room temperature for 5 min, the solution was loaded onto a preconditioned Oasis HLB cartridge (3 cm³/60 mg, P/N: WAT094226, Waters Corp.) via a peristaltic pump, and then 1 mL eluent solution (0.2 M NaOH) was added to elute the sample into a cuvette, to which 0.06 mL of MR and 0.03 mL of ascorbic acid solution was added to fully develop PMB. Finally, the absorbance of PMB was measured at 700 nm using a spectrophotometer.

Alkaline phosphatase (AP) activity

AP activities were measured in the middle of the photoperiod using p-nitrophenylphosphate (pNPP) as a substrate⁵¹. Briefly, 5 mL of culture was incubated with 250 μL of 10 mM pNPP, 675 μL of Tris-glycine buffer (50 mM, pH 8.5) and 67.5 μL of 1 mM MgCl₂ for 2 h under growth conditions. The absorbance of formed p-nitrophenol (pNP) was measured at 410 nm using a spectrophotometer.

PolyP analysis

At the end of the chemostat culturing, T. erythraeum cells were filtered in the middle of the photoperiod onto 3 μm polycarbonate membrane filters (Millipore), flash frozen in liquid nitrogen, and stored at −80 °C until analysis. PolyP was quantified fluorometrically following Martin and Van Mooy²² and Martin et al.²³. Briefly, samples were re-suspended in 1 mL Tris buffer (pH 7.0), sonicated for 30 s, immersed in boiling water for 5 min, sonicated for another 30 s, and then digested by 10 U DNase (Takara), RNase (2.5 U RNase A + 100 U RNase T1) (Invitrogen) and 20 μl of 20 mg mL⁻¹ proteinase K at 37 °C for 30 min. After centrifugation for 5 min at 14,000×g, the supernatant was diluted with Tris buffer according to the range of standards curve, stained with 60 μL of 100 μM 4, 6-diamidino-2-phenylindole (DAPI) per 500 μL of samples, incubated for 7 min and then vortexed. The samples were then loaded onto a black 96-well plate and the absorption of fluorescence at an excitation wavelength of 415 nm and emission wavelength of 550 nm was measured using a PerkinElmer EnSpire® Multimode Plate Reader. PolyP standard (sodium phosphate glass Type 45) was purchased from Sigma-Aldrich. This method gives a relative measure of polyP concentration²³ that is expressed as femto-equivalents of the standard per cell (feq cell⁻¹).

Cellular ATP measurement

Cellular ATP contents were determined when the chemostat cultures reached a steady state. T. erythraeum cells were collected in the middle of the photoperiod using an ATP Assay Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, the sample was lysed and centrifuged, and the supernatant (100 μL) was mixed with ATP detection working reagent (100 μL) and loaded onto a black 96-well plate. The luminescence was measured using a PerkinElmer EnSpire® Multimode Plate Reader.

Intracellular metabolites measurements

NAD(H), NADP(H), and Glu were measured at the end of the chemostat culturing, using the liquid chromatography-tandem quadrupole mass spectrometry (LC–MS/MS) method modified from Luo et al.⁵². Briefly, T. erythraeum cells were gently filtered at the middle of photoperiod onto 3 μm polycarbonate membrane filters (Millipore), rapidly suspended in −80 °C precooled methanol-water (60%, v/v) mixture. After being kept in −80 °C freezer for 30 min, the sample was sonicated for 30 s, centrifuged at 12,000×g and 4 °C for 5 min, and the supernatant was filtered through a 0.2 μm filter (Jinteng®, China) and stored at −80 °C for further LC–MS/MS analysis.

A 2.0 × 50 mm Phenomenex® Gemini 5u C18 110 Å column (particle size 5.2 µm, Phenomenex, USA) was used for the analysis. The mobile phases consisted of two solvents: mobile phase A (10 mM tributylamine aqueous solution, pH 4.95 with 15 mM acetic acid) and mobile phase B (100% methanol), which were delivered using an Agilent 1290 UPLC binary pump (Agilent Technologies, Palo Alto, CA, USA) at a flow rate of 200 µL min⁻¹, with a linear gradient program implemented as follows: hold isocratic at 0% B (0–2 min); linear gradient from 0% to 85% B (2–28 min); hold isocratic at 0% B (28–34 min). The effluent from the LC column was delivered to an Agilent 6490 triple-quadrupole mass spectrometer, equipped with an electrospray ionization source operating in negative-ion mode. NAD, NADH, NADP, NADPH, and Glu were monitored in the multiple reaction monitoring modes with the transition events at m/z 662.3 > 540, 664.3 > 79, 742 > 620, 744 > 79, and 147 > 84, respectively.

RNA extraction, library preparation, and sequencing

At the end of the chemostat culturing, T. erythraeum was collected in the middle of the photoperiod by filtering onto 3 μm polycarbonate membrane filters (Millipore), flash frozen in liquid nitrogen and stored at −80 °C until extraction. Total RNA was extracted using TRIzol® Reagent (Invitrogen) combined with a physical cell disruption approach by glass beads according to the manufacturer’s instructions. Genomic DNA was removed thoroughly by treating it with RNAase-free DNase I (Takara, Japan). Ribosomal RNA was removed from a total amount of 3 µg RNA using Ribo-Zero rRNA Removal kit (Illumina, USA). Subsequently, cDNA libraries were generated according to the manufacturer’s protocol of NEBNext® Ultra^TM Directional RNA Library Prep Kit for Illumina® (NEB, USA). The quality of the library was assessed on the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Libraries were sequenced on an Illumina Hiseq 2500 platform, yielding 136-bp paired-end reads.

RNA-Seq bioinformatics

Clean reads were obtained from raw data by removing reads containing adapter, ploy-N and low-quality read. Qualified sequences were mapped to the Trichodesmium erythraeum IMS101 genome (https://www.ncbi.nlm.nih.gov/nuccore/NC_008312.1) by using Bowtie2-2.2.3⁵³. Differential expression analysis for high/low pCO₂ with P limitation was performed using the DESeq2 R package⁵⁴. The resulting p-values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted p-value < 0.05 were assigned as significantly differentially expressed. The data are deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE181428. GO and KEGG enrichment analyses of differentially expressed genes were implemented by the GOseq R package and KOBAS software respectively. GO terms with corrected p-value < 0.05 were considered significantly enriched by differentially expressed genes.

Quantitative polymerase chain reaction (qPCR) analysis and standard preparation

Extracted RNA was treated with DNase and then reverse-transcribed using random primers by M-MLV reverse transcriptase (BGI, China) to generate cDNA. The primer sequences of each gene were obtained from the literature or designed at the Genscript website and checked for validity using the Primer-Blast tool in NCBI (Supplementary Table 7). The standards for qPCR were generated as described previously⁶. Briefly, cDNA amplicon of the interested genes was PCR amplified, separated by gel electrophoresis, purified, inserted into pMD 18-T vectors (Takara), and used to transform DH5a Escherichia coli competent cells. Sequenced plasmid DNA from positive clones was purified and then quantified using the Qubit DNA HS Assay kit (Invitrogen).

All qPCR reactions were carried out on a fluorescent quantitative instrument CFX 96 TOUCH (Bio-Rad Laboratories). An SYBR Green I master mix (Zhishan Biotech) was used for qPCR in 20 μL reactions containing ~5 μL of diluted cDNA template, 0.4 mM dNTPs, 200 nM of each primer, and 0.05 U Taq polymerase (Tiangen Biotech). The following PCR reactions program was applied: 95 °C for 3 min, followed by 39 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Standards corresponding to between 10³ and 10⁸ copies per well were amplified on the same 96-well plate as the cDNA generated from experimental materials. The amplification efficiencies of PCR were always between 90% and 103% with R² values >0.99. To correct for differences in cDNA synthesis efficiency, the abundance of each transcript was normalized to the abundance of the housekeeping gene FtsZ (cell division protein) transcript.

Calculating P contents in RNA and genomic DNA

Extracted total RNA was exactly quantified using the Qubit RNA HS Assay kit (Invitrogen). According to the approximate average M.W. of nucleotide within polynucleotide, i.e., 320.5 g mol⁻¹ (Thermo Fisher Scientific), the P contents in RNA were estimated. According to the genome size of T. erythraeum IMS101 (i.e., 7,750,108 bp) and taking into account polyploidy in Trichodesmium (i.e., assuming 100 genome copies per cell under P-limited conditions)⁵⁵, the P contents in genomic DNA was estimated.

Statistical analysis

The statistical significance of differences between ambient and acidified treatments was analyzed by two-tailed paired Student’s t-test using SigmaPlot 12.5 (Systat Software, Inc). A significance level of p < 0.05 was applied.

Field experiments

A total of four phosphate amendment experiments and seven ocean acidification experiments were conducted aboard the R/V Dongfanghong 2 and R/V Tan Kah Kee during several cruises to the northern South China Sea between May 2016 and August 2018 (Supplementary Fig. 4).

Phosphate amendment experiments

Experimental setup

The incubation experiments were carried out at stations TM-4, TM-5, S1, and SK2 (Supplementary Fig. 4). Trace metal-clean seawater was collected using a towed fish system in which surface seawater (~5 m) was pumped through Teflon tubing into 10-L polycarbonate carboys directly, using a Teflon diaphragm pump (Sandpiper, USA). Trace metal-clean technique was used in setting up and sampling the experiments. All materials coming in contact with the incubation water were acid-washed in a class-100 cleanroom before use. For the phosphate-amended treatments, NaH₂PO₄ (chelexed and filter-sterilized) was added at a final concentration of 100 nM. For all experiments, triplicate carboys were incubated for 3 days in on-deck, flow-through incubators screened with neutral density screening to ~45% of sea surface irradiance, and subsampling was done in a laminar flow hood.

Sample collection for Trichodesmium genes sequencing and sphX gene transcription

After incubation, 3–4 L of seawater from each one of the triplicate carboys was collected by filtration under low vacuum pressure onto 47 mm 0.2 µm polycarbonate membrane filters (Millipore) for subsequent DNA and RNA extraction. Nucleic acid filtrations were typically completed within 45 min. All filters were flash-frozen in liquid nitrogen and kept at −80 °C until extraction.

Ocean acidification experiments

Experimental setup

The incubation experiments were carried out using either 2–4 L polycarbonate bottles or 10–20 L polycarbonate carboys (Nalgene Labware) at 7 stations including TM-4 and TM-5 where the phosphate amendment experiments were conducted (Supplementary Fig. 4). At station OA-5, a Trichodesmium bloom occurred. For the experiments at stations, OA-2–OA-5, non-trace metal-clean near-surface seawater (~5 m) was collected using either a Teflon diaphragm pump or a Sea-bird CTD-General Oceanic rosette sampler with GO-Flo bottles. For the rest of the experiments (i.e., at stations TM-3–TM-5), the trace metal-clean technique was used in seawater sampling and experimental setup, as described above. For all the experiments, triplicate carboys or bottles were incubated for 1–3 days in on-deck, flow-through incubators screened to ~45% of sea surface irradiance.

Carbonate chemistry manipulation

For the experiments at station OA-5, ultra-pure HCl and NaOH were used to adjust seawater carbonate chemistry. For the experiments at other stations, seawater carbonate chemistry was manipulated by gently bubbling with 0.22 µm-filtered air or CO₂–air mixture generated by CO₂ mixers (Ruihua Instrument & Equipment Ltd.). pH was measured using a pH electrode (Eutech pH 110 meter with Eutech ECFC7352901B probe) calibrated with National Institute of Standards and Technology pH standard buffers. Intercalibrations between the electrode measurements and spectrophotometric pH measurements⁴⁶ were made on seawater samples to arrive at pH_T (pH on the total scale). The measurements of dissolved inorganic carbon (DIC) concentration and the calculations of alkalinity and pCO₂ were done as described above. The carbonate chemistry of the different experiments is shown in Supplementary Table 6.

N₂ fixation rates

N₂ fixation rates were measured using the ¹⁵N₂ gas dissolution method⁵⁶. Briefly, 0.22 µm-filtered surface seawater was degassed as described in Shiozaki et al.⁵⁷. After that, 10 mL 98% pure ¹⁵N₂ gas (Cambridge Isotope Laboratories) was injected into a gas-tight plastic bag containing 1 L of the degassed seawater and allowed to fully equilibrate before use. The percentage of ¹⁵N₂ in the ¹⁵N₂-enriched seawater was validated using a GasBench-IRMS⁵⁸. Following 1–3 days of incubation, a sub-sample from each incubation carboy was transferred into a polycarbonate bottle, and then ¹⁵N₂-enriched seawater was added with the enriched water constituting approximately 2.6% of the total sample volume. Bottles were then returned to the flow-through incubators under the same irradiance of each experiment and incubated for 24 h. After the incubation, particulate matter in seawater from each bottle was filtered onto 25 mm pre-combusted GF/F filters (Millipore). Particulate organic matters collected from the same incubation carboy but without ¹⁵N₂ enrichment were filtered to determine ¹⁵N-PON natural abundance. All filter samples were stored at −20 °C immediately after collection. In the shore-based laboratory, sample filters were dried and analyzed using a Flash HT 2000 elemental analyzer coupled with a Thermo Finnigan Delta V Plus isotope ratio mass spectrometer. The rates of N₂ fixation were calculated according to Mohr et al.⁵⁶.

Soluble reactive phosphate (SRP) analysis

The SRP concentrations were measured using the method as described above in I. Laboratory experiments.

Sample collections for nifH abundance and Trichodesmium ppnK gene transcription

To analyze nifH gene abundance, at most stations, 3–4 L (500 mL at station OA-5) of seawater from each one of the triplicate carboys was collected following the incubation by filtration under low vacuum pressure onto 47 mm 0.2 µm polycarbonate membrane filters (Millipore). At station OA-2, 4.5 L of the surface seawater was directly collected for nifH gene analysis without incubation. For Trichodesmium ppnK gene transcription analysis, 3–4 L of seawater was subsampled from the incubations at stations TM-3, TM-4, TM-5, OA-3, OA-4, and OA-5. All filters were flash-frozen in liquid nitrogen and kept at −80 °C until DNA/RNA extraction.

DNA extraction

To extract DNA, membrane filters were cut into pieces under sterile conditions and then placed in tubes containing 800 µL of sucrose lysis buffer (40 mM EDTA, 50 mM Tris–HCl, and 0.75 M sucrose) for beads beating with 0.1 mm and 0.5 mm glass beads. The cells were broken, agitated for 3 min inside a Fast Prep machine (MP Biomedicals, USA), and frozen in liquid nitrogen 3 times. 5 µL of lysozyme (100 mg mL⁻¹) was then added and the sample was incubated for 1 h at 37 °C. After incubation, the lysates were transferred into a 2-mL Eppendorf tube. Proteins were digested by incubating with 1% sodium dodecyl sulfate (SDS) and proteinase K (250 µg mL⁻¹) at 55 °C for 2 h, and were removed by centrifugation at 12,000×g for 20 min at 4 °C after being treated with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) containing 5 M NaCl. As a result, the sample was separated into three layers. The top aqueous layer that contained genomic DNA was transferred into a new tube, added an equal volume of chloroform: isoamyl alcohol (24:1), and centrifuged at 12,000×g for 20 min at 4 °C. Genomic DNA was purified by precipitation with 100% isopropanol at −20 °C overnight, followed by washing with 70% ethanol and air-drying. The genomic DNA was then eluted into 50 µL TE buffer and stored at −20 °C.

RNA extraction and reverse transcription-PCR

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hildern, Germany) according to manufacturer instructions, with a minor modification to the cell disruption step. Briefly, RLT buffer with 1% of β-Mercaptoethanol and RNA-clean glass beads (0.1 mm diameter) were added and samples were vortexed with a Fast Prep machine (MP Biomedicals, USA). The resulting lysate was processed following the manufacturer’s instructions, including on-column DNase digestion (RNase-free DNase Kit, Qiagen), and then RNA was eluted in RNase-free water. The concentration of RNA was determined using the Qubit RNA HS Assay kit (Invitrogen). Reverse transcription was carried out using M-MLV reverse transcriptase in a 20 µL reaction volume containing 150 ng of random primers, 1 mM dNTP mix, and 10 mM DTT. Complimentary DNA (cDNA) was stored at −20 or −80 °C.

Cloning and sequencing

According to genome sequences of T. erythraeum IMS101 and T. thiebautii H9-4, the degenerate primers used to amplify the high-affinity phosphate binding protein gene sphX and the inorganic polyphosphate/ATP_NAD kinase gene ppnk from Trichodesmium species were designed (Supplementary Table 7). The sphX (783 bp) and ppnK (620 bp) genes were amplified from samples collected at stations TM-4 and TM-5. The amplified products were separated by gel electrophoresis, purified, and cloned for sequencing. Forty positive clones were sequenced for each gene from each station. The cloned sequences were aligned with the target gene sequence in T. erythraeum IMS101 and T. thiebautii H9-4 genomes using Mega software version 7.0 and were then used to generate a maximum-likelihood phylogenetic tree (Supplementary Fig. 5). The sphX and ppnk sequences produced in this study have been submitted to GenBank and are accessible through GenBank accession numbers MZ749754 – MZ749900.

Quantitative qPCR analysis

The clade-specific primers of housekeeping gene rnpB and the Tery-specific sphX primers were listed in Supplementary Table 7. According to our alignments, the clade-specific ppnK primers and the Tten-specific sphX primers were designed at the Genscript website and checked for validity using the Primer-Blast tool in NCBI (Supplementary Table 7). Standard curves for qPCR were constructed using cloned target DNA fragments. Briefly, cDNA amplicon of the genes of interest was PCR amplified, separated by gel electrophoresis, purified, and cloned. Sequenced plasmid DNA from positive clones was purified and quantified using the Qubit DNA HS Assay kit (Invitrogen). All qPCR reactions were carried out on a fluorescent quantitative instrument CFX 96 TOUCH (Bio-Rad, Singapore). A SYBR Green I master mix (Zhishan Biotech) was used for qPCR in 20 μL reactions containing ~5 μL of diluted cDNA template, 0.4 mM dNTPs, 200 nM of each primer, and 0.05 U Taq polymerase (Tiangen Biotech). The following PCR reactions program was applied: 95 °C for 3 min, followed by 39 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. Using 10-fold increments, the standards corresponding to between 10⁰ and 10⁹ copies per well were amplified on the same 96-well plate as the cDNA generated from experimental materials. The amplification efficiencies of PCR were always between 90 and 95% with R² values >0.99. The specificity of the qPCR reactions was confirmed by melting curve analysis, agarose gel electrophoresis, and sequencing analysis. Relative expression of target genes versus rnpB was calculated by dividing the copy numbers of the target gene by the copy numbers of rnpB quantified in the same sample.

nifH Abundance

The qPCR analysis was targeted on the nifH phylotypes of Trichodesmium spp., Richelia sp. (Het-1), unicellular cyanobacteria UCYN-A1, UCYN-A2 and UCYN-B, and a gamma-proteobacterium (γ-24774A11) using previously designed primers^59,60,61. Probes were 5′-labeled with the fluorescent reporter FAM (6-carboxyfluorescein) and 3′-labeled with TAMRA (6-carboxytetramethylrhodamine) as a quenching dye. The nifH standards were obtained by cloning the environmental sequences from previous samples of the South China Sea. The DNA concentrations of nifH standards were determined by Quant-iTTM Picogreens® dsDNA Reagent and Kits (Invitrogen) using a Fluoroskan Ascent FL fluorescence microplate reader (Thermo Scientific). Quantitative PCR analysis was carried out as described previously⁶⁰ with slight modifications. qPCR reactions were run in triplicate for each environmental DNA sample and each standard, using the following thermal cycle program: 50 °C for 2 min, 94 for 10 min, followed by 49 cycles of 95 °C for 15 s, 60 °C for 1 min. The 20 µL reactions contained environmental cDNA or standard (1 µL), forward and reverse primers, and probes. Standards corresponding to between 10¹ and 10⁹ copies per well were amplified on the same 96-well plate. The copy numbers of the target genes in the environmental samples were calculated from the standard curve.

Statistical analysis

Statistical significance of differences between ambient and acidified treatments was tested both on data from each individual experiment and on data from all experiments by one-tailed paired Student’s t-test using SigmaPlot 12.5 (Systat Software, Inc.). A significance level of p < 0.05 was applied.

Model estimations

Contribution to global oceanic N₂ fixation by Trichodesmium

N₂ fixation rate by each diazotrophic group was estimated by the product of nifH-based diazotrophs’ cell abundance and their cell-specific N₂ fixation rate:

where nifH abundance = nifH gene copies L⁻¹, conversion factor of nifH copies to cell count = nifH gene copies cell⁻¹, and cell specific N₂ fixation rate = fmol N cell⁻¹ h⁻¹.

We first used a machine learning algorithm (build in MATLAB_R2021a)—random forest—to estimate the nifH gene abundance of Trichodesmium, UCYN-A, UCYN-B (Crocosphaera), and Richelia (diatom-diazotroph associations) in the global ocean based on Tang and Cassar⁴². Briefly, field-observed volumetric nifH gene abundance was matched to contemporaneous, climatological, or modeled environmental predictors including temperature, salinity, dissolved oxygen, nitrate, phosphate, photosynthetically available radiation (PAR), and iron. Climatological temperature, salinity, dissolved oxygen, nitrate, and phosphate data were obtained from World Ocean Atlas 2018⁶². PAR was measured by SeaWiFS and MODIS satellites, and dissolved iron concentration in the global ocean was modeled by CESM2⁶³. After training random forest models with these environmental properties (Supplementary Fig. 6), we estimated the volumetric nifH gene abundance of four diazotrophic groups in the global ocean using the environmental factors. The conversion factor of nifH copies to cell counts and the cell-specific N₂ fixation rate were obtained from the existing literature for each diazotrophic group (Supplementary Table 8). Average values for each parameter were used in the rate estimate. N₂ fixation was assumed to be active over 12 h daily. Overall, Trichodesmium accounts for approximately 35% of the global oceanic N₂ fixation (Supplementary Fig. 7). We acknowledge the large uncertainties associated with our estimates due to the variation in parameters used in the calculation (e.g., variation in the conversion factor from nifH gene abundance to cell count^55,64 and cell-specific N₂ fixation rate⁶⁵). Nevertheless, our estimated total contribution of Trichodesmium to the global oceanic N₂ fixation, in particular its dominance in tropical and subtropical oceans, is generally within the range of the previous estimates^2,66,67. More targeted observations on Trichodesmium will be required to better constrain its contribution to the global N₂ fixation.

Effects of ocean acidification and P limitation on N₂ fixation by Trichodesmium

We estimated changes in the global oceanic N₂ fixation under the impacts of ocean acidification and/or P limitation by the end of 21st century under the representative concentration pathway (RCP) 8.5 emission scenario. The global oceanic N₂ fixation was simulated using the biogeochemical elemental cycling (BEC) Model embedded in the Community Earth System Model version 1 (CESMv1)⁶⁸, which has been described in detail previously^69,70. Further calculations were conducted using code written with MATLAB_R2018b. As in most Earth system models, there is only one diazotrophic functional group representing all N₂ fixers in BEC. Therefore, N₂ fixation by Trichodesmium is calculated by multiplying the BEC-simulated N₂ fixation by the estimated contribution of Trichodesmium (Supplementary Fig. 7). Note that impacts of acidification on N₂ fixation were not represented in the BEC. An offline mode of BEC was then combined with our laboratory results to illustrate the potential magnitude of impacts of ocean acidification and/or P limitation on the global N₂ fixation by Trichodesmium.

First, we estimated the impacts of ocean acidification without considering P-limitation. As shown in a previous study⁸, when P is not limiting ocean acidification only affects N₂ fixation, not C biomass. Assuming N₂ fixation rates declined linearly with [H⁺] in acidified waters, we interpolated and/or extrapolated impacts of ocean acidification based on the laboratory results and simulated pH for each model grid of CESMv1 from 2081 to 2100:

where ({{{rm {NF}}}}_{{{rm {Tr}}}}^{{{rm {oa}}}}) is N₂ fixation by Trichodesmium at simulated [H⁺] (({left[{{rm {H}}}^{+}right]}_{{rm {s}}})) in the grid under the RCP 8.5 scenario, NF is the BEC-simulated N₂ fixation, R_Tr is the contribution of Trichodesmium to N₂ fixation (Supplementary Fig. 7), and oa of −18.4% is from a previous study⁸ in which C biomass-specific N₂ fixation rate of Trichodesmium declined by 18.4% as seawater [H⁺] increased from ({left[{{rm {H}}}^{+}right]}_{0}) (10^−8.01) to ({left[{{rm {H}}}^{+}right]}_{1}) (10^−7.81) when P is not limiting (Supplementary Table 2). Meanwhile, the simulated climatology of Trichodesmium N₂ fixation without ocean acidification impacts (i.e., ({{{rm {NF}}}}_{{{rm {Tr}}}}={{rm {NF}}}times {R}_{{{rm {Tr}}}})) from 2081 to 2100 is used as the reference. Comparison of the integrated ({{{rm {NF}}}}_{{{rm {Tr}}}}^{{{rm {oa}}}}) with the reference value, i.e., (sum {{{rm {NF}}}}_{{{rm {Tr}}}}^{{{rm {oa}}}}-sum {{{rm {NF}}}}_{{{rm {Tr}}}}), shows impacts of ocean acidification on the global N₂ fixation by Trichodesmium.

Next, we estimated the impacts of ocean acidification on Trichodesmium with the synergistic impacts of P limitation being considered. Our laboratory experiments showed that ocean acidification affects both C biomass and N₂ fixation under P limitation, which was taken into account in our estimations. The limitation strength of various nutrients was calculated in the BEC model, which can be used to identify waters in which Trichodesmium growth was limited by P availability. Since Trichodesmium can use both DIP and DOP for their growth in the model, both forms of P are considered in the limiting factor calculations based on Michaelis–Menten kinetics:

where ({V}_{i}^{{rm {P}}}) is the limiting factor for P, and ({K}_{i}^{{{{{{{rm{PO}}}}}}}_{4}}) is about 5 times lower than ({K}_{i}^{{{{{{rm{DOP}}}}}}}), representing that phosphate is a more preferred P source than DOP. The additional impacts of P limitation were then only applied to Trichodesmium in these P-limiting regions, while the impacts of acidification in P-replete waters were calculated similarly as in Eq. (2). Under P limitation, we assumed C biomass and N₂ fixation rate decreased linearly with [H⁺] based on our laboratory data:

where oap_C and oap_N were set to −39.2% and −10.1%, respectively, based on our laboratory results in which C biomass (i.e., POC:POP) and C biomass-specific N₂ fixation rate of P-limited Trichodesmium decreased by 39.2% and 10.1%, respectively, under acidified conditions (Supplementary Table 2). The synergistic interaction of acidification and P limitation on the global N₂ fixation was calculated by (sum {{{rm {NF}}}}_{{{rm {Tr}}}}^{{{rm {oap}}}}-sum {{{rm {NF}}}}_{{{rm {Tr}}}}).

The large-scale effects of ocean acidification and P limitation on Trichodesmium N₂ fixation illustrated here are first-order estimates largely representing their relative strength in different oceanic regions. Uncertainties existed in our estimates due to the assumption of a linear function and an offline calculation. However, simulations with explicit representations of various diazotrophic groups and their responses to acidification in Earth system models are not feasible without further laboratory results to provide key information for model developments.

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Source: Ecology - nature.com