Mesopelagic N2 Fixation Related to Organic Matter Composition in the Solomon and Bismarck Seas (Southwest Pacific)

Abstract

Dinitrogen (N2) fixation was investigated together with organic matter composition in the mesopelagic zone of the Bismarck (Transect 1) and Solomon (Transect 2) Seas (Southwest Pacific). Transparent exopolymer particles (TEP) and the presence of compounds sharing molecular formulae with saturated fatty acids and sugars, as well as dissolved organic matter (DOM) compounds containing nitrogen (N) and phosphorus (P) were higher on Transect 1 than on Transect 2, while oxygen concentrations showed an opposite pattern. N2 fixation rates (up to ~1 nmol N L-1 d-1) were higher in Transect 1 than in Transect 2, and correlated positively with TEP, suggesting a dependence of diazotroph activity on organic matter. The scores of the multivariate ordination of DOM molecular formulae and their relative abundance correlated negatively with bacterial abundances and positively with N2 fixation rates, suggesting an active bacterial exploitation of DOM and its use to sustain diazotrophic activity. Sequences of the nifH gene clustered with Alpha-, Beta-, Gamma- and Deltaproteobacteria, and included representatives from Clusters I, III and IV. A third of the clone library included sequences close to the potentially anaerobic Cluster III, suggesting that N2 fixation was partially supported by presumably particle-attached diazotrophs. Quantitative polymerase chain reaction (qPCR) primer-probe sets were designed for three phylotypes and showed low abundances, with a phylotype within Cluster III at up to 103 nifH gene copies L-1. These results provide new insights into the ecology of non-cyanobacterial diazotrophs and suggest that organic matter sustains their activity in the mesopelagic ocean.

Fig 1. Stations sampled during the MoorSPICE cruise superimposed on a satellite image of chlorophyll a (Chl-a) concentrations.

Chl-a data were obtained from the National Aeronautics and Space Administration (NASA) Goddard Earth Sciences Data and Information Services Center Giovanni (NASA GES DISC) online database for the month of March 2014. Transect stations are numbered from 1 to 12 and depicted by an open circle. Process stations (where DOM enrichment experiments were made) are numbered from 1 to 4 and depicted by a cross.

Fig 2. Distribution of organic matter compounds along transects 1 and 2.

Transect 1 and Transect 2 (a, b) concentrations of TEP (μg GX Equiv L^-1), (c, d) sum of relative peak intensities of saturated fatty acids without heteroatoms, (e, f) saturated fatty acids with heteroatoms, (g, h) sugars without heteroatoms, (i, j) sugars with heteroatoms, and (k, l) peptides.

Fig 3. Sum of relative peak intensities of N-containing organic compounds.

(a) Transect 1, and (b) Transect 2, and P-containing organic compounds in (c) Transect 1, and (d) Transect 2.

Fig 4. Multivariate ordinations of DOM compound relative peak intensities for 23 samples.

Each point is labeled with the station and depth where the sample was collected (for the geographical location of the stations see Fig 1).

Fig 5. N₂ fixation rates superimposed on oxygen concentrations.

(a) N₂ fixation rates on Transect 1, and (b) Transect 2. Oxygen concentrations refer to the color scale (mL L^-1). The lower black circle indicates the reference size corresponding to 0.6 nmol N L^-1 d^-1.

Fig 6. Response of N₂ fixation rates to sugars and amino acids additions at Process stations 1–4.

Error bars represent the standard deviation of the mean of the triplicates.

Fig 7. Neighbor-joining tree of partial nifH amino acid sequences.

The sequences recovered from transects 1 and 2 are depicted in bold font. The number of clones recovered for each sample is indicated in parenthesis, and the station (st) and depth where the sample was recovered are shown. Clusters according to nifH-based phylogeny are shown to the right of the tree. Phylotypes are labeled as N1-N14 and cross-referenced with accession numbers in S2 Table (note that sequence M6432A05 is not included in the tree due to it being shorter). Reference amino acid sequence names are shown with GenBank accession, site, depth and clone numbers (where available). The sequences M6433A04 (N4), M6411A02 (N6) and M6413A02 (N10) were used for qPCR assays.

Fig 8. NifH copy abundance of the phylotype M6413A02 as determined by qPCR assays.

The lower black circle represents the reference size corresponding to 3.2 log₁₀ copies L^-1 in (a) Transect 1, and (b) Transect 2.