Marta M. Varela

Diazotrophs, microorganisms capable of fertilizing the ocean through biological nitrogen fixation (BNF), are widespread in the oceans, including upwelling bays. However, their ability to respond to short‐lived upwelling pulses, lasting only a few days, remains poorly understood. To investigate diazotrophy across temporal scales, we analyzed 18 months of multidisciplinary observations from two dynamic upwelling bays in North West Iberia, combining weekly year‐round sampling with daily measurements during a 2‐week intensive summer cruise. BNF exhibited pronounced seasonal variability, with the highest rates (1.5 nmol N ) coinciding with increased abundance of the UCYN‐A2 nitroplast during late summer and early autumn. Time–frequency analysis indicated that diazotrophic abundance variability was driven not only by seasonal forcing but also by rapid environmental fluctuations, a pattern corroborated by the daily summer data set. BNF rates peaked during relaxation–downwelling periods, when they contribute with episodic yet substantial inputs of new nitrogen to the euphotic zone. In contrast, both BNF and UCYN‐A2 abundance declined sharply during nutrient‐rich upwelling pulses. Elevated BNF coincided with high abundances by microscopy of Hemiaulus spp. and Rhizosolenia spp., along with Richelia spp. detection by qPCR, presenting the first direct‐detection for diatom‐diazotroph associations in this region. These demonstrate that BNF can episodically become a main source of the new nitrogen supply during upwelling‐relaxation phases, acting as a transient “bridge” that helps sustain productivity between upwelling events. Our study underscores the need to re‐assess the role of diazotrophy in coastal upwelling systems, where its contribution may have been historically underestimated due to coarse temporal sampling.

Microbes in the dark ocean are exposed to hydrostatic pressure increasing with depth. Activity rate measurements and biomass production of dark ocean microbes are, however, almost exclusively performed under atmospheric pressure conditions due to technical constraints of sampling equipment maintaining in situ pressure conditions. To evaluate the microbial activity under in situ hydrostatic pressure, we designed and thoroughly tested an in situ microbial incubator (ISMI). The ISMI allows autonomously collecting and incubating seawater at depth, injection of substrate and fixation of the samples after a preprogramed incubation time. The performance of the ISMI was tested in a high‐pressure tank and in several field campaigns under ambient hydrostatic pressure by measuring prokaryotic bulk ³H‐leucine incorporation rates. Overall, prokaryotic leucine incorporation rates were lower at in situ pressure conditions than under to depressurized conditions reaching only about 50% of the heterotrophic microbial activity measured under depressurized conditions in bathypelagic waters in the North Atlantic Ocean off the northwestern Iberian Peninsula. Our results show that the ISMI is a valuable tool to reliably determine the metabolic activity of deep‐sea microbes at in situ hydrostatic pressure conditions. Hence, we advocate that deep‐sea biogeochemical and microbial rate measurements should be performed under in situ pressure conditions to obtain a more realistic view on deep‐sea biotic processes.

Zooplankton grazing onphytoplankton promotes the release of particulate and dissolved organic matter (DOM) into the water column and therefore plays a key role in organic matter cycling in aquatic systems. Prokaryotes are the main DOM consumers in the ocean by actively remineralizing and transforming it, contributing to its molecular diversification. To explore the molecular composition of zooplankton‐derived DOM and its bioavailability to natural prokaryotic communities, the DOM generated by a mixed zooplankton community in the coastal Atlantic off Spain was used as substrate for a natural prokaryotic community and monitored over a ~ 5‐d incubation experiment. The molecular composition of solid‐phase extracted DOM was characterized via Fourier‐transform ion cyclotron resonance mass spectrometry. After ~ 4 d in the zooplankton‐derived DOM amended incubation, the prokaryotic community demonstrated a 17‐fold exponential increase in cell number. The prokaryotic growth resulted in a reduction in bulk dissolved organic carbon concentration and the zooplankton‐derived DOM was considerably transformed at molecular and bulk elemental levels over the incubation period. The C : N ratio (calculated from the obtained molecular formulae) increased while the functional diversity decreased over the incubation time. In addition, molecular indices pointed to a reduced bioavailability of DOM at the end of the experiment. These findings show that zooplankton excreta are a source of labile organic matter that is quickly metabolized by the prokaryotic community. Therefore, a fraction of carbon is shunted from transfer to secondary consumers similarly to the viral shunt, suggesting that the zooplankton–prokaryotic interactions play an important role in the ocean's carbon cycle.

Abundance, activity and prokaryotic community composition were studied in the euphotic, intermediate and deep waters off the Galician coast (NW Iberian margin) in relation to the optical characterization of dissolved organic matter (DOM). Microbial (Archaea and Bacteria) community structure was vertically stratified. Among the Archaea, Euryarchaeota, especially Thermoplasmata, was dominant in the intermediate waters and decreased with depth, whereas marine Thaumarchaeota, especially Marine Group I, was the most abundant archaeal phyla in the deeper layers. The bacterial community was dominated by Proteobacteria through the whole water column. However, Cyanobacteria and Bacteroidetes occurrence was considerable in the upper layer and SAR202 was dominant in deep waters. Microbial composition and abundance were not shaped by the quantity of dissolved organic carbon, but instead they revealed a strong connection with the DOM quality. Archaeal communities were mainly related to the fluorescence of DOM (which indicates respiration of labile DOM and generation of refractory sub-products), while bacterial communities were mainly linked to the aromaticity/age of the DOM produced along the water column. Taken together, our results indicate that the microbial community composition is associated to the DOM composition of the water masses, suggesting that distinct microbial taxa have the potential to use and/or produce specific DOM compounds.

Summary: Marine Crenarchaeota are among the most abundant groups of prokaryotes in the ocean and recent reports suggest that they oxidize ammonia as an energy source and inorganic carbon as carbon source, while other studies indicate that Crenarchaeota use organic carbon and hence, live heterotrophically. We used catalysed reporter deposition fluorescence in situ hybridization (CARD‐FISH) to determine the crenarchaeal and bacterial contribution to total prokaryotic abundance in the (sub)tropical Atlantic. Bacteria contributed ∼50% to total prokaryotes throughout the water column. Marine Crenarchaeota Group I (MCGI) accounted for ∼5% of the prokaryotes in subsurface waters (100 m depth) and between 10 and 20% in the oxygen minimum layer (250–500 m depth) and deep waters (North East Atlantic Deep Water). The fraction of both MCGI and Bacteria fixing inorganic carbon, determined by combining microautoradiography with CARD‐FISH (MICRO‐CARD‐FISH), decreased with depth, ranging from ∼30% in the oxygen minimum zone to < 10% in the intermediate waters (Mediterranean Sea Outflow Water, Antarctic Intermediate Water). In the deeper water masses, however, MCGI were not taking up inorganic carbon. Using quantitative MICRO‐CARD‐FISH to determine autotrophy activity on a single cell level revealed that MCGI are incorporating inorganic carbon (0.002–0.1 fmol C cell ⁻¹ day ⁻¹ ) at a significantly lower rate than Bacteria (0.01–0.6 fmol C cell ⁻¹ day ⁻¹ ). Hence, it appears that MCGI contribute substantially less to autotrophy than Bacteria . Taking the stoichiometry of nitrification together with our findings suggests that MCGI might not dominate the ammonia oxidation step in the mesopelagic waters of the ocean to that extent as the reported dominance of archaeal over bacterial amo A would suggest.

The variability of the percentage of extracellular dissolved organic nitrogen (DON) release (PER), along with the relationship between DON release and bacterioplankton activity, was examined during five oceanographic cruises, carried out in the upwelling region of the NW Iberian Peninsula, the SW Bay of Biscay, and a latitudinal transect in the Atlantic Ocean (50°N–35°S). Rates of nitrogen uptake, DON release, and bacterial production were measured at 66 stations and sampled between August 1998 and October 2000. The percentage of DON release relative to the gross uptake of ammonium (PER^NH4+) ranged from 3 to 46%, whereas that relative to total nitrogen (NH₄ ⁺ + NO₃ ⁻ + urea) gross uptake (PER^total) varied between 21 and 82%. The highest values for both PER^NH4+ and PER^total were found in oligotrophic oceanic waters (6 mg chlorophyll a m⁻³), such as the continental shelf off the NW Iberian Peninsula, PER held constant as nitrogen uptake increased. These results suggest the dominance of different processes controlling DON release in oceanic and neritic zones. DON release rates accounted for less than 15% of the variability observed in bacterial production rates, suggesting a weak response of bacterioplankton to phytoplankton on short time scales (hours). Furthermore, nitrogen budgets showed an excess of DON release in relation to bacterial requirements.

Ammonium regeneration and dissolved organic nitrogen (DON) release were studied experimentally in the euphotic zone of shelf and oceanic waters of NW Spain in relation to coastal upwelling dynamics and the size-structure of phytoplankton communities. Incubations of plankton labelled with [¹⁵N]ammonium were made during four cruises, two of which also included size-fractionated determinations of chlorophyll a and primary production, and experimental determinations of production rates of dissolved organic carbon (DOC) using ¹⁴C. Inorganic nitrogen concentrations were mainly related to nitrate enrichment by upwelling pulses, while ammonium concentrations were generally low in all situations. Ammonium did not accumulate in the study area, suggesting a daily time scale coupling between regeneration and uptake. In contrast, DON largely exceeded inorganic nitrogen in all situations and generally increased from spring to autumn. Ammonium regeneration was positively correlated with DON release and both rates showed the largest variation in summer, with minimum values during active upwelling and maximum values when upwelling relaxed. Comparison of DON stocks and rates in different shelf areas suggests that DON release near the coast during summer was more persistent in the water than DON release in off-shelf and oceanic areas. The carbon:nitrogen ratio of DOC and DON release rates was highly variable, revealing a large excess of DOC compared with DON. This excess can be attributed to either an underestimate of total DON release (as only release from ammonium was measured) or to an enhanced production of carbon-rich organic substances by diatoms in coastal areas. By considering a broad range of trophic situations, this study reveals a fundamental difference between short term release of DOC and DON by plankton. Physiological processes (such as carbohydrate exudation by diatoms) seem to be the cause of large DOC excess, whereas trophic processes (such as grazing) are more likely the cause of enhanced DON release.