Metabolic preference of nitrate over oxygen as an electron acceptor in foraminifera from the Peruvian oxygen minimum zone.

Benthic foraminifera populate a diverse range of marine habitats. Their ability to use alternative electron acceptors-nitrate (NO3-) or oxygen (O2)-makes them important mediators of benthic nitrogen cycling. Nevertheless, the metabolic scaling of the two alternative respiration pathways and the environmental determinants of foraminiferal denitrification rates are yet unknown. We measured denitrification and O2 respiration rates for 10 benthic foraminifer species sampled in the Peruvian oxygen minimum zone (OMZ). Denitrification and O2 respiration rates significantly scale sublinearly with the cell volume. The scaling is lower for O2 respiration than for denitrification, indicating that NO3- metabolism during denitrification is more efficient than O2 metabolism during aerobic respiration in foraminifera from the Peruvian OMZ. The negative correlation of the O2 respiration rate with the surface/volume ratio is steeper than for the denitrification rate. This is likely explained by the presence of an intracellular NO3- storage in denitrifying foraminifera. Furthermore, we observe an increasing mean cell volume of the Peruvian foraminifera, under higher NO3- availability. This suggests that the cell size of denitrifying foraminifera is not limited by O2 but rather by NO3- availability. Based on our findings, we develop a mathematical formulation of foraminiferal cell volume as a predictor of respiration and denitrification rates, which can further constrain foraminiferal biogeochemical cycling in biogeochemical models. Our findings show that NO3- is the preferred electron acceptor in foraminifera from the OMZ, where the foraminiferal contribution to denitrification is governed by the ratio between NO3- and O2.

Widespread occurrence of nitrate storage and denitrification among Foraminifera and Gromiida.

Benthic foraminifers inhabit a wide range of aquatic environments including open marine, brackish, and freshwater environments. Here we show that several different and diverse foraminiferal groups (miliolids, rotaliids, textulariids) and Gromia, another taxon also belonging to Rhizaria, accumulate and respire nitrates through denitrification. The widespread occurrence among distantly related organisms suggests an ancient origin of the trait. The diverse metabolic capacity of these organisms, which enables them to respire with oxygen and nitrate and to sustain respiratory activity even when electron acceptors are absent from the environment, may be one of the reasons for their successful colonization of diverse marine sediment environments. The contribution of eukaryotes to the removal of fixed nitrogen by respiration may equal the importance of bacterial denitrification in ocean sediments.

Contribution of boulder reef habitats to oxygen dynamics of a shallow estuary.

We assessed the importance of boulder reefs to the oxygen dynamics of a shallow estuary during two growing seasons in 2017 and 2018. Using open-system diel oxygen measurements and benthic and pelagic incubations, we evaluated the relative contribution of pelagic and benthic habitats to the ecosystem metabolism along a depth gradient in two areas, with (Reef) and without (Bare) boulder reefs in the Limfjorden, Denmark. System integrated areal rates of gross primary production (GPP) and ecosystem respiration (ER) both increased with depth in both areas. Benthic contribution to system GPP and ER was highest at shallow depth where it represented 47 and 53% respectively. However, with increasing depth pelagic processes dominated GPP and ER (98 and 94%) even in the Reef area. Although the Reef area had higher biomass of auto- and heterotrophic organisms (macroalgae and macrofauna), benthic GPP was at similar level in both areas, due to a significant contribution from micro-phytobenthic organisms. The Reef area had lower sediment pools of organic matter, nitrogen and phosphorous and was slightly more oxygenated compared to the nearby Bare area. Extreme temperatures and higher levels of nutrients in 2018 caused a marked increase in benthic ER rates resulting in net heterotrophy (NEM = GPP - ER < 0) in 2018 compared to net autotrophy (NEM > 0) in 2017. Under current eutrophic conditions, boulder reefs do not contribute positively to the oxygen dynamics in the estuary. Reoccurring blooms of phytoplankton with high organic matter decomposition combined with high temperatures and dominance of fauna stimulate depletion of oxygen around the reefs. Significant improvements in water clarity are needed to regrow perennial macroalgae and induce net autotrophy. Under current turbid conditions, it is only recommended to establish boulder reefs in shallow (<3 m) parts of the estuary.

Sinks and Sources of Intracellular Nitrate in Gromiids.

A substantial nitrate pool is stored within living cells in various benthic marine environments. The fate of this bioavailable nitrogen differs according to the organisms managing the intracellular nitrate (ICN). While some light has been shed on the nitrate carried by diatoms and foraminiferans, no study has so far followed the nitrate kept by gromiids. Gromiids are large protists and their ICN concentration can exceed 1000x the ambient nitrate concentration. In the present study we investigated gromiids from diverse habitats and showed that they contained nitrate at concentrations ranging from 1 to 370 mM. We used 15N tracer techniques to investigate the source of this ICN, and found that it originated both from active nitrate uptake from the environment and from intracellular production, most likely through bacterial nitrification. Microsensor measurements showed that part of the ICN was denitrified to N2 when gromiids were exposed to anoxia. Denitrification seemed to be mediated by endobiotic bacteria because antibiotics inhibited denitrification activity. The active uptake of nitrate suggests that ICN plays a role in gromiid physiology and is not merely a consequence of the gromiid hosting a diverse bacterial community. Measurements of aerobic respiration rates and modeling of oxygen consumption by individual gromiid cells suggested that gromiids may occasionally turn anoxic by their own respiration activity and thus need strategies for coping with this self-inflicted anoxia.

Nitrate Storage and Dissimilatory Nitrate Reduction by Eukaryotic Microbes.

The microbial nitrogen cycle is one of the most complex and environmentally important element cycles on Earth and has long been thought to be mediated exclusively by prokaryotic microbes. Rather recently, it was discovered that certain eukaryotic microbes are able to store nitrate intracellularly and use it for dissimilatory nitrate reduction in the absence of oxygen. The paradigm shift that this entailed is ecologically significant because the eukaryotes in question comprise global players like diatoms, foraminifers, and fungi. This review article provides an unprecedented overview of nitrate storage and dissimilatory nitrate reduction by diverse marine eukaryotes placed into an eco-physiological context. The advantage of intracellular nitrate storage for anaerobic energy conservation in oxygen-depleted habitats is explained and the life style enabled by this metabolic trait is described. A first compilation of intracellular nitrate inventories in various marine sediments is presented, indicating that intracellular nitrate pools vastly exceed porewater nitrate pools. The relative contribution by foraminifers to total sedimentary denitrification is estimated for different marine settings, suggesting that eukaryotes may rival prokaryotes in terms of dissimilatory nitrate reduction. Finally, this review article sketches some evolutionary perspectives of eukaryotic nitrate metabolism and identifies open questions that need to be addressed in future investigations.

A single-cell sequencing approach to the classification of large, vacuolated sulfur bacteria.

The colorless, large sulfur bacteria are well known because of their intriguing appearance, size and abundance in sulfidic settings. Since their discovery in 1803 these bacteria have been classified according to their conspicuous morphology. However, in microbiology the use of morphological criteria alone to predict phylogenetic relatedness has frequently proven to be misleading. Recent sequencing of a number of 16S rRNA genes of large sulfur bacteria revealed frequent inconsistencies between the morphologically determined taxonomy of genera and the genetically derived classification. Nevertheless, newly described bacteria were classified based on their morphological properties, leading to polyphyletic taxa. We performed sequencing of 16S rRNA genes and internal transcribed spacer (ITS) regions, together with detailed morphological analysis of hand-picked individuals of novel non-filamentous as well as known filamentous large sulfur bacteria, including the hitherto only partially sequenced species Thiomargarita namibiensis, Thioploca araucae and Thioploca chileae. Based on 128 nearly full-length 16S rRNA-ITS sequences, we propose the retention of the family Beggiatoaceae for the genera closely related to Beggiatoa, as opposed to the recently suggested fusion of all colorless sulfur bacteria into one family, the Thiotrichaceae. Furthermore, we propose the addition of nine Candidatus species along with seven new Candidatus genera to the family Beggiatoaceae. The extended family Beggiatoaceae thus remains monophyletic and is phylogenetically clearly separated from other related families.

Distribution, ecology and molecular identification of Thioploca from Danish brackish water sediments.

The distribution of Thioploca populations was investigated in Danish fjords, brackish lakes and coastal waters. Thioploca was found in three geographically distinct populations, where biomasses reached 33.8+/-14.3 g wet weight m(-2) (mean+/-SD). Mats or lawns were not formed at the sediment surfaces and Thioploca biomasses peaked 4-7 cm into the sediment and extended down to 18 cm depth. Morphology and 16S rRNA gene sequences classified all populations as Thioploca ingrica. A sequence divergence of 1.7-2.2% indicated that T. ingrica comprise at least two genotypes. Physiological analysis showed that T. ingrica accumulate nitrate in concentrations of approximately 3 mM and that bicarbonate and acetate are used as a carbon source. The presence of oxygen promoted carbon incorporation, but T. ingrica could survive up to 3 months without an external supply of nitrate or oxygen. Thioploca ingrica populations were exclusively found close to river outlets in a bioturbated sediment with separate sulphidic spots and worm burrow walls containing nitrate and oxygen. It is hypothesized that the subsurface T. ingrica have a special advantage in this heterogeneous environment using their sheath surrounding the bacterial trichomes when navigating between electron donor and acceptor.

Physiology and behaviour of marine Thioploca.

Among prokaryotes, the large vacuolated marine sulphur bacteria are unique in their ability to store, transport and metabolize significant quantities of sulphur, nitrogen, phosphorus and carbon compounds. In this study, unresolved questions of metabolism, storage management and behaviour were addressed in laboratory experiments with Thioploca species collected on the continental shelf off Chile. The Thioploca cells had an aerobic metabolism with a potential oxygen uptake rate of 1760 micromol O2 per dm(3) biovolume per h, equivalent to 4.4 nmol O2 per min per mg protein. When high ambient sulphide concentrations (approximately 200 microM) were present, a sulphide uptake of 6220+/-2230 micromol H2S per dm(3) per h, (mean+/-s.e.m., n=4) was measured. This sulphide uptake rate was six times higher than the oxidation rate of elemental sulphur by oxygen or nitrate, thus indicating a rapid sulphur accumulation by Thioploca. Thioploca reduce nitrate to ammonium and we found that dinitrogen was not produced, neither through denitrification nor through anammox activity. Unexpectedly, polyphosphate storage was not detectable by microautoradiography in physiological assays or by staining and microscopy. Carbon dioxide fixation increased when nitrate and nitrite were externally available and when organic carbon was added to incubations. Sulphide addition did not increase carbon dioxide fixation, indicating that Thioploca use excess of sulphide to rapidly accumulate sulphur rather than to accelerate growth. This is interpreted as an adaptation to infrequent high sulphate reduction rates in the seabed. The physiology and behaviour of Thioploca are summarized and the adaptations to an environment, dominated by infrequent oxygen availability and periods of high sulphide abundance, are discussed.