Quantifying Cyanothece growth under DIC limitation.

The photoautotrophic, unicellular N2-fixer, Cyanothece, is a model organism that has been widely used to study photosynthesis regulation, the structure of photosystems, and the temporal segregation of carbon (C) and nitrogen (N) fixation in light and dark phases of the diel cycle. Here, we present a simple quantitative model and experimental data that together, suggest external dissolved inorganic carbon (DIC) concentration as a major limiting factor for Cyanothece growth, due to its high C-storage requirement. Using experimental data from a parallel laboratory study as a basis, we show that after the onset of the light period, DIC was rapidly consumed by photosynthesis, leading to a sharp drop in the rate of photosynthesis and C accumulation. In N2-fixing cultures, high rates of photosynthesis in the morning enabled rapid conversion of DIC to intracellular C storage, hastening DIC consumption to levels that limited further uptake. The N2-fixing condition allows only a small fraction of fixed C for cellular growth since a large fraction was reserved in storage to fuel night-time N2 fixation. Our model provides a framework for resolving DIC limitation in aquatic ecosystem simulations, where DIC as a growth-limiting factor has rarely been considered, and importantly emphasizes the effect of intracellular C allocation on growth rate that varies depending on the growth environment

Metabolic flux analysis and the NAD(P)H/NAD(P) + ratios in chemostat cultures of Azotobacter vinelandii

Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P) + ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P) + ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions. The results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP + ratio, showing that at the lowest OTR (2.4 mmol L −1 h −1), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L −1 h −1). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L −1 h −1. At the highest OTR condition, a decrease in the NADPH/NADP + ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH). We have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes

Quantifying nutrient throughput and DOM production by algae in continuous culture

© 2020 Freshwater and marine algae can balance nutrient demand and availability by regulating uptake, accumulation and exudation. To obtain insight into these processes under nitrogen (N) and phosphorus (P) limitation, we reanalyze published data from continuous cultures of the chlorophyte Selenastrum minutum. Based on mass budgets, we argue that much of the non‐limiting N and P had passed through the organisms and was present as dissolved organic phosphorus or nitrogen (DOP or DON). We construct a model that describes the production of biomass and dissolved organic matter (DOM) as a function of the growth rate. A fit of this model against the chemostat data suggests a high turnover of the non‐limiting N and P: at the highest growth rates, N and P atoms spent on average only about 3 h inside an organism, before they were exuded as DON and DOP, respectively. This DOM exudation can explain the observed trends in the algal stoichiometric ratios as a function of the dilution rate. We discuss independent evidence from isotope experiments for this apparently wasteful behavior and we suggest experiments to quantify and characterize DON and DOP exudation further

Unusual marine cyanobacteria/haptophyte symbiosis relies on N2 fixation even in N-rich environments

The microbial fixation of N2 is the largest source of biologically available nitrogen (N) to the oceans. However, it is the most energetically expensive N-acquisition process and is believed inhibited when less energetically expensive forms, like dissolved inorganic N (DIN), are available. Curiously, the cosmopolitan N2-fixing UCYN-A/haptophyte symbiosis grows in DIN-replete waters, but the sensitivity of their N2 fixation to DIN is unknown. We used stable isotope incubations, catalyzed reporter deposition fluorescence in-situ hybridization (CARD-FISH), and nanoscale secondary ion mass spectrometry (nanoSIMS), to investigate the N source used by the haptophyte host and sensitivity of UCYN-A N2 fixation in DIN-replete waters. We demonstrate that under our experimental conditions, the haptophyte hosts of two UCYN-A sublineages do not assimilate nitrate (NO3-) and meet little of their N demands via ammonium (NH4+) uptake. Instead the UCYN-A/haptophyte symbiosis relies on UCYN-A N2 fixation to supply large portions of the haptophyte's N requirements, even under DIN-replete conditions. Furthermore, UCYN-A N2 fixation rates, and haptophyte host carbon fixation rates, were at times stimulated by NO3- additions in N-limited waters suggesting a link between the activities of the bulk phytoplankton assemblage and the UCYN-A/haptophyte symbiosis. The results suggest N2 fixation may be an evolutionarily viable strategy for diazotroph-eukaryote symbioses, even in N-rich coastal or high latitude waters