Organic Phosphorus Scavenging Supports Efficient Growth of Diazotrophic Cyanobacteria Under Phosphate Depletion.

Considering the reported significant diazotrophic activities in open-ocean regions where primary production is strongly limited by phosphate, we explored the ability of diazotrophs to use other sources of phosphorus to alleviate the phosphate depletion. We tested the actual efficiency of the open-ocean, N2-fixer Crocosphaera watsonii to grow on organic phosphorus as the sole P source, and observed how the P source affects the cellular C, N, and P composition. We obtained equivalent growth efficiencies on AMP and DL-α-glycerophosphate as compared with identical cultures grown on phosphate, and survival of the population on phytic acid. Our results show that Crocosphaera cannot use all phosphomonoesters with the same efficiency, but it can grow without phosphate, provided that usable DOP and sufficient light energy are available. Also, results point out that organic phosphorus uptake is not proportional to alkaline phosphatase activity, demonstrating that the latter is not a suitable proxy to estimate DOP-based growth yields of organisms, whether in culture experiments or in the natural environment. The growth parameters obtained, as a function of the P source, will be critical to improve and calibrate mathematical models of diazotrophic growth and the distribution of nitrogen fixation in the global ocean.

Independence of a Marine Unicellular Diazotroph to the Presence of NO3.

Marine nitrogen (N2) fixation was historically considered to be absent or reduced in nitrate (NO3-) rich environments. This is commonly attributed to the lower energetic cost of NO3- uptake compared to diazotrophy in oxic environments. This paradigm often contributes to making inferences about diazotroph distribution and activity in the ocean, and is also often used in biogeochemical ocean models. To assess the general validity of this paradigm beyond the traditionally used model organism Trichodesmium spp., we grew cultures of the unicellular cyanobacterium Crocosphaera watsonii WH8501 long term in medium containing replete concentrations of NO3-. NO3- uptake was measured in comparison to N2 fixation to assess the cultures' nitrogen source preferences. We further measured culture growth rate, cell stoichiometry, and carbon fixation rate to determine if the presence of NO3- had any effect on cell metabolism. We found that uptake of NO3- by this strain of Crocosphaera was minimal in comparison to other N sources (~2-4% of total uptake). Furthermore, availability of NO3- did not statistically alter N2 fixation rate nor any aspect of cell physiology or metabolism measured (cellular growth rate, cell stoichiometry, cell size, nitrogen fixation rate, nitrogenase activity) in comparison to a NO3- free control culture. These results demonstrate the capability of a marine diazotroph to fix nitrogen and grow independently of NO3-. This lack of sensitivity of diazotrophy to NO3- suggests that assumptions often made about, and model formulations of, N2 fixation should be reconsidered.

Enhancing PUFA-rich polar lipids in Tisochrysis lutea using adaptive laboratory evolution (ALE) with oscillating thermal stress.

Adaptive laboratory evolution is a powerful tool for microorganism improvement likely to produce enhanced microalgae better tailored to their industrial uses. In this work, 12 wild-type strains of Tisochrysis lutea were co-cultivated under increasing thermal stress for 6 months. Indeed, temperature was oscillating daily between a high and a low temperature, with increasing amplitude along the experiment. The goal was to enhance the polyunsaturated fatty acid content of the polar lipids. Samples were taken throughout the evolution experiment and cultivated in standardized conditions to analyze the evolution of the lipid profile. Genomic analysis of the final population shows that two strains survived. The lipid content doubled, impacting all lipid classes. The fatty acid analyses show a decrease in SFAs correlated with an increase in monounsaturated fatty acids (MUFAs), while changes in polyunsaturated fatty acid (PUFAs) vary between both photobioreactors. Hence, the proportion of C18-MUFAs (18:1 n-9) and most C18-PUFAs (18:2 n-6, 18:3 n-3, and 18:4 n-3) increased, suggesting their potential role in adjusting membrane fluidity to temperature shifts. Of particular interest, DHA in polar lipids tripled in the final population while the growth rate was not affected. KEY POINTS: • Adaptive laboratory evolution on a mix of 12 T. lutea strains led to survival of 2 • Thermal stress impacted cell size, total lipid cell content, and all lipid classes • DHA cell content partitioned to polar lipids tripled throughout the experiment.

How do microalgae perceive light in a high-rate pond? Towards more realistic Lagrangian experiments.

Hydrodynamics in a high-rate production reactor for microalgae cultivation affects the light history perceived by cells. The interplay between cell movement and medium turbidity leads to a complex light pattern, whose forcing effects on photosynthesis and photoacclimation dynamics are non-trivial. Hydrodynamics of high density algal ponds mixed by a paddle wheel has been studied recently, although the focus has never been on describing its impact on photosynthetic growth efficiency. In this multidisciplinary downscaling study, we first reconstructed single cell trajectories in an open raceway using an original hydrodynamical model offering a powerful discretization of the Navier-Stokes equations tailored to systems with free surfaces. The trajectory of a particular cell was selected and the associated high-frequency light pattern was computed. This light pattern was then experimentally reproduced in an Arduino-driven computer controlled cultivation system with a low density Dunaliella salina culture. The effect on growth and pigment content was recorded for various frequencies of the light pattern, by setting different paddle wheel velocities. Results show that the frequency of this realistic signal plays a decisive role in the dynamics of photosynthesis, thus revealing an unexpected photosynthetic response compared to that recorded under the on/off signals usually used in the literature. Indeed, the light received by a single cell contains signals from low to high frequencies that nonlinearly interact with the photosynthesis process and differentially stimulate the various time scales associated with photoacclimation and energy dissipation. This study highlights the need for experiments with more realistic light stimuli to better understand microalgal growth at high cell densities. An experimental protocol is also proposed, with simple, yet more realistic, step functions for light fluctuations.

Continuous selection pressure to improve temperature acclimation of Tisochrysis lutea.

Temperature plays a key role in outdoor industrial cultivation of microalgae. Improving the thermal tolerance of microalgae to both daily and seasonal temperature fluctuations can thus contribute to increase their annual productivity. A long term selection experiment was carried out to increase the thermal niche (temperature range for which the growth is possible) of a neutral lipid overproducing strain of Tisochrysis lutea. The experimental protocol consisted to submit cells to daily variations of temperature for 7 months. The stress intensity, defined as the amplitude of daily temperature variations, was progressively increased along successive selection cycles. Only the amplitude of the temperature variations were increased, the daily average temperature was kept constant along the experiment. This protocol resulted in a thermal niche increase by 3°C (+16.5%), with an enhancement by 9% of the maximal growth rate. The selection process also affected T. lutea physiology, with a feature generally observed for 'cold-temperature' type of adaptation. The amount of total and neutral lipids was significantly increased, and eventually productivity was increased by 34%. This seven month selection experiment, carried out in a highly dynamic environment, challenges some of the hypotheses classically advanced to explain the temperature response of microalgae.

Coupling and uncoupling of triglyceride and beta-carotene production by Dunaliella salina under nitrogen limitation and starvation.

BACKGROUND: Nitrogen starvation and limitation are known to induce important physiological changes especially in lipid metabolism of microalgae (triglycerides, membrane lipids, beta-carotene, etc.). Although little information is available for Dunaliella salina, it is a promising microalga for biofuel production and biotechnological applications due to its ability to accumulate lipid together with beta-carotene. RESULTS: Batch and chemostat experiments with various degrees of nitrogen limitation, ranging from starvation to nitrogen-replete conditions, were carried out to study carbon storage dynamics (total carbon, lipids, and beta-carotene) in steady state cultures of D. salina. A new protocol was developed in order to manage the very high beta-carotene concentrations and to more accurately separate and quantify beta-carotene and triglycerides by chromatography. Biomass evolution was appropriately described by the Droop model on the basis of the nitrogen quota dynamics. CONCLUSIONS: Triglycerides and beta-carotene were both strongly anti-correlated with nitrogen quota highlighting their carbon sink function in nitrogen depletion conditions. Moreover, these two valuable molecules were correlated each other for nitrogen replete conditions or moderated nitrogen limitations (N:C ratio higher than 0.04). Under nitrogen starvation, i.e., for very low N:C ratio, the dynamic revealed, for the first time, uncoupled part (higher triglyceride accumulation than beta-carotene), possibly because of shortage in key proteins involved in the stabilization of lipid droplets. This study motivates the accurate control of the microalgal nitrogen quota in order to optimize lipid productivity.

Long-term adaptive response to high-frequency light signals in the unicellular photosynthetic eukaryote Dunaliella salina.

Productivity of microalgal cultivation processes is tightly related to photosynthetic efficiency, and therefore to light availability at the cell scale. In an agitated, highly turbid suspension,the light signal received by a single phytoplankton cell moving in a dense culture is a succession of flashes. The growth characteristics of microalgae under such dynamic light conditions are thus fundamental information to understand nonlinear properties of the photosynthetic process and to improve cultivation process design and operation. Studies of the long term consequences of dynamic illumination regime on photosynthesis require a very specific experimental set-up where fast varying signals are applied on the long term. In order to investigate the growth response of the unicellular photosynthetic eukaryote Dunaliella salina (Chlorophyceae) to intermittent light exposure, different light regimes using LEDs with the same average total light dose were applied in continuous cultures. Flashing light with different durations of light flashes (△t of 30 s, 15 s, 2 s and 0.1 s) followed by dark periods of variable length (0.67 ≤ L:D ≤ 2) yielding flash frequencies in the range 0.017-5 Hz, were compared to continuous illumination. Specific growth rate, photosynthetic pigments, lipid productivity and elemental composition were measured on two duplicates for each irradiance condition. The different treatments of intermittent light led to specific growth rates ranging from 0.25 to 0.93 day(-1) . While photosynthetic efficiency was enhanced with increased flash frequency, no significant differences were observed in the particular carbon and chlorophyll content. Pigment analysis showed that within this range of flash frequency, cells progressively photoacclimated to the average light intensity.

Effect of gaseous cement industry effluents on four species of microalgae.

Experiments were performed at lab scale in order to test the possibility to grow microalgae with CO2 from gaseous effluent of cement industry. Four microalgal species (Dunaliella tertiolecta, Chlorella vulgaris, Thalassiosira weissflogii, and Isochrysis galbana), representing four different phyla were grown with CO2 enriched air or with a mixture of gasses mimicking the composition of a typical cement flue gas (CFG). In a second stage, the culture submitted to the CFG received an increasing concentration of dust characteristic of cement industry. Results show that growth for the four species is not affected by the CFG. Dust added at realistic concentrations do not have any impact on growth. For dust concentrations in two ranges of magnitude higher, microalgae growth was inhibited.

Photoperiod length paces the temporal orchestration of cell cycle and carbon-nitrogen metabolism in Crocosphaera watsonii.

We analysed the effect of photoperiod length (PPL) (16:8 and 8:16 h of light-dark regime, named long and short PPL, respectively) on the temporal orchestration of the two antagonistic, carbon and nitrogen acquisitions in the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii strain WH8501 growing diazotrophically. Carbon and nitrogen metabolism were monitored at high frequency, and their patterns were compared with the cell cycle progression. The oxygen-sensitive N2 fixation process occurred mainly during the dark period, where photosynthesis cannot take place, inducing a light-dark cycle of cellular C : N ratio. Examination of circadian patterns in the cell cycle revealed that cell division occurred during the midlight period, (8 h and 4 h into the light in the long and short PPL conditions, respectively), thus timely separated from the energy-intensive diazotrophic process. Results consistently show a nearly 5 h time lag between the end of cell division and the onset of N2 fixation. Shorter PPLs affected DNA compaction of C. watsonii cells and also led to a decrease in the cell division rate. Therefore, PPL paces the growth of C. watsonii: a long PPL enhances cell division while a short PPL favours somatic growth (biomass production) with higher carbon and nitrogen cell contents.

Cell cycle implication on nitrogen acquisition and synchronization in Thalassiosira weissflogii (Bacillariophyceae).

The Michaelis-Menten model of nitrogen (N) acquisition, originally used to represent the effect of nutrient concentration on the phytoplankton uptake rate, is inadequate when other factors show temporal variations. Literature generally links diurnal oscillations of N acquisition to a response of the physiological status of microalgae to photon flux density (PFD) and substrate availability. This work describes how the cell cycle also constitutes a significant determinant of N acquisition and, when appropriate, assesses the impact of this property at the macroscopic level. For this purpose, we carried out continuous culture experiments with the diatom Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle exposed to various conditions of light and N supply. The results revealed that a decrease in N acquisition occurred when a significant proportion of the population was in mitosis. This observation suggests that N acquisition is incompatible with mitosis and therefore that its acquisition rate is not constant during the cell cycle. In addition, environmental conditions, such as light and nutrient supply disrupt the cell cycle at the level of the individual cell, which impacts synchrony of the population.

Light-dark (12:12) cycle of carbon and nitrogen metabolism in Crocosphaera watsonii WH8501: relation to the cell cycle.

This study provides with original data sets on the physiology of the unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501, maintained in continuous culture in conditions of obligate diazotrophy. Cultures were exposed to a 12:12 light-dark regime, representative of what they experience in nature and where growth is expected to be balanced. Nitrogen and carbon metabolism were monitored at high frequency and their dynamics was compared with the cell cycle. Results reveal a daily cycle in the physiological and biochemical parameters, tightly constrained by the timely decoupled processes of N(2) fixation and carbon acquisition. The cell division rate increased concomitantly to carbon accumulation and peaked 6 h into the light. The carbon content reached a maximum at the end of the light phase. N(2) fixation occurred mostly during the dark period and peaked between 9 and 10 h into the night, while DNA synthesis, reflected by DNA fluorescence, increased until the end of the night. Consequently, cells in G1- and S-phases present a marked decrease in their C:N ratio. Nitrogen acquisition through N(2) fixation exceeded 1.3- to 3-fold the nitrogen requirements for growth, suggesting that important amounts of nitrogen are excreted even under conditions supposed to favour balanced, carbon and nitrogen acquisitions.

DIEL VARIATIONS OF CARBOHYDRATES AND NEUTRAL LIPIDS IN NITROGEN-SUFFICIENT AND NITROGEN-STARVED CYCLOSTAT CULTURES OF ISOCHRYSIS SP.(1).

The goal of this study was to investigate the time response of two major carbon (C) reserves, respectively neutral lipids (NL) and total carbohydrate (TC), in the Haptophyte Isochrysis sp. growing in nitrogen (N)-sufficient or N-starved conditions and under light:dark (L:D) cycles. Experiments were carried out in a cyclostat culture system that allowed the following of the dynamics of the main cell compounds at both hourly and daily time scales. Under N-sufficient conditions, the L:D cycles cause the population to be synchronized, with most of the cells dividing at the beginning of the dark period. The C-specific growth rate was maximal around midday and negative during the dark period due to respiration processes. NL and TC both accumulated during the day and consumed during the night. We showed that NL and TC are highly dynamic compounds, as more than three quarters of NL and TC accumulated during the light period were consumed during the dark period. In contrast to NL, phospholipid and glycolipid to C ratios remained quite stable during the light/dark cycles. The major effect of N starvation on the NL and TC dynamics was to uncouple their diel variations from the L:D cycle, in two different ways depending on their respective role during short-term acclimation. Whereas the TC per cell ratio increased rapidly to reach a stable value in response to N starvation, NL per cell continued to oscillate, but with a pattern out of phase with the L:D cycle.

NEUTRAL LIPID AND CARBOHYDRATE PRODUCTIVITIES AS A RESPONSE TO NITROGEN STATUS IN ISOCHRYSIS SP. (T-ISO; HAPTOPHYCEAE): STARVATION VERSUS LIMITATION(1).

Partitioning of the carbon (C) fixed during photosynthesis between neutral lipids (NL) and carbohydrates was investigated in Isochrysis sp. (Haptophyceae) in relation to its nitrogen (N) status. Using batch and nitrate-limited continuous cultures, we studied the response of these energy reserve pools to both conditions of N starvation and limitation. During N starvation, NL and carbohydrate quotas increased but their specific growth rates (specific rates of variation, µCAR and µNL ) decreased. When cells were successively deprived and then resupplied with NO3 , both carbohydrates and neutral lipids were inversely related to the N quota (N:C). These negative relationships were not identical during N impoverishment and replenishment, indicating a hysteresis phenomenon between N and C reserve mobilizations. Cells acclimated to increasing degrees of N limitation in steady-state chemostat cultures showed decreasing NL quota and increasing carbohydrate quota. N starvation led to a visible but only transient increase of NL productivity. In continuous cultures, the highest NL productivity was obtained for the highest experimented dilution rate (D = 1.0 d(-1) ; i.e., for non N-limited growth conditions), whereas the highest carbohydrate productivity was obtained at D = 0.67 d(-1) . We used these results to discuss the nitrogen conditions that optimize NL productivities in the context of biofuel production.