Antoine Sciandra

An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures

This method of nitrate determination by ultraviolet absorption spectrometry is based on the measurement of sample absorbance at a single wavelength (220 nm), which was chosen on the basis of the absorption spectra of the main components of artificial seawater in the ultraviolet domain. No reagents are used and no sophisticated instruments are necessary. For standards prepared in artificial seawater, the relationship between absorbance and nitrate concentration is linear up to 500 μmol N L−1 and the detection limit is 1 μmol N L−1. Precision is 1.5%. Urea and amino acids did not interfere at concentrations typical of seawater. The method also measures nitrite, but this interference only becomes important for species which excrete large amounts of nitrite. The method is extremely rapid, simple to implement and does not require the use of toxic chemicals such as cadmium. It should prove useful for monitoring quickly the nitrate concentrations in laboratory cultures of marine phytoplankton.

Modelling neutral lipid production by the microalga Isochrysis aff. galbana under nitrogen limitation

This article proposes a dynamical model of microalgal lipid production under nitrogen limitation. In this model, intracellular carbon is divided between a functional pool and two storage pools (sugars and neutral lipids). The various intracellular carbon flows between these pools lead to a complex dynamic with a strong discrepancy between synthesis and mobilization of neutral lipids. The model has been validated with experiments of Isochrysis aff. galbana (clone T-iso) culture under various nitrogen limitation conditions and under nitrogen starvation. The hysteresis behavior of the neutral lipid quota observed experimentally is accurately predicted.

The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae

Microalgae are currently emerging as one of the most promising alternative sources for the next generation of food, feed, cosmetics and renewable energy in the form of biofuel. Microalgae constitute a diverse group of microorganisms with advantages like fast and efficient growth. In addition, they do not compete for arable land and offer very high lipid yield potential. Major challenges for the development of this resource are to select lipid-rich strains using high-throughput staining for neutral lipid content in microalgae species. For this purpose, the fluorescent dyes most commonly used to quantify lipids are Nile red and BODIPY 505/515. Their fluorescent staining for lipids offers a rapid and inexpensive analysis tool to measure neutral lipid content, avoiding time-consuming and costly gravimetric analysis. This review collates and presents recent advances in algal lipid staining and focuses on Nile red and BODIPY 505/515 staining characteristics. The available literature addresses the limitations of fluorescent dyes under certain conditions, such as spectral properties, dye concentrations, cell concentrations, temperature and incubation duration. Moreover, the overall conclusion of the present review study gives limitations on the use of fluorochrome for screening of lipid-rich microalgae species and suggests improved protocols for staining recalcitrant microalgae and recommendations for the staining quantification.

Nonlinear observers for a class of biological systems: application to validation of a phytoplanktonic growth model

The authors construct nonlinear observers in order to discuss the validity of biological models. They consider a class of systems including many classical models used in biological modeling. They formulate the nonlinear observers corresponding to these systems and prove the conditions necessary for their exponential convergence. They apply these observers on the well-known Droop model which describes the growth of a population of phytoplanktonic cells. The validity of this model is discussed based on the performance of the observers working on experimental data.

Modeling continuous cultures of microalgae colimited by nitrogen and phosphorus

It is well documented that the combination of low nitrogen and phosphorus resources can lead to situations where colimitation of phytoplankton growth arises, yet the underlying mechanisms are not fully understood. Here, we propose a Droop-based model built on the idea that colimitation by nitrogen and phosphorus arises from the uptake of nitrogen. Indeed, since N-porters are active systems, they require energy that could be related to the phosphorus status of the cell. Therefore, we assumed that N uptake is enhanced by the P quota. Our model also accounts for the biological observations that uptake of a nutrient can be down-regulated by its own internal quota, and succeeds in describing the strong contrast for the non-limiting quotas under N-limited and P-limited conditions that was observed on continuous cultures with Selenastrum minutum and with Isochrysis affinis galbana. Our analysis suggests that, regarding the colimitation concept, N and P would be better considered as biochemically dependent rather than biochemically independent nutrients.

The effects of a controlled fluctuating nutrient environment on continuous cultures of phytoplankton monitored by computers

Abstract A completely automatic continuous phytoplankton culturing device is described. This computer-controlled system monitors simultaneously and automatically the basic culture parameters of several chemostats, i.e., limiting nutrient concentrations, cell size spectra, cell densities and temperatures. Culture pH can also be regulated. The dilution rate and the limiting nutrient concentration in the enrichment medium may be programmed to follow different dynamic patterns with time. An example is given with a sinusoidal variation of the dilution rate. Applications are discussed, which concern the advantages to modelling gained by acquiring information from continuously fluctuating systems rather than from nutrient-pulsed systems.

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.

The steady states of continuous cultures with low rates of medium renewal per cell

Abstract Continuous cultures of the chlorophyte Dunalietta tertiolecta R. W. Butcher were stabilized for different sets of dilution rates d and nitrate concentrations so in the renewal milieu. Cell concentrations at equilibrium were compared to the theoretical concentrations obtained using differential equations postulated in Droops model. The experimental data were in agreement with the theoretical when the rate of nutrient renewal per cell exceeded a certain limit R. Coupling different values of d and so where the limit R is not attained —for example, low values of d with high value of so —algal density at equilibrium is inferior to the theoretical density. Algal mass, calculated from cell volume, is also inferior to theoretical mass, although to a lesser extent. It is suggested that when the rate of renewal R (which is probably species specific) is insufficient, the population of cells is inhibited by the emergence of unfavourable factors (e.g. insufficient elimination of toxic excretion, predominant bacterial activity). In these extreme experimental conditions, as the population is not uniquely limited by the limiting factor, the hypotheses supported by the Droops model and the continuous culture theory become non-verifiable.