Nitrogen budgets in microalgae are strongly affected by growth conditions and physiological state of the cultures. As a consequence, protein N (PN) to total N (TN) ratio may be variable in microalgae grown in batch cultures, and this may limit the usefulness of the nitrogen-to-protein conversion factors (N-Prot factors), the most practical way of determining protein content. The accuracy of protein determination by this method depends on the establishment of specific N-Prot factors, and experimental data are needed to fill this gap. Complementing a previous study, the present work was designed to quantify the fluctuations of the main nitrogenous compounds during the growth of 12 species of marine microalgae, as well as to determine N-Prot factors for them. The microalgae were cultured in two experimental conditions: (a) using a N-replete culture medium (initial N concentration, 1.18 mM) and aeration, and (b) with a N-depleted culture medium (initial N concentration, 235 mM) and no aeration. The distribution of intracellular nitrogen was studied by constructing budgets of different nitrogen pools in different growth phases of the cultures. In all species, large variations occurred in the distribution of PN and non-protein N (NPN) in the treatments tested and in different growth phases. Intracellular inorganic nitrogen (NO 3 7 , NO 2 7 and NH 3 + NH 4 +) was the most important NPN component (0.4-30.4% of TN) in all species, followed by nucleic acids (0.3-12.2% of TN), and chlorophylls (0.1-1.8% of TN). The relative importance of NPN was greater in the exponential phase, decreasing during growth. PN ranged from 59.3 to 96.8% of TN. N-Prot factors are proposed for each of the species studied, based on the ratio of amino acid residues to TN, with values ranging from 2.53 to 5.77. Based on current results and on the previous study, we establish an overall average N-Prot factor for all species, treatments and growth phases of 4.78 + 0.62 (n = 354). This study confirms that the use of the traditional factor 6.25 is unsuitable for marine microalgae, and the use of the N-Prot factors proposed here is recommended.

The utilization of nitrogen‐to‐protein conversion factors (N‐Prot factors) is a widely accepted and practical way to determine total protein content. The accuracy of protein determination depends on the establishment of specific N‐Prot factors, since the conventional factor of 6.25 may be unsuitable for all species. This study was designed to determine the concentrations of the main nitrogenous compounds and to establish N‐Prot factors specific for the following marine microalgae: Chlorella minutissima, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana, Nannochloropsis oculata, Phaeodactylum tricornutum, Prorocentrum minimum, Skeletonema costatum, Synechococcus subsalsus, and Tetraselmis gracilis. Cultures were maintained under a 12‐h photoperiod (300 μmol photons·m−2·s−1) at temperatures of 20.0°± 1.0° C (dark) to 23.0°± 2.0° C (light) in Walne’s culture medium without additional external carbon sources. The distribution of intracellular nitrogen was studied by determining total nitrogen (TN, by CHN [carbon, hydrogen, and nitrogen] analysis), protein N (PN, by analysis of total amino acids), and nonprotein N (NPN, determined by analysis of DNA, RNA, chlorophylls (chl) a,b, and c, and intracellular inorganic nitrogen—NO3−, NO2−, and NH3+ NH4+) in logarithmic and stationary growth phases of cultures. Variations occurred in both accumulation and distribution of PN and NPN among the species, as well as in each species during the different growth phases. Inorganic nitrogen compounds were observed to be the most important NPN source (from 6.4 ± 0.1% to 41.8 ± 4.2% of total N) in all species (except D. tertiolecta), followed by nucleic acids (from 0.8 ± 0.1% to 26.1 ± 2.4% of TN) and chlorophylls (from 0.2 ± 0.0% to 3.1 ± 0.3% of TN). Total amino acid residues ranged from 63.1 ± 4.6% up to 88.1 ± 11.2% of TN, which is in agreement with the presence of high NPN concentrations. N‐Prot factors are proposed for each growth phase in the studied species, based on the ratio of amino acid residues to TN, establishing specific N‐prot factors ranging from 3.60 ± 0.27 to 4.99 ± 0.64. The mean N‐Prot factor for all species/growth phases was 4.58 ± 0.11. The present study shows that the use of the traditional factor 6.25 is not suitable for these marine microalgae, and possibly for other species, because it overestimates their actual protein content.

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Marine planktonic algae are frequently exposed to metallic contaminants. Because heavy metals can be assimilated and accumulated by algal cells, they can then be transferred to higher trophic levels of food chains. We studied the effects of cadmium on protein production and the growth of the marine prasinophyte Tetraselmis gracilis (Kylin) Butcher. By means of toxicological assays, we estimated the LC50 of cadmium as 3.2 ppm and 1.8 ppm after 48 h and 96 h of exposure to this heavy metal, respectively. The growth curves and survival percentages of cell cultures in the presence of cadmium were determined, and a proportional reduction of both parameters with increasing metal concentrations was found. When chronically exposed to sublethal concentrations of cadmium, T. gracilis contained high levels of superoxide dismutase (SOD) activity, one of the main enzymes of the cell's antioxidant defense mechanism. Under these growth conditions, total SOD activity in crude extracts was increased by 41% (at 1.5 ppm) and 107% (at 3.0 ppm). Assays of SOD activity in nondenaturing polyacrylamide gels also showed a similar induction by cadmium. These results show that cadmium has potentially toxic properties since it significantly inhibited the growth of T. gracilis at low concentrations and promoted the induction of SOD activity, suggestive of an oxidative stress state. Besides being the first report of SOD in T. gracilis, this work describes experimental evidence of SOD induction by cadmium in this species.

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