DISTRIBUTION OF INTRACELLULAR NITROGEN IN MARINE MICROALGAE: BASIS FOR THE CALCULATION OF SPECIFIC NITROGEN‐TO‐PROTEIN CONVERSION FACTORS

Abstract

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.