Distribution of intracellular nitrogen in marine microalgae: Calculation of new nitrogen-to-protein conversion factors

Abstract

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.

Biochemical characterization of microalgal biomass from freshwater species isolated in Alberta, Canada for animal feed applications

“…This N-to-P factor overlooks the assumption that the protein source contains 16% N and does not adequately take into account the often high content of non-protein nitrogen (NPN) found in microalgae. The NPN content of microalgae has been reported to be 4 to 40% depending upon species, season and growth phase [1,27,58].Assuch,some of the most comprehensive and commonly cited summaries of protein contents of microalgae and other single-celled proteins (both for research and for product labeling) are likely over-estimates. Ultimately, the most accurate method to estimate protein content is by comprehensive analysis of amino acid profile; however, this may be cost-prohibitive and beyond the requirement for some research and industrial applications.…”

“…The two major methods predominantly reported in the literature are spectrophotometric analysis (e.g., Lowry and Bradford) and elemental nitrogen (N) analysis. The method used in this study involving elemental analysis of total N and the use of an 'appropriate' N-to-P conversion factor (e.g., N × 4.78) [27] is the most suitable method and we encourage others to adopt this approach in an effort to standardize protein content estimations of algal biomass. This approach is fully destructive so prior protein extraction steps (e.g., cell wall disruption) are not required and it also eliminates interferences caused by carotenoids and chlorophyll that can make spectrophotometric methods difficult to reproduce.…”

“…Amino acid (AA) concentrations are expressed as mg AA g DW − 1 and g AA 100 g protein − 1 and protein quality was evaluated on the basis of EAA indices [26]. Protein content was calculated using a nitrogen-to-protein conversion factor of N × 4.78 [27] and also by calculating species-specific nitrogen-toprotein conversion factors (N × k) for each species based on their individual amino acid contributions [28]. Fatty acids were extracted by methanolic HCl in-situ transesterification [29] and the corresponding fatty acid methyl esters (FAMEs) were separated and quantified by gas chromatography with flame ionization detection (Omegawax 250 column, Agilent 7890 GC-FID).…”

Snapshot prediction of carbon productivity, carbon and protein content in a Southern Ocean diatom using FTIR spectroscopy

“…Mass spectrometry was conducted by the Australian Rivers Institute (Griffith University, Nathan, QLD, Australia) using an Isoprime Mass Spectrometer (GV Instruments, Manchester, UK) instrument with an EA 3000 inlet (Eurovector, Milan Italy). Protein content was calculated from particulate organic nitrogen using the protein-to-nitrogen conversion factor of 4.78 (±0.62), which has been determined from 12 species of phytoplankton, including 3 species of diatom (Lourenço et al, 2004). This approach is currently the most practical way of determining protein concentration, as it avoids uncertainties associated with extracting proteins from biomass, which can introduce substantial errors (Lourenço et al, 2004).…”

Application of Algae as Cosubstrate To Enhance the Processability of Willow Wood for Continuous Hydrothermal Liquefaction

“…The oxygen content was determined by difference. The content of protein in algal biomass was derived from the elemental analysis using the nitrogen-protein conversion factor (N-factor) 4.58 30 . The N-factor was determined as an average of 10 marine microalgae species with a standard deviation of 0.11.…”