Amino Acids Are an Ineffective Fertilizer for Dunaliella spp. Growth

Autotrophic microalgae are a promising bioproducts platform. However, the fundamental requirements these organisms have for nitrogen fertilizer severely limit the impact and scale of their cultivation. As an alternative to inorganic fertilizers, we investigated the possibility of using amino acids from deconstructed biomass as a nitrogen source in the genus Dunaliella. We found that only four amino acids (glutamine, histidine, cysteine, and tryptophan) rescue Dunaliella spp. growth in nitrogen depleted media, and that supplementation of these amino acids altered the metabolic profile of Dunaliella cells. Our investigations revealed that histidine is transported across the cell membrane, and that glutamine and cysteine are not transported. Rather, glutamine, cysteine, and tryptophan are degraded in solution by a set of oxidative chemical reactions, releasing ammonium that in turn supports growth. Utilization of biomass-derived amino acids is therefore not a suitable option unless additional amino acid nitrogen uptake is enabled through genetic modifications of these algae

TAG content increases under LL (A) and saturated FAs accumulate at 35°C in TAG (B).

<p><i>D</i>. <i>viridis</i> cultures were grown either under LD (dashed lines) or LL (solid lines). All cultures were grown at 25°C for the first 24 hours and then either remained at 25°C (blue lines) or the temperature in the growth chamber was raised to 35°C (red lines). The temperature shift is indicated by the black arrow. Total TAG content was calculated as the sum of the fatty acids from the TAG fraction. Total TAG content was normalized to 1 million cells. The error bars represent the standard deviation from three independent biological replicates. Statistical significance was assessed by unpaired, two-tailed, Student’s <i>t</i>-test. The values with the same letters are not significantly different at 54 hrs (p<0.05).</p

Effect of temperature on the transcript abundance of the TAG pathway genes.

<p>Relative transcript abundance changes for the transcripts of the lipid biosynthesis pathway are provided in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127562#pone.0127562.s020" target="_blank">S7A Table</a>). Compounds include: G-3-P, glycerol-3-phosphate; Lyso-PA, lyso-phosphatidic acid; PA, phosphatidic acid; PG, phosphatidylglycerol, SQDG, sulfoquinovosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol, DGDG, digalactosyldiacylglycel, DAG: diacylglycerol, TAG, triacylglycerol. Enzymes include: GPAT, glycerol-3-P acyltransferase; LPAT, lysophosphatidic acid acyltransferase; PAH, phosphatidate phosphatase; DGAT and DGTT, diacylglycerol acyltransferase; PCT, CDP-diacylglycerol synthase; PGP, phosphatidylglycerolphosphate synthase; SQD1, UDP-sulfoquinovose synthase; SGD2, sulfolipid synthase; MGD1, monogalactosyldiacylglycerol synthase; DGD1, digalactosyldiacyglycerol synthase; PGD1, plastid galactoglycerolipid degradation1; GGGT, galactolipid:galactolipid galactosyltransferase; PDAT1, phospholipid:diacylglycerol acyltransferase; MLDP, major lipid droplet protein; Kcs, β-ketoacyl-CoA synthase; CHAD, 3-hydroxyacyl-CoA dehydrogenase; TER, Trans-2-enoyl-CoA reductase; PCH, Palmitoyl-CoA hydrolase; FAD5, MGDG specific palmitate Δ-7 desaturase; FAD6, ω-6 fatty acid desaturase; FAD7, ω -3 fatty acid desaturase; LCIII, class 3 lipase, FAP, class 3 lipase, LIP, lipase; TAGL, triacylglycerol lipase.</p

Numbers of transcripts responding to changes in light duration and/or temperature at 30, 40 and 54 hours.

<p>The diagrams represent the number of transcripts differentially expressed either only in response to continuous light compared to light:dark cycles at 25°C (25°C-LL/LD) or only in response to increased temperature under continuous light (LL-35°C/25°C).</p

Changes in fatty acid composition of the TAG fraction.

<p>The error bars represent the standard deviation from three independent biological replicates. Statistical significance was assessed by unpaired, two-tailed, Student’s <i>t</i>-test. The values with the same letters are not significantly different at 54 hrs (p<0.05).</p

Amino Acids Are an Ineffective Fertilizer for Dunaliella spp. Growth

Autotrophic microalgae are a promising bioproducts platform. However, the fundamental requirements these organisms have for nitrogen fertilizer severely limit the impact and scale of their cultivation. As an alternative to inorganic fertilizers, we investigated the possibility of using amino acids from deconstructed biomass as a nitrogen source in the genus Dunaliella. We found that only four amino acids (glutamine, histidine, cysteine, and tryptophan) rescue Dunaliella spp. growth in nitrogen depleted media, and that supplementation of these amino acids altered the metabolic profile of Dunaliella cells. Our investigations revealed that histidine is transported across the cell membrane, and that glutamine and cysteine are not transported. Rather, glutamine, cysteine, and tryptophan are degraded in solution by a set of oxidative chemical reactions, releasing ammonium that in turn supports growth. Utilization of biomass-derived amino acids is therefore not a suitable option unless additional amino acid nitrogen uptake is enabled through genetic modifications of these algae

Total chlorophyll (A) and soluble protein (B) content in <i>D</i>. <i>viridis</i> in response to light duration and temperature.

<p>While chlorophyll content increases in response to elevated temperature, soluble protein content in those cells decreases. <i>D</i>. <i>viridis</i> cultures were grown either under LD (dashed lines) or LL (solid lines). All cultures were grown at 25°C for the first 24 hours and then either remained at 25°C (blue lines) or the temperature in the growth chamber was raised to 35°C (red lines). Temperature shift is indicated by the black arrow. Total chlorophyll content, soluble protein and cell counts were measured in three biological replicates with three technical replicates each. The data were normalized to 1 million cells. The error bars represent the standard deviation. Statistical significance was assessed by unpaired, two-tailed, Student’s <i>t</i>-test. The values with the same letters are not significantly different at 54 hrs (p<0.05).</p

Major polar lipid classes responds to photoperiod and temperature changes.

<p>The cellular content of the major polar lipid classes DGDG (grey bars), MGDG (black bars) and PL (striped bars) responded to changes in temperature and light duration. The error bars represent the standard deviation from three independent biological replicates. Statistical significance was assessed by unpaired, two-tailed, Student’s <i>t</i>-test. The values with the same letters are not significantly different at 54 hrs (p<0.05).</p

Cell cycle regulated genes show diurnal regulation under LD, but constant transcript levels in LL.

<p>Lines represent the changes in transcript abundance (RPKM) for each of the genes. Genes are: H2A, histone H2A; CYCA, A-type cyclin; CYCB1, B-type cyclin 1; CYCB2, B-type cyclin 2; cyclin dependent kinase B.</p