Evaluation of Cellular Responses by Chlamydomonas reinhardtii in Media Containing Dairy-Processing Residues Derived from Cheese as Nutrients by Analyzing Cell Growth Activity and Comprehensive Gene Transcription Levels

Abstract

Utilities of whey powder (WP) and whey protein concentrate 34% powder (WPC34) prepared as dairy-processing residues were evaluated using a green alga Chlamydomonas reinhardtii. Analysis of C. reinhardtii growth showed that the strain used WP and WPC34 as nitrogen sources. Its specific growth rate and maximum cell density in WP-containing medium were higher than those in WPC34-containing medium; growth with WPC34 was improved by adding KCl or K₂HPO₄, which content was decreased as a result of WPC34's preparation from WP. Although the lipid contents in media containing dairy-processing residues were 2.72 ± 0.31 wt% and 2.62 ± 0.20 wt% with no significant difference, the composition ratio of fatty acid C14 with WPC34 was higher than that with WP and the composition ratio of the sum of fatty acid-C16 and -C18 with WPC34 tended to be lower than that with WP. Additionally, analyses of gene transcription showed that the transcription level of acetyl-CoA carboxylase biotin carboxyl carrier protein in WPC34-containing medium was lower than that in WP-containing medium, possibly affecting the ratios of the chain lengths of fatty acids. The transcription of genes involved in glycolysis and the TCA cycle was outstandingly lower in algae grown in WPC34-containing medium when compared to those cultivated in the presence of WP, resulting in differences in energy production for cell proliferation.

Keywords: Chlamydomonas reinhardtii; biomass; cellular response; mineral; potassium; transcriptomics; whey; whey retentate.

Figure 1

Preparatory processes of dairy residues and compositions of WPC34 and WP. WPC34 and WP as dairy-processing residue in this study. (a) Flow of preparatory process of WPC34 and WP from raw milk [32,33,34,35]; (b) Composition ratios of carbohydrates, proteins, and lipids as main components and minerals and residue as ash in WPC34 and WP.

Figure 2

Analyses of spectra of photo-absorbances derived from WPC34 and WP in medium. Photo-absorbance spectra of media containing (a) 1 g∙L⁻¹, 3 g∙L⁻¹, 6 g∙L⁻¹, and 10 g∙L⁻¹ of WPC34 and (b) 5 g∙L⁻¹, 10 g∙L⁻¹, 20 g∙L⁻¹, and 30 g∙L⁻¹ of WP were analyzed in the 400–700 nm range. Box plots were drawn with absorbance data of media containing (c) 1 g∙L⁻¹, 3 g∙L⁻¹, 6 g∙L⁻¹, and 10 g∙L⁻¹ of WPC34 and (d) 5 g∙L⁻¹, 10 g∙L⁻¹, 20 g∙L⁻¹, and 30 g∙L⁻¹ of WP at each wavelength. The data used for the box plot analysis were three-time replicates for each concentration of dairy-processing residue (n = 3).

Figure 3

Analyses of time course profiling of OD₇₀₀ related to C. reinhardtii growth in broth containing WPC34 and WP as nutrients. Assimilation activities of C. reinhardtii in media containing WPC34 and WP were analyzed with data obtained by 3–8 time-replicated experiments. Related to assimilating WPC34, (a) time course profiling of OD₇₀₀, (b) maximum OD₇₀₀ corresponding to WPC34 concentration, and (c) doubling time corresponding to WPC34 concentration are shown with closed symbols of diamond (1 g∙L⁻¹), square (3 g∙L⁻¹), triangle (6 g∙L⁻¹), and circle (10 g∙L⁻¹). Related to assimilating WP, (d) time course profiling of OD₇₀₀, (e) maximum OD₇₀₀ corresponding to WP concentration, and (f) doubling time corresponding to WP concentration are shown with open symbols of diamond (5 g∙L⁻¹), square (10 g∙L⁻¹), triangle (20 g∙L⁻¹), and circle (30 g∙L⁻¹), respectively. The data used for analysis were 3–9 time-replicates in each concentration of dairy-processing residue (n = 3–9), and error bars indicate the SD of 3–9 time-replicated experiments.

Figure 4

Analysis of time course profiling of OD₇₀₀ related to C. reinhardtii growth in WPC34 broth containing minerals as additional nutrients. C. reinhardtii grown in 6 g∙L⁻¹ WPC34-containing media with KCl (5.1 mM), K₂HPO₄ (2.6 mM), Na₂HPO₄ (2.2 mM), and KH₂PO₄ (2.6 mM) as additional minerals. (a) Time course profiling of OD₇₀₀ is drawn with symbols of gray circle (KCl), gray square (K₂HPO₄), gray diamond (Na₂HPO₄), gray star (KH₂PO₄), and closed triangle (nothing). (b) Doubling times of OD₇₀₀ in 6 g∙L⁻¹ WPC34-containing media with KCl, K₂HPO₄, Na₂HPO₄, and KH₂PO₄ in 0–96 h are displayed from left to right, respectively. The data were obtained by 3–6 time-replicate experiments, and the error bars indicate the SD of 3–6 repeats.

Figure 5

Lipid contents and compositions in C. reinhardtii cells cultivated in each broth. Lipids of C. reinhardtii cells cultivated in modified TAP adjusted to 6 g∙L⁻¹ WPC34 and 20 g∙L⁻¹ WP for 72 h were measured in terms of contents (a) and composition (b). The data obtained by 6–8 time-replicate experiments display error bars indicating the SD, and they were evaluated with significance tests (*: p < 0.05; **: p < 0.01; ***: p < 0.001; N.S.: not significant).

Figure 6

(a–d) Comparison of transcription levels of the genes whose products participate in glycolysis, TCA cycle, fatty acid synthetic pathway, and fatty acid degradation pathway of C. reinhardtii cells in each broth. Data are shown as relative mRNA transcription levels normalized by the level of ATPS as a housekeeping gene. The transcription levels at growth phase are displayed with a black bar (6 g∙L⁻¹ WPC34), shaded bar (20 g∙L⁻¹ WP), and gray bar (0.4 g∙L⁻¹ NH₄Cl) in the figure. The explanation of relative transcription level was abbreviated as RTL. Error bars indicate SD of 3–8 time-replicate experiments. Same shapes (such as square, triangle etc…) in figure meant the same genes. Gene abbreviations are shown below: (a) in glycolysis. GPI: glucose-6-phosphate isomerase, FBP: fructose-1,6-bisphosphatase I, PFK①: 6-phosphofructokinase 1, PFK②: 6-phosphofructokinase 1, ALDO①: fructose-bisphosphate aldolase, class I, ALDO②: fructose-bisphosphate aldolase, class I, ALDO③: fructose-bisphosphate aldolase, class I, ALDO④: fructose-bisphosphate aldolase, class I, GAPDH: glyceraldehyde 3-phosphate dehydrogenase (phosphorylating), PGK: phosphoglycerate kinase, GPMI: 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, ENO: enolase. (b) in TCA cycle. PC: pyruvate carboxylase, CS①: citrate synthase, CS②: citrate synthase, ACLY①: ATP citrate (pro-S)-lyase, ACLY②: ATP citrate (pro-S)-lyase, ACO: aconitate hydratase, IDH1: isocitrate dehydrogenase, IDH3①: isocitrate dehydrogenase (NAD+), IDH3②: isocitrate dehydrogenase (NAD+), OGDH: 2-oxoglutarate dehydrogenase E1 component, DLD①: dihydrolipoyl dehydrogenase, DLD②: dihydrolipoyl dehydrogenase, DLST①: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase), DLST②: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase), LSC1: succinyl-CoA synthetase alpha subunit, SDH1: succinate dehydrogenase (ubiquinone) flavoprotein subunit, SDH2: succinate dehydrogenase (ubiquinone) iron-sulfur subunit, SDH3: succinate dehydrogenase (ubiquinone) cytochrome b₅₆₀ subunit, FUMA: fumarate hydratase, class I, FUMC: fumarate hydratase, class II, MDH1: malate dehydrogenase, MDH2①: malate dehydrogenase, MDH2②: malate dehydrogenase, MDH2③: malate dehydrogenase. (c) in fatty acid synthetic pathway. ACCA: acetyl-CoA carboxylase carboxyl transferase subunit alpha, ACCB: acetyl-CoA carboxylase biotin carboxyl carrier protein, ACCC: acetyl-CoA carboxylase, biotin carboxylase subunit, ACCD: acetyl-CoA carboxylase carboxyl transferase subunit beta, PLSC: 1-acyl-sn-glycerol-3-phosphate acyltransferase, DPP1: diacylglycerol diphosphate phosphatase/phosphatidate phosphatase, DGAT2: diacylglycerol O-acyltransferase 2. (d) in fatty acid degradation pathway. ACSL①: long-chain acyl-CoA synthetase, ACSL②: long-chain acyl-CoA synthetase, ACSL③: long-chain acyl-CoA synthetase, ACSL④: long-chain acyl-CoA synthetase, ACOX1①: acyl-CoA oxidase, ACOX1②: acyl-CoA oxidase, ACOX1③: acyl-CoA oxidase, ACOX1④: acyl-CoA oxidase, ACOX1⑤: acyl-CoA oxidase, ACADM: acyl-CoA dehydrogenase, MFP2: enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, ACAA1: acetyl-CoA acyltransferase 1, ACAT: acetyl-CoA C-acetyltransferase.

Figure 6

(a–d) Comparison of transcription levels of the genes whose products participate in glycolysis, TCA cycle, fatty acid synthetic pathway, and fatty acid degradation pathway of C. reinhardtii cells in each broth. Data are shown as relative mRNA transcription levels normalized by the level of ATPS as a housekeeping gene. The transcription levels at growth phase are displayed with a black bar (6 g∙L⁻¹ WPC34), shaded bar (20 g∙L⁻¹ WP), and gray bar (0.4 g∙L⁻¹ NH₄Cl) in the figure. The explanation of relative transcription level was abbreviated as RTL. Error bars indicate SD of 3–8 time-replicate experiments. Same shapes (such as square, triangle etc…) in figure meant the same genes. Gene abbreviations are shown below: (a) in glycolysis. GPI: glucose-6-phosphate isomerase, FBP: fructose-1,6-bisphosphatase I, PFK①: 6-phosphofructokinase 1, PFK②: 6-phosphofructokinase 1, ALDO①: fructose-bisphosphate aldolase, class I, ALDO②: fructose-bisphosphate aldolase, class I, ALDO③: fructose-bisphosphate aldolase, class I, ALDO④: fructose-bisphosphate aldolase, class I, GAPDH: glyceraldehyde 3-phosphate dehydrogenase (phosphorylating), PGK: phosphoglycerate kinase, GPMI: 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, ENO: enolase. (b) in TCA cycle. PC: pyruvate carboxylase, CS①: citrate synthase, CS②: citrate synthase, ACLY①: ATP citrate (pro-S)-lyase, ACLY②: ATP citrate (pro-S)-lyase, ACO: aconitate hydratase, IDH1: isocitrate dehydrogenase, IDH3①: isocitrate dehydrogenase (NAD+), IDH3②: isocitrate dehydrogenase (NAD+), OGDH: 2-oxoglutarate dehydrogenase E1 component, DLD①: dihydrolipoyl dehydrogenase, DLD②: dihydrolipoyl dehydrogenase, DLST①: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase), DLST②: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase), LSC1: succinyl-CoA synthetase alpha subunit, SDH1: succinate dehydrogenase (ubiquinone) flavoprotein subunit, SDH2: succinate dehydrogenase (ubiquinone) iron-sulfur subunit, SDH3: succinate dehydrogenase (ubiquinone) cytochrome b₅₆₀ subunit, FUMA: fumarate hydratase, class I, FUMC: fumarate hydratase, class II, MDH1: malate dehydrogenase, MDH2①: malate dehydrogenase, MDH2②: malate dehydrogenase, MDH2③: malate dehydrogenase. (c) in fatty acid synthetic pathway. ACCA: acetyl-CoA carboxylase carboxyl transferase subunit alpha, ACCB: acetyl-CoA carboxylase biotin carboxyl carrier protein, ACCC: acetyl-CoA carboxylase, biotin carboxylase subunit, ACCD: acetyl-CoA carboxylase carboxyl transferase subunit beta, PLSC: 1-acyl-sn-glycerol-3-phosphate acyltransferase, DPP1: diacylglycerol diphosphate phosphatase/phosphatidate phosphatase, DGAT2: diacylglycerol O-acyltransferase 2. (d) in fatty acid degradation pathway. ACSL①: long-chain acyl-CoA synthetase, ACSL②: long-chain acyl-CoA synthetase, ACSL③: long-chain acyl-CoA synthetase, ACSL④: long-chain acyl-CoA synthetase, ACOX1①: acyl-CoA oxidase, ACOX1②: acyl-CoA oxidase, ACOX1③: acyl-CoA oxidase, ACOX1④: acyl-CoA oxidase, ACOX1⑤: acyl-CoA oxidase, ACADM: acyl-CoA dehydrogenase, MFP2: enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, ACAA1: acetyl-CoA acyltransferase 1, ACAT: acetyl-CoA C-acetyltransferase.

Figure 6

(a–d) Comparison of transcription levels of the genes whose products participate in glycolysis, TCA cycle, fatty acid synthetic pathway, and fatty acid degradation pathway of C. reinhardtii cells in each broth. Data are shown as relative mRNA transcription levels normalized by the level of ATPS as a housekeeping gene. The transcription levels at growth phase are displayed with a black bar (6 g∙L⁻¹ WPC34), shaded bar (20 g∙L⁻¹ WP), and gray bar (0.4 g∙L⁻¹ NH₄Cl) in the figure. The explanation of relative transcription level was abbreviated as RTL. Error bars indicate SD of 3–8 time-replicate experiments. Same shapes (such as square, triangle etc…) in figure meant the same genes. Gene abbreviations are shown below: (a) in glycolysis. GPI: glucose-6-phosphate isomerase, FBP: fructose-1,6-bisphosphatase I, PFK①: 6-phosphofructokinase 1, PFK②: 6-phosphofructokinase 1, ALDO①: fructose-bisphosphate aldolase, class I, ALDO②: fructose-bisphosphate aldolase, class I, ALDO③: fructose-bisphosphate aldolase, class I, ALDO④: fructose-bisphosphate aldolase, class I, GAPDH: glyceraldehyde 3-phosphate dehydrogenase (phosphorylating), PGK: phosphoglycerate kinase, GPMI: 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, ENO: enolase. (b) in TCA cycle. PC: pyruvate carboxylase, CS①: citrate synthase, CS②: citrate synthase, ACLY①: ATP citrate (pro-S)-lyase, ACLY②: ATP citrate (pro-S)-lyase, ACO: aconitate hydratase, IDH1: isocitrate dehydrogenase, IDH3①: isocitrate dehydrogenase (NAD+), IDH3②: isocitrate dehydrogenase (NAD+), OGDH: 2-oxoglutarate dehydrogenase E1 component, DLD①: dihydrolipoyl dehydrogenase, DLD②: dihydrolipoyl dehydrogenase, DLST①: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase), DLST②: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase), LSC1: succinyl-CoA synthetase alpha subunit, SDH1: succinate dehydrogenase (ubiquinone) flavoprotein subunit, SDH2: succinate dehydrogenase (ubiquinone) iron-sulfur subunit, SDH3: succinate dehydrogenase (ubiquinone) cytochrome b₅₆₀ subunit, FUMA: fumarate hydratase, class I, FUMC: fumarate hydratase, class II, MDH1: malate dehydrogenase, MDH2①: malate dehydrogenase, MDH2②: malate dehydrogenase, MDH2③: malate dehydrogenase. (c) in fatty acid synthetic pathway. ACCA: acetyl-CoA carboxylase carboxyl transferase subunit alpha, ACCB: acetyl-CoA carboxylase biotin carboxyl carrier protein, ACCC: acetyl-CoA carboxylase, biotin carboxylase subunit, ACCD: acetyl-CoA carboxylase carboxyl transferase subunit beta, PLSC: 1-acyl-sn-glycerol-3-phosphate acyltransferase, DPP1: diacylglycerol diphosphate phosphatase/phosphatidate phosphatase, DGAT2: diacylglycerol O-acyltransferase 2. (d) in fatty acid degradation pathway. ACSL①: long-chain acyl-CoA synthetase, ACSL②: long-chain acyl-CoA synthetase, ACSL③: long-chain acyl-CoA synthetase, ACSL④: long-chain acyl-CoA synthetase, ACOX1①: acyl-CoA oxidase, ACOX1②: acyl-CoA oxidase, ACOX1③: acyl-CoA oxidase, ACOX1④: acyl-CoA oxidase, ACOX1⑤: acyl-CoA oxidase, ACADM: acyl-CoA dehydrogenase, MFP2: enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, ACAA1: acetyl-CoA acyltransferase 1, ACAT: acetyl-CoA C-acetyltransferase.

Figure 6

(a–d) Comparison of transcription levels of the genes whose products participate in glycolysis, TCA cycle, fatty acid synthetic pathway, and fatty acid degradation pathway of C. reinhardtii cells in each broth. Data are shown as relative mRNA transcription levels normalized by the level of ATPS as a housekeeping gene. The transcription levels at growth phase are displayed with a black bar (6 g∙L⁻¹ WPC34), shaded bar (20 g∙L⁻¹ WP), and gray bar (0.4 g∙L⁻¹ NH₄Cl) in the figure. The explanation of relative transcription level was abbreviated as RTL. Error bars indicate SD of 3–8 time-replicate experiments. Same shapes (such as square, triangle etc…) in figure meant the same genes. Gene abbreviations are shown below: (a) in glycolysis. GPI: glucose-6-phosphate isomerase, FBP: fructose-1,6-bisphosphatase I, PFK①: 6-phosphofructokinase 1, PFK②: 6-phosphofructokinase 1, ALDO①: fructose-bisphosphate aldolase, class I, ALDO②: fructose-bisphosphate aldolase, class I, ALDO③: fructose-bisphosphate aldolase, class I, ALDO④: fructose-bisphosphate aldolase, class I, GAPDH: glyceraldehyde 3-phosphate dehydrogenase (phosphorylating), PGK: phosphoglycerate kinase, GPMI: 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, ENO: enolase. (b) in TCA cycle. PC: pyruvate carboxylase, CS①: citrate synthase, CS②: citrate synthase, ACLY①: ATP citrate (pro-S)-lyase, ACLY②: ATP citrate (pro-S)-lyase, ACO: aconitate hydratase, IDH1: isocitrate dehydrogenase, IDH3①: isocitrate dehydrogenase (NAD+), IDH3②: isocitrate dehydrogenase (NAD+), OGDH: 2-oxoglutarate dehydrogenase E1 component, DLD①: dihydrolipoyl dehydrogenase, DLD②: dihydrolipoyl dehydrogenase, DLST①: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase), DLST②: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase), LSC1: succinyl-CoA synthetase alpha subunit, SDH1: succinate dehydrogenase (ubiquinone) flavoprotein subunit, SDH2: succinate dehydrogenase (ubiquinone) iron-sulfur subunit, SDH3: succinate dehydrogenase (ubiquinone) cytochrome b₅₆₀ subunit, FUMA: fumarate hydratase, class I, FUMC: fumarate hydratase, class II, MDH1: malate dehydrogenase, MDH2①: malate dehydrogenase, MDH2②: malate dehydrogenase, MDH2③: malate dehydrogenase. (c) in fatty acid synthetic pathway. ACCA: acetyl-CoA carboxylase carboxyl transferase subunit alpha, ACCB: acetyl-CoA carboxylase biotin carboxyl carrier protein, ACCC: acetyl-CoA carboxylase, biotin carboxylase subunit, ACCD: acetyl-CoA carboxylase carboxyl transferase subunit beta, PLSC: 1-acyl-sn-glycerol-3-phosphate acyltransferase, DPP1: diacylglycerol diphosphate phosphatase/phosphatidate phosphatase, DGAT2: diacylglycerol O-acyltransferase 2. (d) in fatty acid degradation pathway. ACSL①: long-chain acyl-CoA synthetase, ACSL②: long-chain acyl-CoA synthetase, ACSL③: long-chain acyl-CoA synthetase, ACSL④: long-chain acyl-CoA synthetase, ACOX1①: acyl-CoA oxidase, ACOX1②: acyl-CoA oxidase, ACOX1③: acyl-CoA oxidase, ACOX1④: acyl-CoA oxidase, ACOX1⑤: acyl-CoA oxidase, ACADM: acyl-CoA dehydrogenase, MFP2: enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, ACAA1: acetyl-CoA acyltransferase 1, ACAT: acetyl-CoA C-acetyltransferase.