Limits...
High-throughput metabolic screening of microalgae genetic variation in response to nutrient limitation.

Bajhaiya AK, Dean AP, Driver T, Trivedi DK, Rattray NJ, Allwood JW, Goodacre R, Pittman JK - Metabolomics (2015)

Bottom Line: Limitation of nutrients including nitrogen and phosphorus can induce metabolic changes in microalgae, including the accumulation of glycerolipids and starch.These results demonstrate that the PSR1 gene is an important determinant of lipid and starch accumulation in response to phosphorus starvation but not nitrogen starvation.However, the SNRK2.1 and SNRK2.2 genes are not as important for determining the metabolic response to either nutrient stress.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT UK.

ABSTRACT

Microalgae produce metabolites that could be useful for applications in food, biofuel or fine chemical production. The identification and development of suitable strains require analytical methods that are accurate and allow rapid screening of strains or cultivation conditions. We demonstrate the use of Fourier transform infrared (FT-IR) spectroscopy to screen mutant strains of Chlamydomonas reinhardtii. These mutants have knockdowns for one or more nutrient starvation response genes, namely PSR1, SNRK2.1 and SNRK2.2. Limitation of nutrients including nitrogen and phosphorus can induce metabolic changes in microalgae, including the accumulation of glycerolipids and starch. By performing multivariate statistical analysis of FT-IR spectra, metabolic variation between different nutrient limitation and non-stressed conditions could be differentiated. A number of mutant strains with similar genetic backgrounds could be distinguished from wild type when grown under specific nutrient limited and replete conditions, demonstrating the sensitivity of FT-IR spectroscopy to detect specific genetic traits. Changes in lipid and carbohydrate between strains and specific nutrient stress treatments were validated by other analytical methods, including liquid chromatography-mass spectrometry for lipidomics. These results demonstrate that the PSR1 gene is an important determinant of lipid and starch accumulation in response to phosphorus starvation but not nitrogen starvation. However, the SNRK2.1 and SNRK2.2 genes are not as important for determining the metabolic response to either nutrient stress. We conclude that FT-IR spectroscopy and chemometric approaches provide a robust method for microalgae screening.

No MeSH data available.


Related in: MedlinePlus

Screening nutrient limitation conditions. Fresh weight biomass (a, e), chlorophyll fluorescence (Fv/Fm ratio) (b, f), lipid:amide I ratio (c, g) and carbohydrate:amide I ratio (d, h) values derived from FT-IR spectra, of wild type Chlamydomonas reinhardtii grown under different concentrations of N (a–d) and P (e–g) for 7 days. All data are mean ± SE of 3 biological replicates. Asterisks denote significant difference compared to control (7 mM P or 1 mM N) treatments
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Fig1: Screening nutrient limitation conditions. Fresh weight biomass (a, e), chlorophyll fluorescence (Fv/Fm ratio) (b, f), lipid:amide I ratio (c, g) and carbohydrate:amide I ratio (d, h) values derived from FT-IR spectra, of wild type Chlamydomonas reinhardtii grown under different concentrations of N (a–d) and P (e–g) for 7 days. All data are mean ± SE of 3 biological replicates. Asterisks denote significant difference compared to control (7 mM P or 1 mM N) treatments

Mentions: Initial baseline screening was performed on wild type C. reinhardtii to examine and compare the metabolic responses to five decreasing concentrations of P (from 1 mM to 1 µM) or N (from 7 mM to 0.07 mM) at late exponential phase (day seven). As seen previously, reduced nutrient availability led to a reduction in biomass production, with a consistent step-by-step decrease in cell biomass as N concentration decreased until there was very little cell growth at 0.07 mM N (a 92 % decrease) (Fig. 1a). The exception was the 3.5 mM N treatment, which stimulated biomass production. A reduction in P concentration to 0.1 mM P did not cause any significant change in growth, while a reduction to 0.05 mM P started to inhibit growth slightly, although not significantly, suggesting mild-to-moderate P limitation, but biomass production was very significantly reduced following 10 µM and 1 µM P treatment (a 56 and 87 % decrease, respectively) (Fig. 1e). Chlorophyll fluorescence (Fv/Fm ratio) was measured as an indicator of stress and physiological status of the cell. The Fv/Fm ratio profile for both P and N limitation treatment was equivalent to the biomass production profile, with a significant reduction in Fv/Fm ratio in response to severe P or N limitation but severe N limitation appeared to be more stressful to the cells (Fig. 1b, f). Following analysis by FT-IR spectroscopy over the wavenumber range of 1780–950 cm−1, clear variation was apparent in the baseline-corrected spectra from cells exposed to the different P and N treatments (Supplementary Fig. 1). The strong peak at ~1740 cm−1 visible in spectra from both P and N limited cells is indicative of an increase in total lipid whereas overlapped bands between ~1160 and 1036 cm−1 are indicative of carbohydrate increases (Dean et al. 2010; Stehfest et al. 2005). These spectral changes indicated that total lipid and carbohydrate responses were the most pronounced following severe N and P limitation. Quantification of total lipid, and carbohydrate band heights, were normalised by expressing these as a ratio to the amide I band (1655 cm−1). There was increasing accumulation of lipid and carbohydrate in response to 0.7, 0.3 and 0.07 N (Fig. 1c, d) and in response to 1 and 10 µM P (Fig. 1g–h) but the N-limited cells clearly accumulated more lipid and carbohydrate than any of the P-limited cells. For example, the carbohydrate:amide I ratio value increased from 0.19 in 1 mM P/7 mM N cells to 2.76 in 1 µM P cells (14.5-fold increase) but increased to 9.14 in 0.07 mM N cells (48.1-fold increase), while the lipid:amide I ratio value increased from 0.16 in 1 mM P/7 mM N cells to 0.83 in 1 µM P cells (5.2-fold increase) but increased to 3.87 in 0.07 mM N cells (24.2-fold increase).Fig. 1


High-throughput metabolic screening of microalgae genetic variation in response to nutrient limitation.

Bajhaiya AK, Dean AP, Driver T, Trivedi DK, Rattray NJ, Allwood JW, Goodacre R, Pittman JK - Metabolomics (2015)

Screening nutrient limitation conditions. Fresh weight biomass (a, e), chlorophyll fluorescence (Fv/Fm ratio) (b, f), lipid:amide I ratio (c, g) and carbohydrate:amide I ratio (d, h) values derived from FT-IR spectra, of wild type Chlamydomonas reinhardtii grown under different concentrations of N (a–d) and P (e–g) for 7 days. All data are mean ± SE of 3 biological replicates. Asterisks denote significant difference compared to control (7 mM P or 1 mM N) treatments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
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Fig1: Screening nutrient limitation conditions. Fresh weight biomass (a, e), chlorophyll fluorescence (Fv/Fm ratio) (b, f), lipid:amide I ratio (c, g) and carbohydrate:amide I ratio (d, h) values derived from FT-IR spectra, of wild type Chlamydomonas reinhardtii grown under different concentrations of N (a–d) and P (e–g) for 7 days. All data are mean ± SE of 3 biological replicates. Asterisks denote significant difference compared to control (7 mM P or 1 mM N) treatments
Mentions: Initial baseline screening was performed on wild type C. reinhardtii to examine and compare the metabolic responses to five decreasing concentrations of P (from 1 mM to 1 µM) or N (from 7 mM to 0.07 mM) at late exponential phase (day seven). As seen previously, reduced nutrient availability led to a reduction in biomass production, with a consistent step-by-step decrease in cell biomass as N concentration decreased until there was very little cell growth at 0.07 mM N (a 92 % decrease) (Fig. 1a). The exception was the 3.5 mM N treatment, which stimulated biomass production. A reduction in P concentration to 0.1 mM P did not cause any significant change in growth, while a reduction to 0.05 mM P started to inhibit growth slightly, although not significantly, suggesting mild-to-moderate P limitation, but biomass production was very significantly reduced following 10 µM and 1 µM P treatment (a 56 and 87 % decrease, respectively) (Fig. 1e). Chlorophyll fluorescence (Fv/Fm ratio) was measured as an indicator of stress and physiological status of the cell. The Fv/Fm ratio profile for both P and N limitation treatment was equivalent to the biomass production profile, with a significant reduction in Fv/Fm ratio in response to severe P or N limitation but severe N limitation appeared to be more stressful to the cells (Fig. 1b, f). Following analysis by FT-IR spectroscopy over the wavenumber range of 1780–950 cm−1, clear variation was apparent in the baseline-corrected spectra from cells exposed to the different P and N treatments (Supplementary Fig. 1). The strong peak at ~1740 cm−1 visible in spectra from both P and N limited cells is indicative of an increase in total lipid whereas overlapped bands between ~1160 and 1036 cm−1 are indicative of carbohydrate increases (Dean et al. 2010; Stehfest et al. 2005). These spectral changes indicated that total lipid and carbohydrate responses were the most pronounced following severe N and P limitation. Quantification of total lipid, and carbohydrate band heights, were normalised by expressing these as a ratio to the amide I band (1655 cm−1). There was increasing accumulation of lipid and carbohydrate in response to 0.7, 0.3 and 0.07 N (Fig. 1c, d) and in response to 1 and 10 µM P (Fig. 1g–h) but the N-limited cells clearly accumulated more lipid and carbohydrate than any of the P-limited cells. For example, the carbohydrate:amide I ratio value increased from 0.19 in 1 mM P/7 mM N cells to 2.76 in 1 µM P cells (14.5-fold increase) but increased to 9.14 in 0.07 mM N cells (48.1-fold increase), while the lipid:amide I ratio value increased from 0.16 in 1 mM P/7 mM N cells to 0.83 in 1 µM P cells (5.2-fold increase) but increased to 3.87 in 0.07 mM N cells (24.2-fold increase).Fig. 1

Bottom Line: Limitation of nutrients including nitrogen and phosphorus can induce metabolic changes in microalgae, including the accumulation of glycerolipids and starch.These results demonstrate that the PSR1 gene is an important determinant of lipid and starch accumulation in response to phosphorus starvation but not nitrogen starvation.However, the SNRK2.1 and SNRK2.2 genes are not as important for determining the metabolic response to either nutrient stress.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT UK.

ABSTRACT

Microalgae produce metabolites that could be useful for applications in food, biofuel or fine chemical production. The identification and development of suitable strains require analytical methods that are accurate and allow rapid screening of strains or cultivation conditions. We demonstrate the use of Fourier transform infrared (FT-IR) spectroscopy to screen mutant strains of Chlamydomonas reinhardtii. These mutants have knockdowns for one or more nutrient starvation response genes, namely PSR1, SNRK2.1 and SNRK2.2. Limitation of nutrients including nitrogen and phosphorus can induce metabolic changes in microalgae, including the accumulation of glycerolipids and starch. By performing multivariate statistical analysis of FT-IR spectra, metabolic variation between different nutrient limitation and non-stressed conditions could be differentiated. A number of mutant strains with similar genetic backgrounds could be distinguished from wild type when grown under specific nutrient limited and replete conditions, demonstrating the sensitivity of FT-IR spectroscopy to detect specific genetic traits. Changes in lipid and carbohydrate between strains and specific nutrient stress treatments were validated by other analytical methods, including liquid chromatography-mass spectrometry for lipidomics. These results demonstrate that the PSR1 gene is an important determinant of lipid and starch accumulation in response to phosphorus starvation but not nitrogen starvation. However, the SNRK2.1 and SNRK2.2 genes are not as important for determining the metabolic response to either nutrient stress. We conclude that FT-IR spectroscopy and chemometric approaches provide a robust method for microalgae screening.

No MeSH data available.


Related in: MedlinePlus