Gas chromatography for analysis and estimation of 13C at natural abundance level in fatty acids produced from Aurantiochytrium limacinum, a sustainable source of polyunsaturated fatty acid

Abstract

Aurantiochytrium limacinum (A. limacinum) is a promising microbial source of polyunsaturated fatty acids (PUFAs), particularly Docosahexaenoic Acid (DHA, C22:6n-3). In this study, we first optimized the culture conditions of A. limacinum ATCC MYA-1381 (strain SR21). Cell growth was monitored via optical density, cell counts, and glucose concentration. Cells were harvested at exponential and stationary phases, and lipids were extracted using a green method. Fatty Acid Methyl Esters (FAMEs) were prepared and analyzed using Gas Chromatography-Flame Ionisation Detection (GC-FID). At the exponential phase, DHA was the most abundant (65.6% of total fatty acids) followed by palmitic acid (C16:0) at 34.4%. At the stationary phase, Docosapentaenoic acid (DPA, C22:5n-3) and DHA were the most abundant at 45.4% and 33.9%, before respectively. Myristic acid (C14:0), myristoleic acid (C14:1n-9), palmitic acid (C16:0) were present at 4.6%, 6.2% and 9.9%, respectively. Compound-specific isotope analysis (CSIA) using Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) revealed that all FAMEs had negative δ¹³C values, indicating depletion in ¹³C. At the exponential phase, δ¹³C (‰) of C16:0 and DHA were -16.8 ± 0.2 and -18.5‰ ± 0.1‰, respectively. At the stationary phase, δ¹³C (‰) of C14:0, C14:1n-9, C16:0, C22:5n-3 and DHA were -10.6 ± 1.1, -11.3 ± 0.1, -11.1 ± 0.2, -8.3 ± 0.2 and -10.6‰ ± 0.1‰, respectively. Overall, our findings emphasized the importance of A. limacinum as a viable microbial platform for environmentally friendly production of PUFA such as DHA. Also, the study reinforced the utility of CSIA in tracking PUFA metabolic fate, which has latent applications in biomedical research, particularly in neurodegenerative disease frameworks where DHA plays a vital role. Finally, these results may also contribute to understanding isotopic fractionation patterns and metabolic flux variations across different microalgal growth phases.

Keywords: Aurantiochytrium limacinum; CSIA; DHA; GC-C-IRMS; GC-FID; PUFA.

FIGURE 1

Conditions of CO₂ continuous flow for isotope ratios calibration.

FIGURE 2

Multipoint normalization curve for the reporting of true δ¹³C values for GC-IRMS data. FAME (20-carbon) certified reference materials, USGS70, USGS71, and USGS72, were injected periodically during each programmed sequence, totalling at least three injections per run.

FIGURE 3

Cellular absorbance measured by UV-Visible Spectrophotometry at wavelength of 660 nm of the culture of Aurantiochytrium limacinum ATCC MYA-1381 in 200 mL of medium under control condition ¹²C-glucose. (A) Exponential phase culture (B) Stationary phase culture. Values are expressed as means+/-SD (n = 3).

FIGURE 4

Cell counts of Aurantiochytrium limacinum ATCC MYA-1381 in 200 mL of medium under control condition ¹²C-glucose. (A) Exponential phase culture (B) Stationary phase culture. Values are expressed as means+/-SD (n = 3)

FIGURE 5

Glucose consumption at exponential and stationary phase cultures of Aurantiochytrium limacinum ATCC MYA-1381 on the day of harvesting. Values are expressed as means+/-SD (n = 3).

FIGURE 6

Biomass collected from Aurantiochytrium limacinum ATCC MYA-1381 at exponential and stationary phase cultures. Values are expressed as means+/-SD (n = 3).

FIGURE 7

Fatty acid composition of Aurantiochytrium limacinum ATCC MYA-1381 in 200 mL of culture medium under control conditions at exponential (A) and stationary phase (B) using GC-FID analysis. Results are expressed as percentage of FA and expressed as means+/-SD (n = 3).

FIGURE 8

Isotopic signatures of Reference Materials: (A) USGS70, (B) USGS71 and (C) USGS72.

FIGURE 9

GC-IRMS analysis of FAMEs derived from total lipid extracted from Aurantiochytrium limacinum at exponential phase (A) and stationary phase (B). The orange plot shows the signal ratio for m/z 45/44. The blue plot shows the signal for m/z 45 for the duration of the IRMS run.

FIGURE 10

δ¹³C signature of FAME at exponential and stationary phase using GC-C-IRMS analysis. Results are expressed as percentage of FA and expressed as means+/-SD (n = 3).