Qualitative analysis of algal secretions with multiple mass spectrometric platforms

Abstract

Lipid secretions from algae pose a great opportunity for engineering biofueler feedstocks. The lipid exudates could be interesting from a process engineering perspective because lipids could be collected directly from the medium without harvesting and disrupting cells. We here report on the extracellular secretions of algal metabolites from the strain UTEX 2341 (Chlorella minutissima) into the culture medium. No detailed analysis of these lipid secretions has been performed to date. Using multiple mass spectrometric platforms, we observed around 1000 compounds and were able to annotate 50 lipids by means of liquid chromatography coupled to accurate mass quadrupole time-of-flight mass spectrometry (LC-QTOF), direct infusion with positive and negative electrospray ion trap mass spectrometry and gas chromatography coupled to mass spectrometry (GC-MS). These compounds were annotated by tandem mass spectral (MS/MS) database matching and retention time range filtering. We observed a series of triacylglycerols (TG), sulfoquinovosyldiacylglycerols (SQDG), phosphatidylinositols and phosphatidylglycerols, as well as betaine lipids diacylglyceryl-N,N,N-trimethylhomoserines (DGTS).

Fig. 1

The algae UTEX 2341 (Chlorella minutissima) secretes lipid material into the surrounding media. The photo shows a circular yellow secretion around the green cell body. The exudate was collected, extracted and analyzed with different analytical techniques. Bacterial contaminations were not observed by means of a microscopy method.

Fig. 2

Analytical platform to investigate extracellular lipid secretions from algae. Four different mass spectrometric platforms were used to analyze a complex mixture of lipids.

Fig. 3

A GC–MS fatty acid methyl ester (FAME) analysis revealed two major peaks in the secretions. These peaks in the chromatogram were identified as C16:0 (hexadecanoic acid methyl ester) and C18:0 (octadecanoid acid methyl ester) using retention time and mass spectral matching.

Fig. 4

Chip based nanoelectrospray infusion allows for long direct infusion and data dependent MS/MS. Complex lipids were observed in the algal secretions. The tandem mass spectra are then searched in MS/MS libraries.

Fig. 5

Hydrophilic interaction chromatography (HILIC-UPLC) coupled to high resolution QTOF-MSMS reveals a complex mixture of compounds. A series of lipids are annotated using MS/MS scans including triacylglycerols (TG), sulfoquinovosyl-diacylglycerols (SQDG), phosphatidylinositols (PI) and phosphatidylcholines (PC).

Fig. 6

Left panel: low resolution iontrap MS/MS spectrum from precursor 736.61 Da was identified in a MS/MS library search as the betaine lipid DGTS 34:2; [M+H]⁺; DGTS(16:0/18:2). Right panel: high resolution QTOF-MS/MS, the substance with precursor 850.7866821 Da was identified as TG 50:1; [M+NH4]⁺; TG(16:0/16:0/18:1). Precursor mass accuracy was found as −0.00079 Da (0.93 ppm).