Unusual Shorter‐Chain C₃₅ and C₃₆ Alkenones from Commercially Grown Isochrysis sp. Microalgae

Abstract

Unusual shorter-chain alkenones (C 35 and C 36 ) were the dominant alkenones (rather than C 37 and C 38 ) isolated from commercially grown Tisochrysis lutea microalgae. Structures of the alkenones were determined by analyzing the alkenones and their products following a butenolysis reaction by comprehensive two-dimensional gas chromatography coupled to a high-resolution time-of-flight mass spectrometer (GC × GC-TOF HRMS). The major alkenones were identified as (15E,22E)-heptatriaconta-15,22-dien-2-one and (16E,23E)-octatriaconta-16,23-dien-3-one, similar to one previous finding of abundant shorter-chain alkenones in a culture of Emiliania huxleyi CCMP2758, yet distinguishing them from C 35 and C 36 alkenones extracted from Black Sea sediments with different double bond positions. Our data suggest a possible shared trigger (e.g. nutrient stress) that may occur during cultivation resulting in shorter-chain alkenones from both the aforementioned E. huxleyi and T. lutea. Moreover, for T. lutea, short-chain alkenone biosynthesis can be accompanied by lower alkenone content but higher amounts of valuable docosahexaenoic acid (DHA). These results, therefore, extend beyond alkenones and are important to established and emerging algal oil-based industries.Keywords Isochrysis Á Tisochrysis Á Alkenones Á Omega-3 fatty acids Á Butenolysis Á Comprehensive twodimensional gas chromatography

Monitoring Changes to Alkenone Biosynthesis in Commercial Tisochrysis lutea Microalgae

“…Previously, we used a CM reaction with 2-butene (“butenolysis”) to determine double bond positions when analyzing C35/C36 alkenones isolated from 2018 Phytobloom T-Iso (Scheme ). This reaction effectively cleaves alkenones at their carbon–carbon double bonds, producing a predictable mixture of alkenone fragments from which the original alkenone structure can be deduced. Compared to other chemical methods for alkenone double bond position determination, − butenolysis is attractive in giving high yields of more GC-amenable stable alkenone derivatives.…”

“…Since alkenone hexenolysis produced a complex mixture of products that was challenging to analyze by GC–FID/GC–MS, we opted to employ comprehensive two-dimensional gas chromatography (GC × GC). The increased resolution afforded by this technique has been successfully applied to the analysis of highly complex mixtures such as petroleum products, alkenone-containing extracts, and our previous alkenone butenolysis reactions . Coupled to a high-resolution time-of-flight mass spectrometer (TOF HRMS), the enhanced resolution and accurate mass data allow for a rigorous determination of alkenone structure.…”

“…Alkenones and FAs were isolated and purified from the 2021 and 2023 Phytobloom T-Iso , as previously described. , Briefly, extraction with hexanes by Soxhlet produced a dark green near-black oily solid called algal oil. The latter was then redissolved in methanol/DCM (2:1, 10 × volume of algal oil) and treated with KOH (50% w/w) at 60 °C for 3 h. The resulting saponified acylglycerols were selectively partitioned into water, and the alkenone-containing neutral lipids were extracted into hexanes.…”

“…Secondary metathesis was not observed during our previous studies on alkenone upgrading by CM with methyl acrylate (Scheme 3). 25 In this case, the double bonds in the CM products are significantly different (i.e., conjugated with C� O) than those in the starting alkenones and less prone to secondary metathesis, 47 which is not the case for hexenolysis of CM products. As a result of these large structural differences between the starting alkenones and CM products, the progress of alkenone/methyl acrylate CM reactions can be tracked by 1 H NMR.…”

“…In 2018, however, unusual C35 and C36 alkenones were isolated as the major alkenones from a 4 kg batch of Phytobloom (Figure ). After rigorous structure determination, we deduced that their structure matched those produced by the only other species of algae known to make primarily shorter-chain alkenones (Emiliania huxleyi CCMP2758). The observed shift in the composition and relative abundance of alkenones provide an opportunity to explore algae phylogeny and taxonomy, , alkenone biosynthesis, and determine the impacts on paleothermometry and other alkenone-based technologies.…”