A novel protocol for extracting 3-hydroxy fatty acids from soil with microwave-assisted acid digestion

Abstract

In recent years, bacterial 3-hydroxy fatty acids (3-OH-FAs) have gained increased interest in paleoclimate reconstruction due to their ubiquitous distributions and potential as paleoclimate proxies in palaeo- and modern environmental samples. However, the current extraction method for 3-OH-FAs is time-consuming and not feasible for samples with low lipid concentrations, which greatly hinders the broad application of 3-OH-FA-based proxies. Therefore, it is vital to improve the extraction method to shorten the extraction time and increase the extraction efficiency and stability. Here, we report a novel extraction protocol with microwave-assisted acid digestion for 3-OH-FAs extraction. The operating conditions (such as HCl concentration, heating temperature, and irradiation time) of this novel protocol are optimised via orthogonal array experimental design by analysing 3-OH-FAs from surface soil samples collected from Mt. Yujia, central China. The optimum digestion condition is recommended to be 3 mol/L HCl heated at 130 °C for 55 min. The yield of 3-OH-FAs obtained by the optimised microwave-assisted acid digestion method is about twice as much as that of the traditional acid digestion method, with relatively better reproducibility. Moreover, we re-treated the same soil samples previously reported by Wang et al. (2021) with the optimised microwave-assisted acid digestion method and found that the distribution of 3-OH-FAs and calculated 3-OH-FA-based proxies are identical to those extracted by the traditional acid digestion method. Furthermore, a comparative investigation was conducted on other lipids, such as n-alkanes, fatty acids (FAs), and glycerol dialkyl glycerol tetraethers (GDGTs), among several prevalent methods, including ultrasonic extraction, accelerated solvent extraction, conventional microwave-assisted extraction, traditional acid digestion and microwave-assisted acid digestion established in this study. Our findings show that different extraction methods may produce varying amounts of lipids and values of lipid-based proxies. Consequently, the differences caused by different extraction methods should be considered in comparison and palaeoclimate reconstruction.