A new and improved protocol for extraction of intact polar membrane lipids from archaea

Abstract

Knowledge of intact polar membrane lipids (IPLs) is central to investigations of the physiology of archaea in cultures and their prevalence in environmental samples. IPLs are typically extracted using modifications to the ‘Bligh and Dyer’ protocol developed decades before the chemical complexity of IPLs was fully appreciated. Recent studies have exposed both bias and a low extraction efficiency for archaeal lipids obtained by this technique. Here, we tested different solvent mixtures, mechanical rupture techniques and chemical detergents/enzymatic lysis solutions for their potential to increase the extraction efficiency of archaeal IPLs. Experiments were based on an iterative approach using Sulfolobus acidocaldarius biomass as a model organism and incrementally evaluating single-step modifications. Direct acid hydrolysis of biomass was used as a benchmark to assess the recovery of the archaeal lipids. After this initial phase, the resultant protocol was tested on various cultured archaea and environmental samples. Tests with different solvent mixtures revealed the highest S. acidocaldarius lipid yields when samples were extracted with a monophasic solvent system that included a trichloroacetic acid buffer. Freeze-thaw cycles before extraction enhanced the extraction efficiency by 1.8 times, while other mechanical rupture techniques showed no increase. Cetrimonium bromide, a quaternary ammonium surfactant, used in combination with freeze–thaw cycles resulted in up to 8 times higher lipid yields in S. acidocaldarius and other archaeal test cultures compared to experiments without this pretreatment. Extractions of environmental samples revealed contrasting results: while sediments showed little to no increase in archaeal lipid yields, suspended particulate organic matter yielded elevated abundances compared to the Bligh and Dyer extraction protocol.