Comparison of extraction methods for quantitative analysis of core and intact polar glycerol dialkyl glycerol tetraethers (GDGTs) in environmental samples

Abstract

Archaeal membranes are composed of core lipids called glycerol dialkyl glycerol tetraethers (GDGTs) that contain polar head groups (intact GDGTs) in living cells. The reliability of Archaeal membrane lipids in studies of paleogeochemistry, population distributions, and physiological state depend on efficient extraction and detection of these lipids. We compare existing methods for extraction of core and intact GDGTs so that they can be used as quantitative indicators of living archaeal biomass. We used an active culture of the marine archaeon, Nitrosopumilus maritimus, to document limitations of methods in current use and as a reference for methodological improvement. We found that all extractable GDGTs in the culture were intact and that acid hydrolysis is the only way to quantitatively recover GDGTs from exponentially growing cells. Because acid hydrolysis removes polar head groups, it is not suitable for analysis of intact lipids. In contrast to the culture, high recovery was achieved for intact and core lipids from environmental samples, including water column particles, sediments, and soils. The high recovery from environmental samples and the large portion of core GDGTs relative to intact GDGTs suggest that lipids in the environment are largely derived from dead or dying cells. Because previous studies measured core GDGTs without acid hydrolysis, lipids contained in living cells were not detected and thus these results do not reflect viable biomass. We present a method to accurately quantify living and relict GDGTs by choosing the most efficient extraction protocol and measuring core GDGTs in extracts before and after acid hydrolysis. Here we set out to develop a method to use I-GDGTs as quantitative tracers of viable cell abundance in the environment Sturt et al. 2004). Using traditional extraction techniques on cultures of the mesophilic marine crenarchaeota, Nitrosopumilus maritimus, we discovered that lipid yields per cell varied considerably depending on the extraction technique used. Whereas it is well known that membrane lipids of archaeal cells can be difficult to extract, there are no recent reports that quantify the efficiency of currently used extraction techniques. Moreover, researchers often give no rationale for choosing one extraction protocol over another. As applications of both core and intact GDGTs grow, it is important to compare and unify GDGT extraction techniques. We searched the literature for GDGT extraction methods and measured the efficiency and degree of polar head group loss for each method using a culture of exponentially growing N. maritimus that contained only I-GDGTs. We then analyzed samples from environments where GDGT-based proxies are commonly measured, including marine water column particles, marine sediments, and soils.We evaluated five basic techniques: solvent extraction with mild acid to enhance the lyses of archaeal cells (e.g., Bligh and Dyer 1959;Nishihara and Koga 1987), accelerated solvent extraction (ASE) (e.g., Huguet et al. 2007b; Kim et al. 2008), ...

Comparison of extraction and work up techniques for analysis of core and intact polar tetraether lipids from sedimentary environments

“…2). This is in agreement with observations by Huguet et al (2010) and indicates that ASE is not an effective method for IPL analysis, most likely due to the high (100 °C) temperature applied, leading to partial destruction of the IPLs, or to the use of diatomaceous earth to which part of the IPLs may have adsorbed. The error of the 'subtraction-method' was substantially higher than that of the separation method.…”

“…2; Huguet et al, 2010), Bligh-Dyer extraction is essential for determining the full suite of IPLGDGT head groups. This is particularly relevant as GDGTs with a phospho head group are predicted to be more labile and so may be a more suitable marker for living Archaea.…”

“…The IPL fraction was hydrolyzed for 3 h under reflux in 2N methanolic HCl to release the IPL-derived GDGTs (Pitcher et al, 2009b). Another aliquot of 30% was analyzed by way of a 'subtraction technique' as described by Huguet et al (2010). It was divided into two equal parts.…”

“…For the extracts, concentration was determined for CL-GDGTs and IPL-derived GDGTs using the analytical protocols based on chromatographic separation over SiO 2 (Pitcher et al, 2009) and the 'subtraction' method (Huguet et al, 2010). Concentrations of all GDGTs studied (Fig.…”

Marine ammonia-oxidizing archaeal isolates display obligate mixotrophy and wide ecotypic variation

“…These distinctive differences are directly relevant to their possible contribution to nitrification in different marine environments-including lower salinity coastal regions, the photic zone of the upper water column, and the increasing acidification of ocean waters associated with climate change. (8,38,39). Predominance of AOA was consistent with generally submicromolar concentrations of ammonia at these stations, sufficient to support AOA such as SCM1 but not known AOB (24).…”