# The Cell Membrane of _Sulfolobus_ spp.--Homeoviscous Adaption and Biotechnological Applications
## 4 Extraction and Lipid Analysis  
In a lot of cases, contradictory results can be a consequence of different extraction or detection methods. Exemplarily, it has been shown that the lack of a lipid class in the membrane structure was due to the extraction method, rather than an actual observation [96; 97]. The organic extraction of lipids from biological tissue samples was first described by [PERSON] et al. in 1957. It consists of chloroform/methanol/water, in a ratio of 8:4:3 (_v/v/v_). The idea was that after homogenization in chloroform and methanol mixture, water is added and thus, forms a new aqueous phase. The polar upper phase consisting of methanol and water then contains the hydrophilic compounds, whereas the lower chloroform phase holds the lipids. The recovery of phospholipids and neutral lipids is rather efficient with this method [98; 99]. [PERSON] and [PERSON] modified this method to a volumetric ratio of 2:2:1.8 of chloroform/methanol/water, thereby reducing the amounts of chloroform and methanol. In general, the extraction procedure is the same as by [PERSON] et al. However, other groups already showed that this system has limitations, especially towards neutral lipids. Modification to the protocol has been already suggested by [PERSON] and [PERSON] themselves. However, their proposed re-extraction steps were not commonly applied [100; 101]. Other alterations included the addition of HCl for the better recovery of total fatty acids and polyunsaturated fatty acids [102] and acetic acid for oxidized fatty acids [103]. However, the application of acid hydrolysis removes the polar headgroup of the GDGTs, and consequently, it is only possible to recover core GDGTs [104]. The quantitative recovery of each lipid class is influenced by many variables. Especially, the choice of extraction solvent is important, as it determines the lipid class that is extracted. Non-polar solvents, such as hexane, extract neutral lipids. The polar methanol is used to extract charged phospholipids. More polar glycolipids need a mixture, for example, of methanol and acetone. Additionally, the pH and ion strength of the solvent mixture can also have an impact on the recoveries. The recoveries are greatly dependent on the extraction efficiencies and determining the extraction recovery is unfortunately difficult [105]. The mentioned articles regarding the observation of homeoviscous adaption in Sulfolobales mainly used methanol and chloroform in varying ratios as extraction solvents [42; 43; 91; 93]. [PERSON] et al. and [PERSON] et al. used methanol in combination with dichloromethane, which has similar physical and chemical properties as chloroform, but exhibits less carcinogenic and hazardous characteristics, and was shown to be an effective alternative [39; 79; 106]. [PERSON] et al. used methyl-tert-butyl-ether (MTBE) and methanol in a ratio of 1:1 (_v_/_v_). MTBE's low density causes the formation of the upper phase, including the extracted lipids, facilitating their collection when compared to the methanol/chloroform solvent system [78; 107]. As [PERSON] et al. and [PERSON] et al. applied similar cultivation strategies in _S. acidocaldarius_ and subsequently determined the RI number, which seemed to be generally lower in the first publication, one might wonder if the difference can be attributed to the used solvents. [PERSON] et al., who used the common ratio of 2:1 of chloroform:methanol, discovered higher average cyclization numbers of around 4.6, consistent with the observations in _S. tokdaii_ and _S. islandicus_ by [PERSON] et al. [42; 43; 78]. These results already indicate that a uniform lipid extraction method has to be established, in order to facilitate comparison as well as draw conclusions of the results in their entirety. An additional factor of discrepancy concerning the found lipid classes is the detection method. TLC results are sometimes difficult to interpret, and the method should therefore only be used as separation step prior to an identification. TLC separates the components of the mixture by its polarity.