Biphytane (or bisphytane) is a C₄₀ isoprenoid produced from glycerol dialkyl glycerol tetraether (GDGT) degradation. As a common lipid membrane component, biphytane is widely used as a biomarker for archaea. In particular, given its association with sites of active anaerobic oxidation of methane (AOM), it is considered a biomarker of methanotrophic archaea. It has been found in both marine and terrestrial environments.

Glycerol dialkyl glycerol tetraethers (GDGT) are major membrane lipids synthesized by archaea and some bacteria. In particular, isoprenoid GDGTs are characterized by isoprenoid carbon chains connected to the glycerol molecules by ether bonds. Biphytane is produced by the chemical cleavage of the ether bonds within isoprenoid GDGT (GDGT-0). It is composed of isoprene units bound by ether bonds with six isoprene units (or two phytanes) linked together by a head-to-head linkage.

Biphytane can be found in cyclic forms containing one to three pentacyclic rings when derived from isoprenoid GDGTs with such biosynthetically cyclized isoprenoid carbon skeletons. In most analyzed samples from the environment, the acyclic form with biphytane as the isoprenoid carbon chain is typically the most abundant form. Hence, in this article, biphytane is used to refer to the acyclic form unless stated otherwise.

As it occurs within GDGT, biphytane has been detected in the water column, marine sediments, hydrothermally-influenced sediments, cold seep sediments dominated by anaerobic oxidation of methane activity, and limestone. Though it had been primarily studied in aquatic settings, recent studies have also started investigating terrestrial environments, such as peat bogs where the source of biphytane was identified as methanogenic peat archaea. Studies have reported the detection of biphytane in petroleum as well.

While early studies had considered GDGTs (and hence biphytane) to be biomarkers of extremophilic archaea, both indirect and direct evidence of GDGT originating from archaea of mesophilic marine environments or lacustrine environments with non-extreme pH and salinity have been available since the late 1970s. Because biphytane in particular has been widely detected in sties of active AOM activity, it is considered a biomarker of methanotrophic archaea.

Analogous to sterols in eukaryotic membranes, GDGT plays a similar role in improving the rigidity of archaeal cell membranes. Supporting this, it has been reported that thermophiles increase the degree of cyclization with increasing growth temperatures to further improve membrane fluidity.

Typically, biphytane measurement is performed as an indirect analysis of GDGT. When chemically deriving biphytane from such ether lipids, the ether bonds are first cleaved using hydrogen iodide (HI), boron trichloride (BCl₃), or boron tribromide (BBr₃) that produces alkyl halides. Then, the alkyl halides are either reduced to saturated hydrocarbons using HI/NaSCH₃ or LiAlH₄ or converted to methylthioesthers with NaSCH₃. The obtained saturated or derivatized hydrocarbons can subsequently be separated and measured using standard gas chromatography-mass spectrometry (GC-MS) procedures.

Alternatively, direct analysis of GDGT can be done with liquid chromatography but, when further structural characterization is required, MS fragments characteristic of biphytane can be obtained via high-performance liquid chromatography linked to tandem mass spectrometry (HPLC-MS/MS).