Steady-state and time-resolved spectroscopy of F420 extracted from methanogen cells and its utility as a marker for fecal contamination

Methanogenic bacteria, which are common inhabitants of the animal digestive tract, contain the fluorescent compound F420 (coenzyme 420), a 7,8-didemet hyl-8-hydroxy-5-deazariboflavin chromophore. F420 was characterized as an i nitial step in determining if this compound would be useful as a fluorescen t marker for the detection of fecal and ingesta contamination. Using a sing le anion exchange chromatographic process, F420 was separated from other ce ll components of a Methano-brevibacter sp. cell culture. The extent of sepa ration was determined spectroscopically. To aid in the development of possi ble techniques for the detection of fecal contamination using F420 as a mar ker, further spectroscopic investigation of F420 was conducted using steady -state and time-resolved fluorescence methods. The fluorescence lifetime of F420 in an elution buffer of pH 7.5 was found to be 4.2 ns. At higher pH v alues, the fluorescence decay, F(t), was best described by a sum of two exp onentials: at pH 13, P(t) = 0.31 exp(-t/4.20 ns) + 0.69 exp(-t/1.79 ns). Fu rther investigation using front-faced fluorescence techniques' has shown th at-emission from F420 can be collected efficiently from samples of methanog en cell cultures as well as from fecal material.